University of Southern California Dissertations and Theses

The function of BS69 in mouse embryogenesis and embryonic stem cell differentiation

(USC Thesis Other)

The function of BS69 in mouse embryogenesis and embryonic stem cell differentiation

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content Copyright 2015 Marie Rippen

THE FUNCTION OF BS69 IN MOUSE EMBRYOGENESIS
AND EMBRYONIC STEM CELL DIFFERENTIATION

by

Marie Rippen

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)

August 2015

ii

Acknowledgements

Looking back over the past six years, I know that I would not have become the scientist
that I am today without the guidance of my advisor, Dr. Krzysztof Kobielak. Kris, the
lessons that I learned in your laboratory were invaluable and will influence my career. I
thank you for teaching me the value of dogged persistence in the face of repeated failures
and the importance of stopping to look at the big picture. I am also grateful to my committee
members, Dr. Robert Maxson and Dr. David Warburton. Thank you for your support,
especially toward the end. Your encouragement and advice helped me push through the
last few months to finish this dissertation.

I was fortunate to benefit from many professors’ input over the years and I sincerely thank
them. Dr. Agnieszka Kobielak, thank you for contributing your ideas, technical skills, and
a lot of time to help me get through the most difficult parts of my project. Dr. Franscesca
Mariani, thank you for always making time to assist me, especially with troubleshooting
experiments. Dr. Qilong Ying, thank you for your expertise that was essential to some of
my experiments. Dr. Yibu Chen, thank you for teaching me more about bioinformatics than
just what was necessary for my project.

I am incredibly grateful to my lab mates, without whom I may not have finished my degree.
Eve Kandyba and Yvonne Leung, thank you for always being ready for “tea time” and for
commiserating when I felt stuck. Also, thank you for teaching me to figure things out for
myself and to not be afraid of making mistakes. Thank you, Keerthi Boddupally, for being
a great source of support and friendship. I admire your composure in even the most stressful
iii

situations. Although only in the lab for one year, I thank Jenny Shevchenko for always
taking the initiative and maintaining a great work ethic—I often forgot that you were still
a high school student.

An amazing source of friendship and sanity throughout this journey has been my Granada
family. You all listened to my difficulties and helped me forget them, even if it was just
for a few hours. Thank you for constantly reminding me to laugh and enjoy life, especially
during the most difficult parts of my project. I particularly want to thank my partner in
crime, Brigitte Ryerson. Brigitte, you heard more about the goings-on of a laboratory than
I think anyone would ever want to know, and you are still my friend! All joking aside, you
encouraged me to try things that I didn’t think I could do, and that has carried over to other
areas of my life.

Finally, I want to thank the most important reason that I was able to finish my PhD: my
family. Mom, thank you for letting me talk your ear off in the middle of the day whenever
I needed to and for being genuinely interested in whatever I’m doing. Dad, thank you for
reminding me that you’re proud of me no matter what and for having really awesome
conversations about everything from science to politics. Emily, thank you for being an
example of real strength and determination and for getting angrier than I do when life
doesn’t go my way.

I truly appreciate everyone who helped me through this process. Because of you, I
accomplished the goal that I’ve pursued since childhood—to become a scientist.
iv

Table of Contents

Acknowledgements ............................................................................................................. ii
List of Tables ..................................................................................................................... vi
List of Figures ................................................................................................................... vii
List of Abbreviations ......................................................................................................... ix
Abstract ..............................................................................................................................xv
Chapter 1: Introduction ........................................................................................................1
1.1 Regenerative Medicine .............................................................................................. 1
1.2 Pre-implantation Embryogenesis .............................................................................. 3
1.3 Axis Formation .......................................................................................................... 5
1.4 Gastrulation ............................................................................................................... 9
1.5 BS69 ........................................................................................................................ 13
Chapter 2 : BS69 is Required for the Formation of Mesendoderm During
Gastrulation ........................................................................................................................23
2.1 Abstract ................................................................................................................... 23
2.2 Introduction ............................................................................................................. 24
2.3 Results ..................................................................................................................... 27
2.4 Discussion ............................................................................................................... 44
Chapter 3 : BS69 is Required for the Formation of Mesendoderm from Mouse
Embryonic Stem Cells In Vitro, but is Dispensable for Neuroectoderm
Differentiation ....................................................................................................................50
3.1 Abstract ................................................................................................................... 50
3.2 Introduction ............................................................................................................. 51
3.3 Results ..................................................................................................................... 53
3.4 Discussion ............................................................................................................... 71
Chapter 4 : Long-Term Consequences of BS69 Function .................................................78
4.1 Abstract ................................................................................................................... 78
4.2 Introduction ............................................................................................................. 79
4.3 Results ..................................................................................................................... 81
4.4 Discussion ............................................................................................................. 105

v

Chapter 5 : Concluding Remarks .....................................................................................110
5.1 Conclusions ........................................................................................................... 110
5.2 Proposed Model of BS69 Function in Germ Layer Specification ........................ 113
5.3 New Hypotheses for BS69 Function in Embryonic Development ....................... 114
5.4 A Putative Mechanism of BS69 Function in Early Embryogenesis ..................... 118
5.5 Future Directions ................................................................................................... 121
Chapter 6 : Materials and Methods ..................................................................................126
6.1 Animals ................................................................................................................. 126
6.2 Genotyping ............................................................................................................ 126
6.3 Embryo Isolation ................................................................................................... 127
6.4 RNA Probe Preparation ......................................................................................... 128
6.5 Whole Mount In Situ Hybridization...................................................................... 129
6.6 BS69 KO mESC Line Establishment .................................................................... 129
6.7 EB Formation ........................................................................................................ 130
6.8 Flow Cytometry..................................................................................................... 131
6.9 RNA Isolation and RTPCR ................................................................................... 131
6.10 β-galactosidase detection .................................................................................... 132
6.11 Immunohistochemistry ........................................................................................ 133
6.12 RNA-sequencing ................................................................................................. 135
6.13 Bioinformatics ..................................................................................................... 135
References ........................................................................................................................136
Appendices .......................................................................................................................151
Appendix 1: RNA-sequencing analysis data: BS69 KO mESCs vs Control mESCs . 151
Appendix 2: RNA-sequencing analysis data: BS69 KO day 3 EBs vs Control day 3
EBs .............................................................................................................................. 154
Appendix 3: RNA-sequencing analysis data: BS69 KO day 5 EBs vs Control day 5
EBs .............................................................................................................................. 159

vi

List of Tables

Table 1.1 Mutations that affect gastrulation. ..................................................................... 8

Table 1.2 Functional categorization of known BS69 binding partners. .......................... 17

Table 6.1 RNA antisense probe list. ............................................................................... 128

Table 6.2 PCR primer list. ............................................................................................. 132

Table 6.3 Immunofluorescence primary antibody list. ................................................... 134

Table 6.4 Immunofluorescence secondary antibody list. ............................................... 134

vii

List of Figures

Figure 1.1 Early development in the mouse embryo. ......................................................... 4
Figure 2.1 The BS69 KO is embryonic lethal at E7.5. ..................................................... 29
Figure 2.2 The endogenous pattern of BS69 expression in early post-implantation
embryos. .................................................................................................................... 31
Figure 2.3 BS69 is expressed throughout the embryo at E9.25 with highest expression
in the neural tube and anterior brain. ...................................................................... 33
Figure 2.4 BS69 expression is concentrated in the epiblast at E7.5. ............................... 34
Figure 2.5 BS69 KO E7.5 embryos undergo apoptosis at a higher rate than controls. .. 36
Figure 2.6 BS69 KO embryos do not form a primitive streak and prematurely specify
neuroectoderm. ......................................................................................................... 38
Figure 2.7 BS69 KO embryos do not maintain pluripotency or initiate expression of
posteriorizing genes. ................................................................................................. 40
Figure 2.8 BS69 KO embryos do not appear to pheno-copy Wnt pathway mutants. ....... 43
Figure 3.1 BS69 KO mESCs are able to sustain pluripotency in media containing both
LIF and 2i. ................................................................................................................ 55
Figure 3.2 BS69 KO mESCs can be maintained in a pluripotent state. ........................... 57
Figure 3.3 BS69 KO mESCs cycle at a similar rate as control mESCs. .......................... 58
Figure 3.4 BS69 KO mESCs can form EBs to similar sizes as controls. ......................... 61
Figure 3.5 BS69 KO Embryoid bodies are able to differentiate into epiblast but not
primitive streak cells. ................................................................................................ 63
Figure 3.6 The number of differentially expressed genes (DEGs) increases over time. .. 65
Figure 3.7 In mESCs there are few enriched Gene Ontology categories. ....................... 67
Figure 3.8 A large proportion of mESC DEGs are down-regulated, predicted, and less
than 100kb away from another mESC DEG. ............................................................ 68
Figure 3.9 Wnt pathway, germ layer formation, mesoderm formation, and gastrulation
related genes are down-regulated in BS69 KO day 3 EBs. ...................................... 70
Figure 3.10 One quarter of BS69 KO day 3 EB DEGs are also differentially expressed
in SMAD4 null day 4 EBs. ........................................................................................ 77
viii

Figure 4.1 Day 5 DEGs confirm trends from mESC and day 3 RNA-sequencing. .......... 83
Figure 4.2 Precocious BS69 KO EBs differentiation into mature neurons in vitro. ........ 85
Figure 4.3 BS69 KO embryoid bodies retain higher expression of early neuroectoderm
markers than controls. .............................................................................................. 88
Figure 4.4 BS69 KO EBs do not show precocious specification of immature neurons. .. 90
Figure 4.5 Decreased efficiency of mesoderm differentiation in BS69 KO EBs. ............. 92
Figure 4.6 BS69 KO embryoid bodies recover Bmp and Tgfβ pathway ligand
expression. ................................................................................................................ 94
Figure 4.7 BS69 KO EBs show delayed expression of primitive streak markers. ............ 96
Figure 4.8 β-catenin is expressed in BS69 KO day 5 EBs. .............................................. 98
Figure 4.9 BS69 KO EBs differentiate into endothelium at a similar rate as controls. . 100
Figure 4.10 BS69 KO EBs proliferation and apoptosis rates are similar to controls. .. 102
Figure 4.11 BS69 KO embryoid bodies contain phosphorylated Smad1/5/8 and
express Bmp target genes........................................................................................ 104
Figure 5.1 Model of BS69 function in early embryogenesis. ......................................... 113
Figure 5.2 Putative mechanism of BS69 function. ......................................................... 120

ix

List of Abbreviations
2i 2 chemical inhibitors
Acvr2a/2b activin receptor IIA and IIB
Al allantois
Anks1 ankyrin repeat and SAM domain containing 1
AP anterior-posterior
AVE anterior visceral endoderm
β-cat catenin (cadherin associated protein), beta 1 (β-catenin)
β-gal beta galactosidase
Bmp bone morphogenetic protein
BRAM1 Bmp receptor 1A associated molecule 1
Bromo bromodomain
BSA bovine serum albumin
CDKN1A cyclin-dependent kinase inhibitor 1A
Cer1 cerberus 1 homolog
ChE chorion ectoderm
ChIP-seq chromatin immunoprecipitation sequencing
CHIR CHIR99021
Cripto teratocarcinoma-derived growth factor 1 (Tdgf1)
cTnT cardiac troponin-T
DAPI 4',6-diamidino-2-phenylindole
DEG differentially expressed gene
x

DEPC diethylpyrocarbonate
DET differentially expressed transcript
DIG digoxygenin
Dkk1 dickkopf homolog 1
DMEM Dulbecco's Modified Eagle's Media
dNTP deoxyribonucleotide
DVE distal visceral endoderm
E embryonic day
E1A adenovirus E1A protein
E2F6 E2F transcription factor 6
EB embryoid body
EBNA2 Epstein-Barr virus EBNA2 oncoprotein
E-cad cadherin 1 (Cdh1, E-cadherin)
EDTA Ethylenediaminetetraacetic acid
EED embryonic ectoderm development
EFTUD2 elongation factor Tu GTP binding domain containing 2
EMSY chromosome 11 open reading frame 30
EMT epithelial to mesenchymal transition
Eomes eomesodermin
EP400 E1A binding protein 400
Epi epiblast
ETS2 v-ets avian erythroblastosis virus E26 oncogene homolog 2
ExE extraembryonic ectoderm
xi

ExM extraembryonic mesoderm
EZH2 enhancer of zeste 2 polycomb repressive complex 2
FBS fetal bovine serum
Fgf fibroblast growth factor
Fgfr fibroblast growth factor receptor
Gapdh glyceraldehyde-3-phosphate dehydrogenase
Gata6 GATA binding protein 6
GSK3β glycogen synthase kinase 3 beta
GTKI gene-trap knock-in
HDAC1 histone deacetylase 1
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
Hesx1 homeobox gene expressed in ES cells
Hhex hematopoietically expressed homeobox
Hnf3β forkhead box A2 (Foxa2)
IACUC Institutional Animal Care and Use Committee
ICM inner cell mass
Id2 inhibitor of DNA binding 2
Ki67 antigen identified by monoclonal antibody Ki 67
KO knockout
Lefty left right determination factor
LIF leukemia inhibitory factor
LMP1 Epstein-Barr virus latent membrane protein 1
MABT maleic acid buffer, 0.1% Tween-20
xii

Map4k4 mitogen-activated protein kinase kinase kinase kinase 4
MAPK8 mitogen activated protein kinase 8
MEF mouse embryonic fibroblast
MEK mitogen activated protein kinase kinase
Mes mesoderm
mESC mouse embryonic stem cell
MF-20 myosin, heavy chain 1E, skeletal muscle
MGA MGA-MAX dimerization protein
MOPS 1M 3-(N-morpholino)propan sulfonic acid
MYB v-myb avian myeloblastosis viral oncogene homolog
MYBL2 v-myb avian myeloblastosis viral oncogene homolog-like 2
MYND myeloid Nervy and DEAF1
N-cad cadherin 2 (Cdh2, N-cadherin)
NCBI National Center for Biotechnology Information
NCOR1 nuclear receptor corepressor 1
NFKB1 nuclear factor of kappa light polypeptide gene enhancer in
B-cells 1

NLS nuclear localization signal
Oct4 POU domain, class 5, transcription factor 1 (Pou5f1)
p38 mitogen-activated protein kinase 14 (Mapk14)
p38IP suppressor of Ty 20 (Supt20)
PBS phosphate buffered saline
PBT phosphate buffered saline, 0.1% Tween-20
xiii

PCR polymerase chain reaction
Pcsk6 proprotein convertase subtilisin/kexin type 6
PD03 PD0325901
PE primitive endoderm
Pecam1 platelet/endothelial cell adhesion molecule 1
PEE proximal epiblast enhancer
PHD plant homeodomain
PIAS1 protein inhibitor of activated STAT 1
PRC2 polycomb repressive complex 2
pSmad phosphorylated Smad
PWWP proline, tryptophan, tryptophan, proline
Rex zinc finger protein 42 (Zfp42)
RNA-seq RNA sequencing
RTPCR reverse-transcriptase PCR
Six3 sine oculis-related homeobox 3
SMA actin, alpha 2, smooth muscle, aorta (Acta2)
Smad SMAD family member
SMARCA SWI/SNF related, matrix associated, actin dependent
regulator of chromatin, subfamily a

Snail snail family zinc finger 1 (Snai1)
Sox SRY (sex determining region Y)-box
SUMO small ubiquitin-like modifier
SUZ12 suppressor of zeste 12 homolog
xiv

TCF3 transcription factor 3
Tgfβ transforming growth factor, beta
TICAM1 toll-like receptor adaptor molecule 1
TP53 tumor protein 53
TRADD TNFRSF1A-associated via death domain
TRAF6 TNF receptor-associated factor
Tuj1 tubulin, beta 3 class III (Tubb3)
TUNEL terminal deoxynucleotidyl transferase dUTP nick end
labeling

U5 snRNP U5 small nuclear ribonucleic protein
UBE2I ubiquitin-conjugating enzyme E2I
VE visceral endoderm
WMISH whole mount in situ hybridization
Wnt wingless-type MMTV integration site family
X-gal 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside
ZHX1 zinc-fingers and homeoboxes 1

xv

Abstract
The ultimate goal of pluripotent stem cell research is to devise new and better treatments
for disease. Specifically, the ability to regenerate tissues and organs to replace those that
have been damaged has been the hope of many researchers and patients alike. Although
there has been significant progress toward this goal, the efficient and safe generation of
many tissues from pluripotent stem cells is still beyond our ability. The embryo is the
blueprint for the derivation of every tissue, so the more accurately we can imitate this in
vitro, the better the future patient outcomes will be. In this dissertation, I aim to increase
the understanding of early embryogenesis and illuminate a previously undiscovered role
for the gene BS69, which has never before been implicated in development. To accomplish
this objective, I first examine the role of BS69 in early mouse embryogenesis. Next, I utilize
the robustness of mouse embryonic stem cell technology to determine whether BS69
functions correspondingly in vitro and to uncover its global effects on gene transcription.
Finally, by studying the differentiation capacity of cells that lack BS69, I determine its
long-term effects on development. Here, I show that BS69 is required for embryonic
development. BS69 facilitates the differentiation of epiblast into mesendoderm during
gastrulation by preventing the premature differentiation of neuroectoderm. Although this
phenotype shares striking similarity to the effects of Bmp pathway abrogation, I have been
able to derive mouse embryonic stem cells that lack BS69, suggesting that Bmp signaling
is intact. I find that BS69 is not required for mouse embryonic stem cell pluripotency or
differentiation into epiblast, but it is necessary for the in vitro differentiation of
mesendoderm. Furthermore, BS69 is required for the efficient differentiation of mesoderm-
derived tissues and prevents the expansion and persistence of early neuroectoderm. Upon
xvi

direct analysis of Bmp signaling in the absence of BS69, I find that the Bmp pathway is
active. Although more work remains to clarify the mechanism by which BS69 functions,
my findings suggest that it is required for gastrulation. Through revealing a previously
unknown function of BS69, this dissertation has added to the understanding of early
embryonic development. Future studies that investigate the effect of BS69 on the derivation
efficiency of specific tissues in vitro may uncover its usefulness in stem cell research and
regenerative medicine.

1

Chapter 1: Introduction
1.1 Regenerative Medicine
Since the first isolation of human embryonic stem cell in 1998, the promise of regenerative
medicine has inspired scientists, doctors, and patients to invest in this research (Thomson
et al, 1998). This dedication has paid off in many ways: We have new in vitro disease
models, ways to test drugs, and the potential for cell replacement therapies (Braam &
Mummery, 2010; Dimos et al, 2008; Kiskinis & Eggan, 2010; Moretti et al, 2010).
Therapies using stem cells to derive transplantable tissues have been by far the slowest to
progress. In the year 2000, it was predicted that hospitals would stock replacement hearts
grown from stem cells within a decade, and transplant waiting lists would be a thing of the
past (Stover, 2000). But after years of research, the only stem cell therapies in use are bone
marrow transplantation and autologous epidermal transplantation, techniques that predate
the isolation of human embryonic stem cells by decades (Gallico et al, 1984; MATHE et
al, 1965). Other therapies are in clinical trials, such as mesenchymal stem cells for use in
multiple sclerosis and retinal pigment epithelium cells for use in macular degeneration, but
it may be many years before patients benefit from them (Megaw & Dhillon, 2014; Schwartz
et al, 2015; Xiao et al, 2015).

Obstacles, both expected and unforeseen, have lined the path toward using stem cells in
the clinic. One issue that has arisen repeatedly is our lack of understanding of normal
development. Without a clear view of how pluripotent cells differentiate into different
tissues in the embryo, the matter of deriving specific cell types is slow and error-ridden.
2

For example, to treat heart disease, cardiac progenitor cells are an attractive target for use
in therapies. However, how they develop in the embryo is not fully understood, which has
led to an inability to differentiate them in great enough numbers for therapeutic use (Sahara
et al, 2015). Inefficiency of cell derivation has plagued regenerative medicine, improving
over time but still not robust enough to generate consistently high yields (Mummery et al,
2012). Additionally, cardiomyocytes that have been derived from pluripotent cells in vitro
caused arrhythmias when transplanted into monkeys (Chong et al, 2014). This should not
be surprising, because if we do not fully understand how the blueprint, the embryo, makes
mature cells, we cannot know if the ones we generate are correct. Embryonic development
is the blueprint for the differentiation of pluripotent cells, but despite decades of research
there are still gaps in our knowledge. In order to move forward, we must clarify the
signaling pathways, epigenetic patterns, and morphogenetic movements that contribute to
embryogenesis.

3

1.2 Pre-implantation Embryogenesis

Although we do not have a complete picture of how the embryo forms, research going back
to at least the 1960’s has contributed to our current understanding of the process
(Diczfalusy, 1969). Since then, studies in multiple species including Xenopus, zebrafish,
and mouse have unveiled numerous molecules and mechanisms that are involved.

In mice, gestation lasts for approximately 20 days, compared to roughly 270 days in human.
After fertilization, the mouse zygote concludes its first division by 1.5 days, and by 2.5
days it reaches the 8-cell stage (Gilbert, 2003). The next stage, called the morula, is a
compact ball of cells that begins to cavitate on the third day to form a blastocoele, marking
the transition to the blastocyst stage (Gilbert, 2003). The blastocyst is fully formed on
embryonic day (E) 3.5 and consists of two types of cells: A single outer layer of cells
called the trophectoderm and a group of cells within called the inner cell mass (ICM)
(Gilbert, 2003). The pluripotent ICM will give rise to all of the embryonic tissues plus
some of the extraembryonic tissues, while the trophectoderm produces solely
extraembryonic cell types (Gilbert, 2003).

4

Figure 1.1 Early development in the mouse embryo. A. Blastocyst stage. B. Egg cylinder
stage; no clear anterior-posterior specification. C. Pre-streak stage; anterior-posterior axis
defined by anterior visceral endoderm location. D. Gastrulation; anterior-posterior axis is
morphologically marked by the primitive streak (Arkell & Tam, 2012).

5

1.3 Axis Formation

Between E4.0 and E4.5, the blastocyst hatches and sheds the zona pellucida, the remaining
outer covering of the oocyte. Following hatching, the embryo implants into the uterine wall
as the ICM differentiates into the epiblast and the primitive endoderm (PE) (Gilbert, 2003).
The PE proliferates as a single cell layer that spreads over the inside of the trophectoderm
and the outside of the epiblast (Tam & Loebel, 2007). The PE cells that coat the epiblast
become visceral endoderm (VE), while the epiblast elongates and cavitates to form the egg
cylinder (Tam & Loebel, 2007). By this time, the proximal-distal axis has been established
with the ectoplacental cone at the proximal end, followed by the extraembryonic ectoderm
(ExE), and finally the epiblast (also called embryonic ectoderm) at the distal end (Tam &
Loebel, 2007; Tam et al, 2006).

Formation of the anterior-posterior (AP) axis is a central event in early post-implantation
development. However, the mechanism of AP axis formation has been elusive. For many
years, the AP axis was thought to originate when the distal visceral endoderm (DVE)
migrated proximally to form the anterior visceral endoderm (AVE) and thereby defined the
anterior of the embryo (Rivera-Perez, 2007). However, recent work has suggested that the
AP axis may develop at peri-implantation with the asymmetric expression of left right
determination factor 1 (Lefty1) in the ICM (Takaoka et al, 2011). Lefty1, a transforming
growth factor beta (Tgfβ) family member, is expressed asymmetrically in a small number
of GATA binding protein 6 (Gata6)-negative cells in the ICM beginning at E3.5 and in a
few Gata6-positive primitive endoderm cells at E4.0 (Takaoka et al, 2011). At E4.5, only
the Gata6-positive cells still express Lefty1, and these are the progenitors of the DVE
6

(Takaoka et al, 2011). The DVE is a group of VE cells at the embryo’s distal-most tip that
develops around E5.5. It is thought that their specification may depend on Nodal signaling
to promote epiblast proliferation, increasing the length of the egg cylinder until the
presumptive DVE is out of range of ExE inhibitory signals (Mesnard et al, 2006). However,
interactions between the VE and epiblast that have yet to be determined may prove this
hypothesis incorrect. Once the DVE is specified, it expresses hematopoietically expressed
homeobox (Hhex), cerberus 1 homolog (Cer1), and Lefty1 in response to Nodal signaling
from the epiblast (Mesnard et al, 2006). Nodal signals through its co-receptor,
teratocarcinoma-derived growth factor 1 (Tdgf1, or Cripto), which acts through the TGFβ
pathway intermediate SMAD family member 2 (Smad2) to activate transcription of its
targets (Ding et al, 1998; Yeo & Whitman, 2001). Cer1 and Lefty1 then inhibit Nodal
expression, forming a negative feedback loop (Branford & Yost, 2002). Many studies
suggest that DVE cells migrate and to become the AVE (Miura et al, 2010; Rivera-Pérez
et al, 2003; Srinivas et al, 2004). However, recent work shows that while DVE cells do
indeed migrate, other VE cells follow them and become AVE when they arrive at the
anterior-proximal border of the embryo by approximately E6.0 (Takaoka et al, 2011).
Nevertheless, not all researchers are convinced of this finding, suggested by the continued
citation of studies which show that the DVE contributes directly to the AVE (Rivera-Pérez
& Hadjantonakis, 2014). What is not disputed is that the AVE comprises an essential
signaling center in the embryo, expressing factors that inhibit genes required for primitive
streak formation. Expression of Lefty1, Cer1, and dickkopf homolog 1 (Dkk1) in the AVE
functions to inhibit Nodal, bone morphogenetic protein 4 (Bmp4), and wingless-type
MMTV integration site family member 3 (Wnt3) signaling from the ExE and proximal
7

epiblast (Fossat et al, 2012; Perea-Gomez et al, 2002). These signals are consequently
limited to the posterior of the embryo where a complex feed-forward loop reinforces their
expression. Nodal from the epiblast up-regulates Bmp4 expression in the ExE where both
proteins are activated by the proteases Furin and proprotein convertase subtilisin/kexin type
6 (Pcsk6), which cleave their respective pro-domains (Beck et al, 2002). Once activated,
Bmp4 induces the expression of Wnt3 in the proximal epiblast (Ben-Haim et al, 2006).
Wnt3 then activates Nodal’s proximal epiblast enhancer (PEE), thereby increasing its
proximal concentration and completing the feed-forward loop (Ben-Haim et al, 2006).
These posterior ligands diffuse to form distinct gradients across the embryo. These
gradients are essential for the specification of tissues prior to and during gastrulation.

8

Table 1.1 Mutations that affect gastrulation. Adapted from (Tam & Loebel, 2007). See
review for individual citations.

9

1.4 Gastrulation

The function of gastrulation is to begin the specification of different tissues within the
developing embryo (Wolpert, 1992). The first tissues that form are the three germ layers,
from which all other tissues arise (Tam & Behringer, 1997). The ectoderm, which generates
the epidermis, central nervous system, and neural crest, develops from the anterior epiblast
(Hemmati-Brivanlou & Melton, 1997). Mesoderm, which forms the muscles, skeleton,
connective tissue, adipose tissue, circulatory system, lymphatic system, dermis,
genitourinary system, and notochord, is derived from a bipotential precursor called the
mesendoderm (Wilkinson et al, 1990). As the name suggests, the other derivative of
mesendoderm is endoderm, from which the gastrointestinal system, liver, pancreas, lungs,
and the epithelial linings of glands are formed (Ang et al, 1993). Mesendoderm forms by
the ingression of posterior epiblast cells through a region called the primitive streak that
forms during gastrulation (Tam & Behringer, 1997; Tam & Loebel, 2007). Once through
the primitive streak, the mesendoderm cells migrate anteriorly and acquire patterning to
become definitive endoderm, axial mesoderm, paraxial mesoderm, heart mesoderm,
cranial mesoderm, and extraembryonic mesoderm (Tam & Behringer, 1997).

AP patterning sets the stage for gastrulation, in which factors including Bmp4, Nodal, and
Wnt3 are essential. The highest concentration of these factors is at the posterior epiblast-
ExE border, and this is where the primitive streak initiates. In order for epiblast cells to
become migratory—a process called epithelial-to-mesenchymal transition (EMT)—cell-
cell adhesions must be inhibited (Sun et al, 1999). Fibroblast growth factor 8 (Fgf8),
regulated in part by Wnt signaling, plays an integral role in initiating EMT (Ciruna &
10

Rossant, 2001; Hierholzer & Kemler, 2010). Acting through its receptor, fibroblast growth
factor receptor 1 (Fgfr1), Fgf8 activates the expression of Snail (Ciruna & Rossant, 2001).
Snail then down-regulates the expression of E-cadherin, a major component of the cadherin
junctions that attach epiblast cells to one another (Cano et al, 2000). In addition, p38
mitogen-activated kinase (p38) and p38-interacting protein (p38IP), which are activated by
mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), are also required to
down-regulate E-cadherin (Zohn et al, 2006). However, this is a separate pathway from
Fgf8 and Snail signaling (Zohn et al, 2006). In p38IP mutant embryos, both E-cadherin and
Snail expression were normal (Zohn et al, 2006). Without the down-regulation of E-
cadherin, mesendoderm cells cannot migrate and the embryo does not survive (Ciruna &
Rossant, 2001; Zohn et al, 2006). Another gene was more recently discovered as a
requirement for E-cadherin down-regulation in the embryo: Eomesodermin (Eomes). A
target of Nodal signaling, Eomes is expressed in the ExE and mid-to-anterior primitive
streak during gastrulation (Ciruna & Rossant, 1999). Interestingly, Eomes expression in
the epiblast prior to gastrulation is required for the down-regulation of E-cadherin, but does
not affect the levels of Fgf8 or Snail (Arnold et al, 2008). This effect is cell-autonomous,
as shown by chimera formation assay in which Eomes-null cells contributed to the VE,
epiblast, and ExE, but do not undergo EMT (Arnold et al, 2008). However, it was shown
that the requirement for Eomes is transient and dependent upon the environment within the
embryo itself. Eomes-null explants were able to undergo EMT in vitro and migrate
similarly to controls, and if Eomes was conditionally knocked out during of gastrulation,
embryos were able to down-regulate E-cadherin and survive to term (Arnold et al, 2008).
This shows that Eomes is required prior to the start of EMT, but is dispensable for its
11

maintenance. Therefore, although EMT has long been recognized as a requirement for
gastrulation, it is a complex process that is still not fully understood.

The process of specifying definitive endoderm from its precursor also requires Eomes
(Arnold et al, 2008). In human embryonic stem cells, EOMES is the first known endoderm
marker to be up-regulated during definitive endoderm differentiation (Teo et al, 2011).
Forkhead box a2 (Foxa2, also known as HNF3β) expression is then up-regulated in the
anterior primitive streak, which contributes to definitive endoderm and notochord
determination (Ang et al, 1993). Moreover, HNF3β is required for the elongation of the
primitive streak and the subsequent formation of anterior primitive streak-derived tissues,
including the node. The node is a structure at the distal-most part of the embryo that marks
the anterior limit of the primitive streak. Thought to be analogous to Hensen’s node in the
chick embryo, it is a signaling center and precursor of axial mesoderm including the
notochord, as well as some definitive endoderm. Additionally, the node delineates a post-
primitive streak region of Nodal expression (Zhou et al, 1993).

As the primitive streak forms, regions along the proximal-distal axis are specified to
generate different types of mesoderm. At the beginning of gastrulation, the proximal-most
mesendoderm contributes to extraembryonic mesoderm (ExM) (Tam & Behringer, 1997).
Next, heart and cranial mesoderm are specified at mid-streak stage, but soon migrate in an
anterior-proximal direction while the axial mesoderm continues to be displaced toward the
distal tip of the embryo as the primitive streak extends (Tam & Behringer, 1997). Lateral
mesoderm and paraxial mesoderm are specified just distal of ExM (Tam & Behringer,
12

1997). The pattern in which these tissues form is affected by the levels of signaling factors
along both the proximal-distal and AP axes.

When all the cells that are fated to become mesendoderm have moved through the primitive
streak, the remaining epiblast cells become the ectoderm. Ectoderm is then specified into
neuroectoderm, surface ectoderm, and neural crest, a multipotent precursor of cranial
mesoderm, pigment cells, and other cell types. Surface ectoderm is specified posterior and
proximal to the neuroectoderm, where it is induced by Bmp signaling that is thought to
prevent the neural fate (Di-Gregorio et al, 2007; Wilson & Hemmati-Brivanlou, 1995). The
anterior-most neuroectoderm cells contribute to the forebrain, followed by midbrain and
hindbrain (Arkell & Tam, 2012). Early markers of neuroectoderm can be seen at E7.5,
including SRY (sex determining region Y)-box (Sox1), sine oculis-related homeobox 3
(Six3), and homeobox gene expressed in ES cells (Hesx1), three genes that are important
for anterior neural development (Lagutin et al, 2003; Lee et al, 2013; Martinez-Barbera et
al, 2000; Oliver et al, 1995; Pevny et al, 1998). In part, they restrict the activity of posterior
signaling factors like Bmps and Wnts to allow neuroectoderm development to take place
(Arkell & Tam, 2012).

When gastrulation is complete, the body plan of the embryo has been established including
ectoderm, mesoderm, and definitive endoderm. Subsequent developmental events follow
this plan to generate all tissues in the embryo. Therefore, understanding gastrulation is of
central importance in the study of development.

13

1.5 BS69

As previously described, Wnt signaling is essential to early embryogenesis.
Catenin (cadherin associated protein) beta 1 (β-catenin), the effector of canonical Wnt
signaling, is required for the transcription of developmental regulators that promote
gastrulation (Haegel et al, 1995; Huelsken et al, 2000). Transcription factor 3 (TCF3) binds
β-catenin and acts as a transcriptional repressor of Wnt/β-catenin target genes, which is
required for normal axis specification and gastrulation (Molenaar et al, 1996). Embryos
that lack TCF3 generate duplicate primitive streaks (Merrill et al, 2004). Interestingly, a
yeast-two-hybrid screen has suggested that TCF3 can bind the protein BS69 (Kobielak,
unpublished results). The discovery of a new TCF3 binding partner was particularly
intriguing because TCF3 plays such an integral role in gastrulation.

Although BS69 was discovered in 1995, there are relatively few publications that have
focused on this gene, officially named ZMYND11 (Hateboer et al, 1995). Furthermore, no
study has examined the role of BS69 in an animal model. BS69 was first discovered as a
binding partner of the adenovirus E1A protein (E1A) in 1995 (Hateboer et al, 1995).
Structurally, the BS69 protein was found to have multiple functional domains including a
Bromodomain, PWWP domain, plant homeodomain (PHD), myeloid Nervy and DEAF1
(MYND) domain, and a nuclear localization signal (NLS) (Hateboer et al, 1995; Masselink
& Bernards, 2000). Early studies have focused on BS69’s MYND domain, located near its
C-terminus. MYND is a dual zinc-finger domain that has been described in numerous
proteins and is conserved among species from Drosophila to human (Feinstein et al, 1995;
Lutterbach et al, 1998). Unlike many zinc-finger domains that are DNA-binding, MYND
14

has been shown to mediate protein-protein interactions (Matthews et al, 2009). BS69 was
thought to have a shorter cytoplasmic splice variant containing the MYND domain called
BMP receptor 1A interacting molecule 1, or BRAM1, due to its interaction with the
specific BMP receptor. It consists of amino acids 377-562 of BS69 with 12 additional N-
terminal amino acids (Kurozumi et al, 1998). It was later shown that these additional amino
acids could be mapped to the anks1 gene on human chromosome 6 and BRAM1 cDNA
was likely a coincidental recombination of Anks1 and BS69 cDNA during library
construction (Velasco et al, 2006). However, before this discovery, BRAM1 was used to
test the sufficiency of BS69’s MYND domain for protein-protein interactions.

Figure 1.2 Functional domains in the BS69 protein. BS69 is 602 amino acids in length and
contains a plant homeodomain (PHD), Bromodomain (Bromo), PWWP domain (PWWP),
Myeloid, Nervy, and DEAF-1 domain (MYND), and a nuclear localization signal (NLS).

15

BS69 was found to bind various transcription factors and cofactors including nuclear
receptor corepressor 1 (NCOR1), v-myb avian myeloblastosis viral oncogene homolog
(MYB), and v-myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2)
(Ladendorff et al, 2001; Masselink & Bernards, 2000; Masselink et al, 2001). BS69 binds
NCOR1, leading to repression of gene transcription (Masselink & Bernards, 2000).
Although binding was mediated by the BS69 MYND domain, BRAM1 was insufficient
for gene repression, suggesting that the N-terminal functional domains are required
(Masselink & Bernards, 2000). Similarly, BS69’s MYND domain binds MYB and
MYBL2, and causes repression of their target genes (Ladendorff et al, 2001; Masselink et
al, 2001). However, BRAM1 alone can inhibit MYBL2 associated gene activation
(Masselink et al, 2001). In 2002, researchers found a conserved motif, PXLXP, among
proteins that bind to BS69’s MYND domain (Ansieau & Leutz, 2002). This motif is present
in the earliest-discovered BS69 binding partner, the adenoviral protein E1A, as well as the
Epstein-Barr virus EBNA2 oncoprotein (EBNA2), MGA-MAX dimerization protein
(MGA), chromosome 11 open reading frame 30 (also known as EMSY), and zinc-fingers
and homeoboxes 1 (ZHX1) (Ansieau & Leutz, 2002; Hughes-Davies et al, 2003; Ogata-
Kawata et al, 2007). Although in most of these cases BS69 binding causes transcriptional
repression, it was found that in the case of ZHX1, it results in cell-type dependent
transcriptional activation (Ogata-Kawata et al, 2007). Additionally, BS69 transcription
cofactor activity was seen to affect cellular senescence in human primary fibroblasts;
knockdown results in elevated levels of cyclin-dependent kinase inhibitor 1A (CDKN1A)
in addition to several senescence markers including formation of senescence-associated
heterochromatin foci and senescence-associated β-galactosidase (β-gal) activity (Zhang et
16

al, 2007). Knockdown of either tumor protein 53 (TP53) or CDKN1A rescues the cells
from BS69 knockdown-induced premature senescence. BS69 was found to form
complexes with both TP53 and E1A binding protein 400 (EP400). Although BS69 was not
shown to bind either TP53 or EP400 directly, its association with the CDKN1A promoter
was found to be indirectly mediated by TP53 (Zhang et al, 2007).

17

Table 1.2 Functional categorization of known BS69 binding partners.

18

Although studies have clearly demonstrated that BS69 is a transcription cofactor, it was
surprisingly found to serve additional functions. BS69 acts in the mitogen activated protein
kinase 8 (MAPK8) pathway through binding Epstein-Barr virus latent membrane protein
1 (LMP1) and TNF receptor-associated factor 6 (TRAF6). BS69 binds LMP1 through its
MYND domain and TRAF6 by amino acids closer to the N-terminus, serving as an adaptor
for signal transduction (Wan et al, 2006). Another example of BS69’s cytoplasmic function
is its translocation from nucleus to cytoplasm caused by toll-like receptor adaptor molecule
1 (TICAM1) (Takaki et al, 2009). TICAM1 is an adaptor molecule that is necessary to
activate nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1).
BS69 has been found to translocate out of the nucleus upon TICAM1 stimulation or
overexpression and colocalize with it in “speckles.” Moreover, BS69 translocation
increased NFKB1 activation (Takaki et al, 2009). BS69 also affects the MAPK8 pathway
through LMP1. LMP1-induced NFKB1 activation is reduced by BS69 expression in a
dose-dependent manner (Ikeda et al, 2009). BS69 knockdown by siRNA results in
increased LMP1-induced NFKB1 activation. BS69 does not interact with TNFRSF1A-
associated via death domain (TRADD) which is a downstream molecule of LMP.
However, TRADD fails to bind LMP1 in the presence of BS69, indicating that it physically
blocks interaction (Ikeda et al, 2009). It was later found that BS69 can interact with other
TRAF proteins including TRAF3, and bind a separate region of LMP1 that mediates the
non-canonial NFKB1 pathway. Thus, through its ability to bind LMP1, BS69 is involved
in three related pathways.

19

To understand BS69’s functions, studies also sought to understand the protein’s behavior
and post-translational modifications. A study found that BS69 forms oligomers that appear
as speckles in the nucleus (Yu et al, 2009). The oligomerization is dependent on not only
the MYND domain, but also the E1A-binding domain. The authors also saw that full-length
BS69 oligomerizes and localizes in the nucleus better than any truncated construct,
showing that the NLS and N-terminal functional domains are important (Yu et al, 2009).
Concerning post-translational modification, the authors found that BS69 is sumoylated by
protein inhibitor of activated STAT 1 (PIAS1) and ubiquitin-conjugating enzyme E2I
(UBE2I) in a PHD and MYND domain dependent manner (Yu et al, 2009). UBE2I is the
only know small ubiquitin-like modifier (SUMO) E2 enzyme and PIAS1 directly binds
BS69 and increases its sumoylation, suggesting that it is a SUMO E3 enzyme (Yu et al,
2009). Interestingly, this study also found that overexpression of BS69 inhibits both muscle
and neuronal differentiation. BS69 caused decreased levels of muscle markers and neurite
outgrowths in myoblasts and rat pheochromocytomacells, respectively (Yu et al, 2009).
Overexpression of BS69 constructs with mutations in the PHD domains, however, are
unable to abrogate muscle or neuronal differentiation (Yu et al, 2009). Since the BS69
mutants that cannot be sumoylated can still inhibit differentiation, this suggests that the
SUMO modification is not correlated with this function of BS69 (Yu et al, 2009).

Perhaps not as surprising as finding cytoplasmic functions for a nuclear protein, another
study revealed that BS69 can interact with chromatin modifying proteins. First, the authors
found that there are an additional 40 amino acids at the N-terminus of BS69 that were not
previously recognized, revealing that BS69 has 602 amino acids rather than 562 as was
20

previously thought (Velasco et al, 2006). The authors analyzed four isoforms of BS69 and
showed the full-length isoform is the most abundant, and all localize to the nucleus
(Velasco et al, 2006). Full-length BS69 associates with mitotic chromosomes and
chromatin. It interacts specifically with SWI/SNF related matrix associated actin
dependent regulator of chromatin subfamily A member 4 (SMARCA4), SWI/SNF related,
matrix associated actin dependent regulator of chromatin subfamily a member 2
(SMARCA2), histone deacetylase 1 (HDAC1), enhancer of zeste 2 polycomb repressive
complex 2 subunit (EZH2), and E2F transcription factor 6 (E2F6). SMARCA4 and
SMARCA2 are both ATP-dependent helicases that are part of the SWI/SNF chromatin
remodeling complex. HDAC1 removes acetyl groups from histone-based lysines as well
as other substrates including TP53. As a part of polycomb repressive complex 2 (PRC2),
EZH2 is involved in repressing gene transcription through the addition of methyl groups
to histone 3 lysine 27 (H3K27). E2F6 is the sole transcription factor in this list of chromatin
modifiers, but it is known to interact with chromatin modifying complexes. This study also
found that EZH2 and E2F6 both bind the BS69 MYND domain, but at different residues
(Velasco et al, 2006). E2F6 is also able to immunoprecipitate EZH2, suggesting that BS69,
EZH2, and E2F6 could bind as a ternary complex (Velasco et al, 2006). However, the
authors did not study how BS69 interaction affected the function of each protein.

Conversely, association studies on various types of cancer have shown that BS69 may
function as a tumor suppressor (Giefing et al, 2011; Römer et al, 2013; Wen et al, 2014).
Additionally, BS69 deletion has been implicated in intellectual disability (Cobben et al,
2014; DeScipio et al, 2012). Although these studies suggest potential functions of BS69,
21

they do not back up the associations with mechanistic data. Recently, a major breakthrough
in the study of BS69 was published by Wen and colleagues. This study showed that BS69
specifically binds to histone 3 variant 3.3 at trimethylated lysine 36 (H3.3K36me3) (Wen
et al, 2014). The authors showed this through chromatin immunoprecipitation sequencing
(ChIP-seq) and through the crystal structure of the interaction (Wen et al, 2014). They
showed that BS69 binds H3.3K36me3 through a pocket-like structure composed of its
bromodomain, PHD domain, and a previously unrecognized zinc-finger residing between
the aforementioned domains (Wen et al, 2014). Because histone variant 3.3 (H3.3) is
inserted into gene bodies during active transcription, the authors tested whether it is
involved in RNA polymerase II activity (Elsaesser et al, 2010). They found that BS69
represses RNA polymerase II elongation (Wen et al, 2014). Finally, because BS69 has been
suggested to be a tumor suppressor, they tested the effects of overexpression on cancer
cells. They found that increased BS69 expression inhibited tumor cell growth whereas
H3.3K36me3-binding deficient BS69 could not, showing that this interaction is essential
(Wen et al, 2014). A following study also suggested that BS69 works as a reader of
H3.3K36me3, but disputed the specific function of this binding. Instead, the authors argue
that BS69 is involved in intron retention through an interaction with elongation factor Tu
GTP binding domain containing 2 (EFTUD2), a member of the U5 small nuclear
ribonucleic protein (U5 snRNP) spliceosome (Guo et al, 2014). To determine this, they
knocked down BS69 and performed RNA sequencing. The results showed an increase in
the number of intron retention events, which is interesting because it is the least common
type of alternative splicing in animals (Guo et al, 2014). Furthermore, the intron retention
22

events were mostly bound by BS69, suggesting that it has a direct role in promoting this
type of splicing (Guo et al, 2014).

In summary, BS69 has known roles as a transcription cofactor, adaptor protein, and
chromatin modification reader. Its interaction with TCF3, a Wnt pathway transcription
cofactor, suggests that it may be involved in early development, similar to other proteins
in Wnt signaling (Kobielak, unpublished results). Therefore, here we have undertaken the
task of determining BS69’s role in mouse development, hypothesizing that it will affect
early embryogenesis. By utilizing a BS69 knockout (BS69 KO) mouse, we show that this
gene is required for embryonic survival and for gastrulation. We use the in vitro approach
of deriving mouse embryonic stem cells (mESCs) to understand what tissues are able to
differentiate without BS69. We find that while BS69 is not required for pluripotency or
self-renewal, it is necessary for mesendoderm differentiation. Furthermore, the lack of
BS69 permits the premature differentiation of neuroectoderm, similar to the phenotype of
Bmp signaling deficiency. However, we demonstrate that the lack of BS69 does not disrupt
the Bmp pathway, suggesting that its down-regulation is not prerequisite for neural
differentiation.

23

Chapter 2: BS69 is Required for the Formation of
Mesendoderm During Gastrulation
2.1 Abstract
The stage in embryogenesis when the three primary germ layers, ectoderm, mesoderm, and
endoderm, are established is called gastrulation. Mesendoderm, a bipotential precursor of
mesoderm and endoderm, forms when epiblast cells undergo EMT and migrate to form a
third layer of cells in the posterior embryo. This region where mesendoderm first forms is
called the primitive streak, and it extends progressively toward the distal end of the embryo.
The remaining epiblast cells form the ectoderm. Here, we use a gene-trap knock-in (GTKI)
mouse that expresses β-gal under the endogenous BS69 promoter to discover the function
of BS69 in embryogenesis. We demonstrate that BS69 is required for embryonic
development beyond E7.5. BS69 KO embryos are smaller than controls and lack a
primitive streak. BS69 is expressed throughout the embryonic tissues of E5.5 to E9.25
embryos, and is found in the ExM of E7.5 embryos. BS69 KO embryos are resorbed by
approximately E8.0, but show only a minor increase in apoptosis at E7.5 and no difference
in proliferation from controls. Finally, we show that BS69 KO embryos do not express
mesendoderm markers or initiate gastrulation, and they prematurely specify
neuroectoderm.

24

2.2 Introduction
During embryonic development, gastrulation is a pivotal event because this is when the
animal’s body plan is first established. The embryonic axes are set, tissue specification
begins, and the primary germ layers are formed. Ectoderm, mesoderm, and endoderm are
the three primary germ layers and the precursors of all somatic tissues. Ectoderm gives rise
to ectoderm and the central nervous system; mesoderm produces muscles, bones, heart,
and the circulatory system, among others; and endoderm is responsible for the digestive
tract, associated organs, and glands (Ang et al, 1993; Hemmati-Brivanlou & Melton, 1997;
Wilkinson et al, 1990). During gastrulation, these three layers are specified. The ectoderm
is derived from the epiblast cells that remain epithelial. Mesoderm and endoderm are
specified together as mesendoderm. These cells are derived from the epiblast, which
undergoes EMT in the posterior of the embryo (Tam & Loebel, 2007). As the epiblast cells
become mesenchymal, they migrate to form a new layer in between the epiblast and VE.
Once they have entered this transitory region between layers, the cells are specified as
mesendoderm. The region itself is known as the primitive streak, and is the first
morphological sign that the embryo is undergoing gastrulation (Downs & Davies, 1993).
Over time, the primitive streak extends from the proximal to the distal ends of the
embryonic tissues, and the location at which the mesendoderm cells enter the streak
determines their fates as either mesoderm or endoderm.

The process of specifying tissues and altering morphology is mediated by a signaling
network that is set up before gastrulation starts. Two signaling centers are the main known
regulators of this network: the ExE and the AVE. At the border between the
25

extraembryonic and embryonic tissues, the ExE expresses the genes Bmp4, Furin, and
Pcsk6, which are instrumental in preparing the proximal epiblast for gastrulation (Beck et
al, 2002; Ben-Haim et al, 2006; Mesnard et al, 2011). Furin and Pcsk6 activate Bmp4,
which turns on the gene Wnt3 in the proximal epiblast. Wnt signaling then activates
Brachyury, and both are required to specify mesendoderm (Liu et al, 1999; Wilkinson et
al, 1990; Yamaguchi et al, 1999). Nodal, which earlier helps to specify the proximal-distal
axis in the epiblast, is also required to activate these signals (Brennan et al, 2001; Conlon
et al, 1994; Robertson, 2014). Meanwhile, the AVE expresses antagonists of Bmp4, Wnt3,
and Nodal, preventing their expression in the anterior of the embryo (Meno et al, 1999;
Tam & Loebel, 2007). Wnt signaling helps to activate Fgf8 expression, which initiates
EMT by activating Snail (Cano et al, 2000; Ciruna & Rossant, 2001). Snail down-regulates
E-cadherin, a protein that is involved in cell-cell adhesion (Cano et al, 2000). Additionally,
p38 and Eomes are required for E-cadherin down-regulation, though neither function
through the Fgf pathway (Arnold et al, 2008; Zohn et al, 2006). As gastrulation progresses
the mesendoderm is patterned depending on the location at which the cells pass through
the primitive streak (Tam & Behringer, 1997). The anterior-most part of the streak
contributes to definitive endoderm and is marked by HNF3β as it extends distally (Ang et
al, 1993; Dufort et al, 1998). Once the streak reaches the distal-most part of the embryo,
the node is specified and expresses Nodal (Zhou et al, 1993).

BS69’s role in development has never been studied, but its interaction with the Wnt
pathway transcription cofactor TCF3 suggests that it may be important during gastrulation
(Kobielak, personal communication). BS69’s MYND domain has been shown to interact
26

with transcription factors and act as a corepressor (Ansieau & Leutz, 2002; Ladendorff et
al, 2001; Masselink et al, 2001). Therefore, this domain is likely required for any putative
BS69 function during gastrulation. To study BS69 in gastrulation, our strategy was to
disrupt BS69 gene transcription in the mouse and analyze the resulting phenotype.

27

2.3 Results

2.3.1 BS69 is Essential for Early Embryonic Development
To understand the biological role of the BS69 gene, first we studied its function in vivo. In
order to address this in embryonic development, we used the GTKI method to insert a
cassette containing β-gal, a splice-acceptor sequence, and a poly-adenylation sequence into
the intron between exons 10 and 11 of the BS69 gene (Bay Genomics Consortium). This
interrupted transcription, preventing transcription of the full length BS69 mRNA,
specifically stopping before the MYND domain (Figure 2.1A). The selected clone of
embryonic stem cells with proper disruption of the BS69 gene has been confirmed by
mRNA/cDNA sequencing. The engineered embryonic stem cells were then injected into
isolated E3.5 blastocysts from C57BL/6 and implanted into the uterus of a pseudo-pregnant
female to generate a chimeric mouse. In the F0 generation we received several mice with
different strengths of chimerism. The strongest chimeras were subsequently bred to obtain
an F1 generation of heterozygotes, which we confirmed the high frequency of germline
transmission. Subsequently, the F2 generation was bred from an F1 heterozygote mating.
The genotype was confirmed by polymerase chain reaction (PCR) using primers for the
inserted LacZ gene. This genotyping method can identify the presence of an allele
containing the inserted cassette, but cannot distinguish between heterozygous and
homozygous mice. To determine if a mouse completely lacked the intact BS69 transcript,
we isolated the mRNA and reverse-transcribed it into cDNA for analysis. We then designed
primers in the exons flanking the GTKI cassette insertion site and used them to perform
PCR on the cDNA (Figure 2.1B). Because the cassette interrupts BS69 transcription the
mouse with LacZ inserted into both alleles does not yield a 379 base pair (bp) RTPCR
28

product (Figure 2.1C). Interestingly, in the F2 generation there were no mice born that were
homozygous for the knock-in (BS69 KO), suggesting that they were not able to survive to
term. We genotyped the viable offspring and observed a Mendelian ratio that correlates
with an embryonic lethal homozygous mutation (data not shown). Therefore, we started to
examine embryos at successively earlier stages of embryonic development to determine
the latest stage to which the embryo is still alive. We found that BS69 KO embryos were
not viable past embryonic day (E) 7.5.

29

Figure 2.1 The BS69 KO is embryonic lethal at E7.5. A. Schematic diagram of BS69 and
the gene-trap knock-in construct used to generate BS69 KO mice. The TRAP β-gal cassette
was inserted between exons 10 and 11. Arrows indicate locations of primers. B. Control
and BS69 KO E7.5 embryos from the same litter. C. RTPCR product from E7.5 embryo
cDNA using BS69 primers across the site of TRAP β-gal insertion. GAPDH is a loading
control. ExE=extraembryonic ectoderm, Em=embryonic tissue, PS=primitive streak. See
Table 6.2 for primer sequences.

30

Additionally, phenotypic differences were apparent at E7.5, but not at E6.5. BS69 KO
embryos were significantly smaller than the heterozygous and wild type embryos,
especially in the distal region that forms the tissues of the embryo proper (Figure 2.1B).
The epiblast appears to be radially symmetrical in most mutants, though some are concave
on one side at the embryonic/extraembryonic border, similar to control embryos.
Additionally, there is no morphological evidence of a primitive streak. The extraembryonic
tissues appear relatively more normal in size than the embryonic tissues, though defects
were still discernable (Figure 2.1B). There is no visible allantois or other morphological
sign of anterior-posterior polarity in the BS69KO. We did not observe any phenotypic
differences between the heterozygous and wild type mice during embryogenesis or as
adults, which suggests that BS69 is a haplosufficient gene. Thus, in our further studies we
used either genotype as a control and used the β-gal expression in the heterozygotes to
track the endogenous expression of BS69 during different stages of development. We
stained whole embryos from E5.5 to E9.25 with 5-bromo-4-chloro-3-indolyl-beta-D-
galacto-pyranoside (X-gal) to visualize the endogenous pattern of BS69 expression (Figure
2.2). We found that BS69 was expressed in the embryonic tissue in all stages tested. At
E5.5, BS69 expression was specifically seen in the embryonic region, distal of the
ectoplacental cone and extraembryonic region (Figure 2.2A).
31

Figure 2.2 The endogenous pattern of BS69 expression in early post-implantation
embryos. A-F. Whole BS69 heterozygous embryos from E5.5 to E9.25 were stained with
X-gal to show the location of β-gal expressed under the endogenous BS69 promoter.
Anterior is to the left in all images. ExE=extraembryonic ectoderm, Em=embryonic tissue.

32

At E7.0 and E7.5 the region of BS69 expression also included the newly formed
extraembryonic mesoderm, specifically the allantois (Figure 2.2C, D). Successive stages
also appeared to express BS69 throughout the embryonic tissues. Serial transverse sections
of an E9.25 embryo showed that expression was ubiquitous at this stage (Figure 2.3). The
sections also revealed that the highest BS69 expression was in the neural tube and brain
(Figure 2.3 arrows and arrowheads, respectively). Transverse sections of a BS69
heterozygous E7.5 embryo stained with X-gal revealed that BS69 was expressed in the
early embryonic ectoderm, or epiblast, and weaker expression has been seen in the
embryonic and extraembryonic mesendoderm (Figure 2.4C). The VE, allantois, and
chorion ectoderm appeared to lack BS69 expression (Figure 2.4C, E). At E7.5 the tissue
with the highest level of BS69 expression was the epiblast (Figure 2.4C).

33

Figure 2.3 BS69 is expressed throughout the embryo at E9.25 with highest expression in
the neural tube and anterior brain. Top: diagram showing relative locations of sections.
A-P. Transverse sections of an E9.25 BS69 heterozygous embryo stained with X-gal.
Arrows indicate neural tube. Arrowheads indicate forebrain. Anterior is to the top in all
images.

34

Figure 2.4 BS69 expression is concentrated in the epiblast at E7.5. Left: Whole-mount X-
gal stained E7.5 BS69 heterozygous embryo showing the approximate location of
transverse sections A-F. A-F: 10uM transverse sections of an E7.5 BS69 heterozygous
embryo. Ant = anterior, Post = posterior, Epi=epiblast, Mes=mesoderm, ChE=chorionic
ectoderm, Al=allantois. Anterior is to the upper left in all images.

35

2.3.2 BS69 is Required for Mesendoderm Formation
To explain the difference in size between the control and BS69 KO embryos, we examined
them for markers of proliferation and apoptosis. (Figure 2.5) We performed
immunofluorescent staining on sections of E7.5 control and KO embryos using an antibody
against Ki67, a protein that is expressed in proliferating cells. We also used terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to mark cells undergoing
the DNA fragmentation that is characteristic of apoptosis. Ki67 staining appears to be
present at a similar level in all cells of both the control and KO embryos (Figure 2.5A).
TUNEL marks a greater number of cells in the KO embryo than the control as seen in both
the sagittal and transverse sections (Figure 2.5B, C). This suggests that the reason the KO
embryos are smaller than the controls is that they have begun to undergo cell death by E7.5,
which is supported by evidence of resorption in E8.0 litters (Figure 2.5D).The BS69 KO
mouse is not only smaller than the control at E7.5, but also has distinct morphological
differences. The main visual indicator of gastrulation, the primitive streak, is notably absent
in the BS69 KO embryos (Figure 2.1B, Downs and Davies, 1993). Therefore, we inquired
as to whether gastrulation occurs in BS69 KO embryos. To do this, we performed whole-
mount in situ hybridization (WMISH) on E7.5 embryos with antisense RNA probes for
genes that are specifically expressed during gastrulation. We found that Wnt3, which is
required for the initiation of gastrulation and formation of the primitive streak, is not
expressed at E7.5 in the BS69 KO embryo (Figure 2.6A). It is known that canonical Wnt
signaling is required for mouse gastrulation from studies in which members of the
canonical pathway were knocked out—such mutant embryos do not survive past
gastrulation (Huelsken et al, 2000; Liu et al, 1999).
36

Figure 2.5 BS69 KO E7.5 embryos undergo apoptosis at a higher rate than controls. A:
Ki67 staining of sagittally sectioned E7.5 control and KO embryos. B: TUNEL staining of
sagittally sectioned E7.5 control and KO embryos. C: TUNEL staining of transversely
sectioned E7.5 control and KO embryos. D. E8.0 control and KO embryos from the same
litter. TUNEL-positive cells are indicated by arrowheads. Anterior is to the left in all
images.

37

To confirm a lack of canonical Wnt signaling, we then looked at Brachyury, a canonical
Wnt target gene that is expressed throughout the embryonic mesendoderm in the primitive
streak and in migrating mesoderm (Yamaguchi et al, 1999). We found that Brachyury is
also not expressed at E7.5 in the BS69 KO embryo (Figure 2.6B). Then we examined
specific anterior mesendoderm markers: Nodal and HNF3β. Nodal, though expressed
throughout the epiblast at earlier stages of development, marks the node at the distal tip of
the E7.5 embryo where definitive endoderm and the notochord will form (Conlon et al,
1994). HNF3β marks the anterior-most part of the primitive streak as it moves distally,
which is the region of mesendoderm destined to contribute to definitive endoderm, the
predecessor of foregut endoderm (Ang et al, 1993; Weinstein et al, 1994). Neither gene
was expressed in the KO, suggesting that no mesendoderm, whether specified for future
mesoderm or endoderm, was present (Figure 2.6C, D). Another gene known to be
expressed in the primitive streak and required for gastrulation is Bmp4. Bmp4 is also
expressed in extraembryonic ectoderm prior to primitive streak formation, as well as during
earlier pre-implantation stages of embryonic development (Lawson et al, 1999). Therefore,
we performed WMISH with a Bmp4 probe to find out whether its expression was affected
in the BS69 KO embryo. Our results show that Bmp4 is still expressed in the mutant
embryo, but only in the extraembryonic ectoderm (Figure 2.6E). There is no embryonic
Bmp4 expression in the KO whereas it is expressed in a posterior-to-anterior gradient in
the proximal embryonic mesoderm of the control (Figure 2.6E). Lack of Bmp signaling
has been seen to cause increased anterior gene expression, so we looked at Hesx1, a gene
transcribed in early anterior neuroectoderm (Di-Gregorio et al, 2007). We found that its
expression was expanded posteriorly and distally in the BS69 KO embryo (Figure 2.6F).
38

Figure 2.6 BS69 KO embryos do not form a primitive streak and prematurely specify
neuroectoderm. Whole mount in situ hybridization of E7.5 control (top) and KO (bottom)
embryos using antisense RNA probes. A. Wnt3 probe. B. Brachyury probe. C. Nodal
probe. D. HNF3β probe. E. Bmp4 probe. F. Hesx1 probe. Arrowheads indicate areas of
positive expression for each gene. Anterior is to the left in all images.

39

One possible explanation was that mesendoderm specification was initiated but not
maintained, resulting in a failure of primitive streak formation as in the Wnt3 epiblast-
specific knockout (Tortelote et al, 2013). To test this, we looked at gastrulation gene
expression in the pre/early streak E6.5 embryo. Neither Wnt3 nor Brachyury are expressed
in the BS69 KO E6.5 embryo, which suggests a complete lack of mesendoderm and
primitive streak specification (Figure 2.7A, B).

Lack of Wnt and Bmp signaling have both been shown to cause embryonic lethality at
E7.5, but previous studies demonstrate that abrogation of each pathway causes a different
defect (Di-Gregorio et al, 2007; Liu et al, 1999). The Wnt3 mutant fails to differentiate
past the epiblast stage and instead retains pluripotency markers (Liu et al, 1999). In
contrast, knocking out the Bmpr1a gene, a receptor required for canonical Bmp signaling,
results in premature differentiation of the epiblast into neuroectoderm (Di-Gregorio et al,
2007). Therefore, we examined a pluripotency marker, Oct4, in the E6.5 embryo at which
time it is normally expressed throughout the epiblast. Our BS69 KO embryo showed
decreased expression of Oct4 (Figure 2.7C). In addition, the expansion of Hesx1 in the
BS69 KO at E7.5 suggests that the BS69 phenotype more closely resembles the phenotype
of the Bmpr1a knockout and does not fully reflect features of the Wnt3 knockout.

40

Figure 2.7 BS69 KO embryos do not maintain pluripotency or initiate expression of
posteriorizing genes. Whole mount in situ hybridization of E6.5 control (top) and KO
(bottom) embryos using antisense RNA probes. A. Wnt3 probe. B. Brachyury probe. C.
Oct4 probe. Arrowheads indicate areas of positive expression for each gene. Anterior is to
the left in all images.

41

Because determining the state of the epiblast in the BS69 KO is critical to our
understanding of the gene’s function, to confirm Oct4 down-regulation we analyzed its
expression at the protein level. We showed that it was down-regulated in the KO embryo
and only has a small number of positive cells in the proximal epiblast, in contrast to the
control, which is positive throughout the epiblast (Figure 2.8B). This, together with our
previous observation that the anterior neuroectoderm gene Hesx1 was expanded in the
BS69 KO embryo, is indicative of early neuroectoderm differentiation. Additionally, the
presence of Sox2, a marker of pluripotent and early neuroectoderm lineage cells,
throughout the epiblast of the KO corroborates our findings (Figure 2.8C). In the control,
Sox2 expression begins to be down-regulated in the posterior epiblast as the primitive
streak expands (Figure 2.8C). To further determine whether Wnt signaling plays a role, we
examined the embryos for presence of β-catenin. As shown in Figure 2.8D, a high level of
β-catenin is present at the cell membranes in the KO, in a similar pattern to the control. β-
catenin is known to be involved in cell-cell adhesion when it is localized to the cell
membrane. During primitive streak formation, cell-cell adhesion is down-regulated to
allow epiblast cells to undergo EMT. Because EMT is a hallmark of gastrulation, we
examined the embryos for down-regulation of another cell-cell adhesion molecule: E-
cadherin. At the protein level, we see that there is a population of E-cadherin negative cells
in the posterior of the control embryo, but no such population in the KO (Figure 2.8A).
This suggests that EMT is not occurring in the KO embryo.

Our data, both at the RNA and protein levels, show that the genes required for
mesendoderm specification and primitive streak formation are not expressed in the BS69
42

KO embryo. Additionally, the genes that must be down-regulated in order to initiate
gastrulation are still expressed throughout the epiblast of the BS69 KO. These results
demonstrate that BS69 is required for the formation of mesendoderm in the mouse gastrula.

43

Figure 2.8 BS69 KO embryos do not appear to pheno-copy Wnt pathway mutants.
Immunofluorescent staining of sagittally sectioned E7.5 control (top) and KO (bottom)
embryos. A. E-cadherin antibody; arrow indicates region without E-cadherin staining in
the control. B. Oct4 antibody. C. Sox2 antibody; arrowhead indicates region of decreased
Sox2 staining in the control. D. β-catenin antibody. 4',6-diamidino-2-phenylindole (DAPI)
serves as a counterstain. Anterior is to the left in all images.

44

2.4 Discussion

2.4.1 Summary
We have created the first BS69 KO mouse using gene-trap knock-in technology (Figure
2.1). Our studies of the KO show that BS69 is required for embryogenesis. Without it,
embryos are not viable past E7.5, are much smaller than controls, and lack a morphological
primitive streak (Figure 2.1B). As shown by X-gal staining of whole-mount embryos
heterozygous for the BS69 knock-in, BS69 is expressed throughout the embryonic tissues
from E5.5 through E9.25 (Figure 2.2) At E9.25, the expression is throughout all tissues but
is highest in the neural tube and brain precursors (Figure 2.3) This increased expression in
neuroectoderm precursors can be traced back to the E7.5 embryo, in which BS69 is most
highly expressed in the epiblast (Figure 2.4C).

To understand the specific defects in the BS69 KO embryo, we first analyzed the difference
in size. Using Ki67 staining, we found that cells in the BS69 KO embryo do proliferate
(Figure 2.5A). TUNEL showed that more cells in the KO are undergoing apoptosis at E7.5,
but because the difference was not drastic, we cannot conclude that cell death is the main
cause of the KO’s smaller size and subsequent resorption (Figure 2.5B-D). To address the
failure of primitive streak formation, we analyzed the expression of genes that are required
for this process. As shown by the lack of Wnt3, Brachyury, Nodal, and HNF3β expression
as well as down-regulation of Bmp4 in the E7.5 BS69 KO embryo, mesendoderm cells do
not form (Figure 2.6A-E). A lack of Wnt3 and Brachyury mRNA at E6.5 shows that BS69
KOs do not initiate the process of gastrulation (Figure 2.7A, B). Rather, we found that
BS69 KO embryos differentiate into early neuroectoderm while down-regulating
45

pluripotency genes. Our analysis showed expanded Hesx1 expression at E7.5 and
decreased Oct4 expression at E6.5 (Figure 2.6F, 2.7C). We confirmed that the Oct4 protein
level is decreased in E7.5 BS69 KO embryos, which shows that the epiblast is no longer
pluripotent (Figure 2.8B). Additionally, we found that Sox2, which marks pluripotent cells
and neuroectoderm, was symmetrically expressed in the KO, suggesting that the entire
epiblast had acquired an anterior fate (Figure 2.8C). E-cadherin, a protein that is down-
regulated in the primitive streak, was found at cell-cell junctions throughout the BS69 KO
embryo, demonstrating a lack of EMT (Figure 2.8A) Finally, we saw that β-catenin, which
also participates in cell-cell adhesion, was localized to the cell membrane in BS69 KO
embryos (Figure 2.8D). Together, this data shows that the primitive streak does not form
in the BS69 KO. Therefore, we conclude that BS69 is required for gastrulation in the mouse
embryo.

2.4.2 Signaling Pathway Analysis
The phenotype of embryonic lethality during or just prior to gastrulation is true for a myriad
of different mutations in signaling pathways. The Fgf, Bmp, Wnt, and Nodal pathways are
all required for successful gastrulation. (Conlon et al, 1994; Liu et al, 1999; Mishina et al,
1995; Yamaguchi et al, 1994) Perhaps the least dramatic mutation from this list is that of
Fgf8. These mutants begin gastrulation and can form mesoderm but the cells are unable to
migrate after they pass through the prospective primitive streak (Sun et al, 1999). Knocking
out the Fgf receptor 1 (Fgfr1) causes ectopic expression of E-cadherin in mesendoderm
cells within the primitive streak, showing that Fgf signaling is required for EMT to occur.
(Ciruna & Rossant, 2001) In this study they observed a decrease in Snail transcript in the
46

absence of Fgfr1, suggesting that it is a target of Fgf signaling in the gastrulating embryo.
Snail functions to down-regulate E-cadherin at the gene level, which would explain the
phenotype of the Fgfr1 KO mouse. Another function of Fgf signaling in the early embryo
is to up-regulate Wnt3a and the T-box transcription factors Brachyury and Tbx6--these
genes are all down-regulated in the Fgfr1 KO. (Ciruna & Rossant, 2001) A possible cause
for these effects is ectopic E-cadherin expression. The Wnt signaling effector β-catenin is
sequestered at the cell membrane with E-cadherin in Fgfr1 mutants. This hypothesis is
supported by the ability of anti-E-cadherin antibodies to restore cytoplasmic β-catenin
levels and Brachyury expression in the Fgfr1 KO. (Ciruna & Rossant, 2001) Thus, the Fgf
pathway plays an integral role in gastrulation. Interestingly, BS69 KO embryos do not
down-regulate E-cadherin, similar to the Fgfr1 mutant (Figure 2.8A). However, they do
not have the accumulated mesoderm cells that are seen when the Fgf pathway is disrupted.
Because Fgfs are down-regulated in the primitive streak of Wnt, Nodal, and Bmp mutants,
this suggests that Fgf signaling is downstream of these pathways. Therefore, BS69 could
similarly function upstream of the Fgf pathway.

Canonical Wnt signaling is a known requirement for gastrulation and mesendoderm
formation. Mice with mutations in canonical Wnt/β-catenin pathway components die
around E7.5 and do not show signs of a primitive streak (Haegel et al, 1995). However,
they also do not show signs of differentiation into neuroectoderm. Wnt/β-catenin pathway
mutants retain pluripotency markers such as Oct4, which suggests that β-catenin signaling
is required for differentiation into both the mesendoderm and neuroectoderm lineages
(Huelsken et al, 2000). Although the BS69 KO embryo lacks Wnt3 expression, it down-
47

regulates Oct4, which suggests that the defect is not solely in Wnt signaling (Figure 2.6A,
2.7A, C). In contrast, Bmp pathway mutants are gastrulation-lethal but do not retain
pluripotency markers (Di-Gregorio et al, 2007; Mishina et al, 1995). Like Wnt mutants,
they cannot form mesendoderm, but they differentiate into neuroectoderm instead. This
suggests that the Bmp pathway is required for both the maintenance of pluripotency and
the differentiation of mesendoderm. Because the Bmpr1A mutant lacks Wnt3 expression
while Bmp4 is expressed in the ExM of Wnt3 mutants, Bmp signaling may be upstream of
Wnt signaling in the embryo (Di-Gregorio et al, 2007; Liu et al, 1999). In regards to the
BS69 KO embryo, the phenotype most closely resembles a Bmp pathway mutation. The
BS69 KO embryo is lethal at E7.5 and expresses neuroectoderm markers but not
mesendoderm or pluripotency markers (Figure 2.1, 2.6, 2.7). Bmp4 is expressed in the ExE
of BS69 KOs, so if BS69 is involved in the Bmp pathway it is probably downstream of
ligand expression and processing (Figure 2.6E).

Both Bmp4 and Nodal are required for Wnt expression in the embryo. As we have already
examined the Bmp pathway, we now turn to Nodal. In the Nodal mutant mouse, the embryo
arrests prior to primitive streak formation, lacks mesendoderm, and has expanded
neuroectoderm specification (Brennan et al, 2001; Conlon et al, 1994). Surprisingly, Nodal
signaling in the embryo that is required for mesendoderm formation is independent of
Smad2, the loss of which produces expanded posterior marker expression and lack of
neuroectoderm gene expression (Brennan et al, 2001). Cripto, a Nodal co-receptor, is
required for normal mesendoderm specification, but some primitive streak markers are still
found in Cripto-null embryos (Ding et al, 1998). The main defect in Cripto-null embryos
48

is a lack of anterior-posterior axis formation, but expression of some neuroectoderm and
mesendoderm genes are present in the ExE. This suggests that not only can Nodal function
independently from Smad2, but this pathway may also be independent of Cripto. To
complicate our understanding of Nodal function in the embryo, the double knockout of
Smad2 and SMAD family member 3 (Smad3) produces a phenotype that does resemble
the Nodal mutant, except the embryo is smaller (Dunn et al, 2004). In fact, the closest
resemblance is to the SMAD family member 4 (Smad4) mutant and the double knockout
of activin receptor IIA and IIB (Acvr2a/2b) (Sirard et al, 1998; Song et al, 1999). Other
TGFβ-family members are known to bind Acvr2a/2b and signal by phosphorylated
Smad2/3 interaction with Smad4, so this phenotype could reflect the inhibition of multiple
convergent pathways (Cheng et al, 2003). To compare these mutants to the BS69 KO
embryo, the Smad4 mutant is more severe and has a visible size difference by E5.5,
whereas we do not see a change in the BS69 KO until E7.5 (Figure 2.1B). The
ActIIA/ActIIB double knockout was not analyzed until E7.5, so we cannot compare the
sizes at earlier stages. At E7.5, the embryonic region of the ActIIA/ActIIB double knockout
is similar to the BS69 KO, but the ExE displays a stronger phenotype with distinct
malformations (Song et al, 1999). Returning to the Nodal mutant, the ExE shows a stronger
defect than in the BS69 KO (Conlon et al, 1994). Nodal null embryos are distinguishable
from controls as early as E5.5, whereas BS69 KOs are not morphologically different until
E7.5 (Camus et al, 2006). Additionally, Nodal null embryos do not stop proliferating once
morphological differences become apparent. Rather, both the epiblast and visceral
endoderm continue to grow. Therefore, we require additional data to determine whether
BS69 is involved in Nodal signaling.
49

Out of the multitude of pathways involved in gastrulation, we have shown that BS69 is
unlikely to be directly involved in either Fgf or Wnt signaling and may play a role in Bmp
or Nodal signaling. Both the Bmp and Nodal pathways are activated earlier than the Fgf or
Wnt pathways in the post-implantation embryo, so it is plausible that BS69 acts upstream
of both the Fgf and Wnt pathways. To determine the function of BS69, we require a tool
with which to analyze the transcriptome of BS69 KO cells in various states of
differentiation.

50

Chapter 3: BS69 is Required for the Formation of
Mesendoderm from Mouse Embryonic Stem Cells In
Vitro, but is Dispensable for Neuroectoderm
Differentiation
3.1 Abstract
Because the BS69 KO mouse embryo dies at E7.5, our ability to further assess BS69
function in vivo is limited. Therefore, we employ mESC technology to generate an in vitro
model of early embryonic development. mESCs have been shown to mimic the ICM, and
when differentiated, mimic the differentiation of tissues in vivo. This has allowed us to
show that BS69 is not required for mESC self-renewal and does not affect the rate of cell
proliferation. BS69 KO mESCs express pluripotency markers similarly to controls and can
differentiate into epiblast. However, BS69 does affect the expression of some genes in
mESCs, though their functions are mostly unknown. BS69 is required for the expression
of mesendoderm markers in vitro, and therefore our data supports our in vivo findings.

51

3.2 Introduction
Embryonic stem cells are a robust in vitro system in which to study early development that
has been used for almost a quarter of a century (Evans & Kaufman, 1981; Martin, 1981).
Whereas the ICM of the blastocyst contains only a small number of cells, mESCs can be
expanded to produce as many cells as necessary for an experiment. Additionally, the
characteristics that delineate stemness have been well-defined in past years, making it
relatively straight-forward to determine if a particular mESC line is pluripotent (Pelton et
al, 2002; Ying et al, 2008). Three transcription factors were implicated early in embryonic
stem cell research to be required for pluripotency: Nanog, Oct4, and Sox2 (Masui et al,
2007; Mitsui et al, 2003). These factors are now known to function in the epiblast as well,
which diminishes their power as markers of mESCs. However, genes such as Rex1 have
been shown to decrease in expression upon mESC to epiblast differentiation, while Fgf5
expression increases (Pelton et al, 2002). Therefore, it is possible to determine whether
mESCs are truly pluripotent or have begun to differentiate.

In addition to being essential tools for studies on pluripotency, mESCs are also valuable
for understanding how tissues differentiate in the embryo. When placed in non-adherent
culture in media that does not support pluripotency, mESCs begin to differentiate into
embryoid bodies (EBs), groups of mESC-derived cells that attempt to recapitulate
embryogenesis (Leahy et al, 1999). These EBs form derivatives of all three germ layers
and can even generate contractile cardiomyocytes and mature neurons. EB formation has
been seen to mimic embryonic development at early stages as well. At three days after the
beginning of EB formation (day 3), EBs begin to form an AP axis that is dependent upon
52

Wnt signaling (ten Berge et al, 2008). Day 3 of EB formation corresponds to E6.5, which
is also the stage when Wnt signaling is initiated and the AP axis established. Additionally,
EBs can differentiate according to treatment with factors that are expressed in vivo during
embryonic development (Yuasa et al, 2005). Thus, EBs can serve as a tool to examine
embryogenesis without the limitations of the embryo itself.

We have used this tool in order to examine whether BS69 functions the same in vitro as in
vivo, and to acquire a global picture of the genes that are affected in the KO. We show that
BS69 KO mESCs can be established and that they express pluripotency markers.
Furthermore, we confirm that BS69 KO EBs recapitulate the phenotype of BS69 KO
embryos.

53

3.3 Results
3.3.1 BS69 is not required for the in vitro establishment and maintenance of
pluripotent mESCs.
Although we observed weak expression of BS69 in the epiblast during early embryonic
development in control embryos at E5.5-E6.5, there is no obvious defect in the
development of the BS69 KO embryo until gastrulation (Figure 2.2A, B). Therefore, it
appears that BS69 may not be required until that stage. To test this hypothesis, we tried to
establish an in vitro model by isolating mESCs. To derive BS69 KO and control mESCs,
we isolated E3.5 blastocysts from the uterus of a pregnant female. We then placed the
blastocysts in plates with mouse embryonic fibroblasts (MEFs) where they hatched from
the zona pellucida and attached to the feeder layer. Once the ICMs of the blastocysts grew
to an appropriate size, we manually subcloned them by removing the out-growths from the
surrounding trophectoderm. Subsequently, we trypsinized them and plated each
disassociated ICM in a separate well with MEFs. Initially, we used leukemia inhibitory
factor (LIF)-containing media, as recommended by a previous study (Williams et al, 1988).
However, neither the control nor the BS69 KO cells retained mESC morphology and we
could not propagate the lines. Most likely our difficulties growing and maintaining them
as pluripotent mESCs were because both the control and the BS69 KO blastocysts were on
a background that includes C57/BL6, a mouse line which is known to be refractory and
does not grow in the standard LIF-containing mESC media (Umehara et al, 2007).
Therefore we tried to culture our mESCS using the 2-chemical inhibitor (2i) media system,
which contains PD0325901 (PD03) and CHIR99021 (CHIR), inhibitors of mitogen-
activated protein kinase-kinase (MEK) and glycogen synthesis kinase 3-beta (GSK3β)
respectively (Ying et al, 2008). The colonies’ morphology had partially improved in this
54

media with a higher incidence of rounded, phase-bright edges, but there were still more
irregularly shaped colonies than seen in mESCs from non-refractory strains. We tried a
variety of different combinations of culture conditions to determine the best media in which
to propagate our mESCs (Figure 3.1). Based on colony morphology, we determined that a
combination of LIF and the 2i system gave the best results (Figure 3.1A). We were able to
propagate these cell lines on MEFs and they continued to perform well on gelatin-coated
plates using media supplemented with LIF and 2i. We did not observe significant
differences between the control and BS69 KO mESCs for any of the culture conditions
tested (Figure 3.1). Moreover, the BS69 KO blastocysts and inner cell masses were
morphologically indistinguishable from controls (data not shown).
55

Figure 3.1 BS69 KO mESCs are able to sustain pluripotency in media containing both LIF
and 2i. A-H. mESCs grown for two days in mESC media with only the pluripotency
sustaining factors listed. Cells were grown on 0.01% gelatin-coated tissue culture dishes.
56

We established two BS69 KO lines from two independent biological samples. We
confirmed by RTPCR that these two cell lines lacked BS69 transcript whereas the controls
had a band at 379bp (Figure 3.2B). Morphologically, the BS69 KO mESC cell lines
exhibited compact colonies with phase-bright edges, indicative of pluripotent cells (Figure
3.2A). To check if BS69 KO mESCs maintain pluripotency, first we stained them for the
pluripotency marker alkaline phosphatase and demonstrated that they are positive for this
enzyme (Figure 3.2D).
Next, we performed RTPCR for additional markers of pluripotency to determine if these
BS69 KO putative embryonic stem cell lines were similar in gene expression to the control
lines. We saw that the BS69 KO and control lines expressed similar levels of Nanog and
Rex1, two commonly used markers of pluripotency (Figure 3.2C; (Kooistra et al, 2010;
Mitsui et al, 2003). Rex1 in particular is only expressed in embryonic stem cells but not
epiblast stem cells; (Adjaye et al, 2005; Pelton et al, 2002). Both BS69 KO mESCs lines
were also similar to control mESCs in their lack of Fgf5 expression, which is one of the
first markers of mESC differentiation that is up-regulated in epiblast stem cells (EpiSCs)
(Pelton et al, 2002). Finally, we analyzed whether BS69 KO mESCs cycle at a similar rate
to control mESCs using PI incorporation and flow cytometry. We detected the intensity of
PI fluorescence per cell and correlated that to its phase in the cell cycle. We saw that the
percentage of cells in s-phase was similar for both the control and KO cell lines (Figure
3.3).

57

Figure 3.2 BS69 KO mESCs can be maintained in a pluripotent state. A. Phase-contrast
images of BS69 KO and control mESCs plated on MEFs. B. RTPCR Genotyping with
primers for BS69 exon 9 to exon 11 with GAPDH as a loading control. C. RTPCR with
primers for Nanog, Rex1, Fgf5, and Brachyury with GAPDH as a loading control. D.
Phase-contrast images of alkaline-phosphatase stained control (C1 and C2) and KO (KO1
and KO2) mESCs.

58

Figure 3.3 BS69 KO mESCs cycle at a similar rate as control mESCs. Histogram of flow
cytometry cell cycle analysis of propidium iodide (PI) stained control and BS69 KO
mESCs. Number of cells (Y-axis) is plotted against PI fluorescence (X-axis). S = DNA
synthesis phase (s-phase).

59

Taken together, these data suggest that the mESC lines formed from BS69 KO blastocysts
are not affected by the genetic disruption of BS69 and are able to proliferate and maintain
their pluripotency in vitro.

60

3.3.2 BS69 is Required for the Formation of Mesendoderm from Mouse Embryonic
Stem Cells In Vitro, but is Dispensable for Neural Differentiation

Our in vivo data we demonstrated that BS69 is required for the formation of mesendoderm
during gastrulation. Thus, we would like to further address whether BS69 is required for
the differentiation of mesendoderm in vitro, and if so, whether it works in the same way in
our derived mESC lines as in vivo. To test this we used the EB formation assay, in which
mESCs are placed in non-adherent culture and pluripotency supplements are withdrawn
from the media. We used the hanging-drop method to culture our EBs because it allowed
us to specify the number of cells per EB. Based on (Mansergh et al, 2009), we used
approximately 1000 cells per hanging drop, as this was found to generate the highest
number of differentiated cell types. In the hanging drops, the cells aggregated and
differentiated in absence of LIF, PD03, and CHIR (Mansergh et al, 2009).

During EB formation over the first 4 days, the sizes of control and BS69 KO EBs were
comparable, suggesting similar proliferation rates (Figure 3.4). To analyze the differences
between the cell lines, we took samples after 3 days of differentiation which corresponds
to approximately E6.5 of mouse in vivo development (Leahy et al, 1999; Mansergh et al,
2009). In day 3 EBs we looked at markers for gastrulation to check if our in vitro assay
recapitulates our findings in vivo and thus would be a reasonable assay to address the
function of BS69 in gastrulation and germ lineage specification.

61

Figure 3.4 BS69 KO mESCs can form EBs to similar sizes as controls. Images taken over
4 days of EB growth in hanging drops.

62

We used RTPCR to analyze the mRNA from Day 3 EBs and saw that they do differentiate
into epiblast. Rex1 is one of the first pluripotency markers to become down-regulated at
the embryonic stem cell to epiblast transition, and by day 3 the KO EBs have little to none,
as do the controls (Figure 3.5A;(Rogers et al, 1991). Concurrently, Fgf5 is up-regulated in
the epiblast, without difference between the control and KO EBs (Figure 3.5A). Next, we
looked at markers of gastrulation that are known to be expressed at E6.5 like: Wnt3 and
Brachyury. We observed that the KO cell lines do not express either gene after 3 days of
differentiation, confirming our in vivo findings (Figure 3.5A). Since we performed the
RTPCR on mRNA isolated from pooled EBs, we performed WMISH for Wnt3 and
Brachyury on the day 3 EBs to determine if there were any differences in gene expression
levels among individual EBs (Figure 3.5B). We did not find any EBs that expressed either
Wnt3 or Brachyury at day 3, which confirmed our RTPCR results (Figure 3.5). We
demonstrated that the early neuroectoderm gene Hesx1 was expanded in the BS69 KO
embryo and Sox2 was concomitantly present throughout the epiblast, indicating premature
neuroectoderm differentiation.

63

Figure 3.5 BS69 KO Embryoid bodies are able to differentiate into epiblast but not
primitive streak cells. A. RTPCR of BS69 KO and control mESCs compared to day 3 EBs
for Rex1, Fgf5, Brachyury, and Wnt3 with GAPDH as a loading control. B. WMISH of
day 3 control and KO EBs using antisense RNA probes against Wnt3 and Brachyury.

64

Thus far, these studies suggest that BS69 does not play an essential role until mesendoderm
formation begins, since we have not found significant differences in gene expression,
morphology, or growth rates between control and BS69 KO mESCs. Therefore, we next
addressed the function of BS69 in regulating global gene expression. We used next
generation RNA sequencing to acquire a global picture of expression in mESCs and day 3
EBs. We chose these specific time points because they represent pluripotent cells in the
blastocyst and the embryo just prior to gastrulation (E6.5). Our sequencing analysis
revealed 106 DEGs between BS69 KO and control mESCs (Figure 3.6A). Because our
RTPCR analysis of the BS69 KO mESCs compared to controls did not uncover any
differences in gene expression, even this relatively low number of DEGs was unexpected.
Interestingly, most of the genes—87 out of 106—in this list were down-regulated (Figure
3.6B).

65

Figure 3.6 The number of differentially expressed genes (DEGs) increases over time. A.
Venn diagram showing the total number of DEGs determined by RNA sequencing analysis
of BS69 KO and control mESCs and day 3 EBs. B. Tabulated DEGs per sample with
numbers down-regulated versus up-regulated.

66

Gene ontology analysis using the DAVID Bioinformatics Resources 6.7 Functional
Annotation Tool (DAVID) resulted in few enriched categories, the highest of which was
among the down-regulated genes: translational initiation (Figure 3.7). However, the
majority of the functions of these genes are not yet known as their status is listed as
predicted on the National Centers for Biotechnology Information Gene database website
(Pruitt et al, 2013).

We observed that the majority of the genes in the mESC DEG list were predicted genes all
of which were down-regulated (Figure 3.8). Surprisingly, we also noticed that almost half
of all the mESC DEGs are within 100kb of another gene that is also on the list of DEGs
(Figure 3.8). While not obviously causal, it could suggest interesting hypotheses about how
BS69 could potentially function in mESCs. We will discuss the significance of this
correlation at the end of this chapter.

67

Figure 3.7 In mESCs there are few enriched Gene Ontology categories. All GO_BP_FAT
terms from DAVID Functional Annotation of mESC differentially expressed genes. A.
Down-regulated gene categories are in blue (Count: 2, EASE: 0.1). B. Up-regulated gene
categories are in red (Count: 2, EASE: 0.1). (Ashburner et al, 2000; Dennis et al, 2003).

68

Figure 3.8 A large proportion of mESC DEGs are down-regulated, predicted, and less
than 100kb away from another mESC DEG. Light blue: 82% of mESC DEGs are down-
regulated. Medium blue: 61% of mESC DEGs are predicted. Dark blue: 49% of mESC
DEGs are within 100kb of another gene on this list. Red: 18% of genes are up-regulated.

69

Our day 3 EB DEG analysis confirmed our in vivo WMISH and RTPCR results, with gene
expression of Wnt3, Nodal, and HNF3β decreased, among others (Figure 3.9A). Strikingly,
the total number of differentially expressed genes at day 3 was limited to just 171;
furthermore, most of these genes (114) were down-regulated (Figure 3.9). Gene ontology
analysis using the DAVID software shows that the main pathways affected in the down-
regulated differentially expressed genes at day 3 are related to Wnt signaling, gastrulation,
and mesoderm formation (Figure 3.9A). The last two of the top 10 categories are related to
neural tube closure and neuron maturation, but many of the genes in these categories are
also associated with mesoderm differentiation. The number of up-regulated differentially
expressed genes is relatively low and the gene ontology analysis shows few significantly
enriched pathways (Figure 3.9B).

70

Figure 3.9 Wnt pathway, germ layer formation, mesoderm formation, and gastrulation
related genes are down-regulated in BS69 KO day 3 EBs. A. Top ten of 32 total
GO_BP_FAT terms from DAVID Functional Annotation of day 3 EB down-regulated
differentially expressed genes (Count: 5, EASE: 0.01). B. All GO_BP_FAT terms from
DAVID Functional Annotation of day 3 EB up-regulated differentially expressed genes
(Count: 5, EASE: 0.01). (Ashburner et al, 2000; Dennis et al, 2003).

71

3.4 Discussion

3.4.1 Summary
To study the function of BS69 in more depth, we derived mESC lines from BS69 KO and
control blastocysts at E3.5. BS69 KO mESCs react to varied growth conditions similarly
to controls and demonstrate ideal morphology with the same media additives as controls
(Figure 3.1). In media containing LIF and 2i, BS69 KO mESCs proliferated and expressed
the pluripotency markers Nanog and Rex1 (Ying et al, 2008). BS69 KO mESCs did not
express Fgf5 or Brachyury, markers of epiblast and mesendoderm differentiation,
respectively (Figure 3.2C). Furthermore, they stained positively for alkaline phosphatase,
a common test for pluripotency (Figure 3.2D). To determine whether BS69 functions the
same in vitro as in vivo, we used the EB formation assay to differentiate the mESCs for 3
days, which is comparable to an E6.5 embryo. Day 3 KO EBs grew to a similar size as
controls and could differentiate into epiblast but not mesendoderm, similar to our in vivo
findings (Figure 3.4, 3.5). Therefore, we used RNA-sequencing to examine the full
transcriptome of BS69 KO mESCs and day 3 EBs in comparison to controls (Figure 3.6).
We found that at both time points there are more down-regulated genes in the BS69 KO
than up-regulated. Interestingly, the down-regulated genes in BS69 KO mESCs contained
a high proportion of predicted genes that were less than 100kb away from another
differentially expressed gene (Figure 3.8). At day 3, gene ontology analysis showed that
Wnts and mesoderm-related genes were down-regulated in the KO, including genes that
we had already confirmed to be down in the BS69 KO embryo (Figure 3.9). Thus, BS69 is
not required in mESCs, but it is needed for normal mesendoderm differentiation in vitro,
which correlates with our findings in vivo.
72

3.4.2 Effects on mESC Gene Transcription
Based on our preliminary analyses, BS69 KO mESCs appeared to be essentially identical
to controls. Therefore, we were surprised to find that BS69 alters the transcription of 106
genes in mESCs. Even more intriguing was the prevalence of down-regulated, predicted
DEGs that cluster within the genome. Out of the 106 DEGs, 49% had these three
characteristics. This suggests that these specific genes may not be targeted for regulation,
but rather the entire region of the chromosome that they occupy. Because they are down-
regulated in BS69 KO mESCs, that suggests that BS69 may be required to allow or
encourage transcription of these regions of DNA. For that to be true, BS69 would likely be
involved in chromatin remodeling or modification, which is known to be important for
differentiation (Chen & Dent, 2014). There is precedence for BS69 interaction with
chromatin modifying proteins, but no evidence to suggest that BS69 itself catalyzes
chromatin modifications or remodifications (Velasco et al, 2006). For now, we will leave
this as an unanswered question to be addressed in the future.

One specific DEG in mESCs was particularly notable: left right determination factor 2
(Lefty2). Lefty2 is expressed in the blastocyst as well as in mESCs, and is not down-
regulated upon differentiation (Kim et al, 2014). In BS69 KO mESCs we found that Lefty2
was down-regulated, which could potentially affect differentiation. Lefty2 knockout E7.5
mouse embryos have expanded primitive streaks and excess mesoderm, which is opposite
of the phenotype that we see in BS69 KO embryos (Meno et al, 1999). This appears to be
contradictory, but in vitro experiments suggest differently: Lefty2 knockout mESCs form
excess neuroepithelium when differentiated in a teratoma formation assay—suggestive of
73

our E7.5 BS69 KO embryos, which form expanded neuroectoderm (Kim et al, 2014).
However, not enough is known to suggest potential interactions between BS69 and Lefty2.
It may be that Lefty2 down-regulation is an indirect effect in BS69 KO mESCs.

74

3.4.3 Signaling Pathway Analysis
As we discussed in Chapter 2, our in vivo data revealed the function of BS69 in
gastrulation, but did not clearly correlate with a signaling pathway. Our in vitro data
supports and adds to what we found in vivo. We suggested previously that the Fgf pathway
is likely to be down-stream of BS69 function, which is supported by our RNA-sequencing
data. Fgf8 is down-regulated in BS69 KO day 3 EBs. Many Wnt ligands are also down-
regulated in these EBs along with known Wnt target genes such as Axin2 and Brachyury
(Figure 3.9A, Data not shown). Additionally, pluripotency genes are not up-regulated,
which suggests that Wnts are not the primary target of BS69. At day 3, only one Bmp is
down-regulated: Bmp7 (Figure 3.9A). Bmp2 and Bmp4 are also known to be expressed
in EBs, and their absence from the list of differentially expressed genes suggests that they
are able to be expressed at a similar level to controls. Therefore, BS69 does not appear to
be up-stream of all Bmp ligand expression. Our in vivo data suggested that Nodal could be
directly affected by BS69, and it is down-regulated in day 3 BS69 KO EBs (Figure 3.9A).
However, known Nodal target genes such as Cer1 and Lefty1 are not down-regulated in
day 3 BS69 KO EBs, so BS69 is not required for Nodal signaling. Although there is no
obvious defect in BS69 KO mESCs, they still can help us to determine BS69’s function.
The fact that we were able to establish mESC lines without BS69 can help us to eliminate
possible mechanisms. This is because the Bmp pathway is required for mESC proliferation;
Bmpr1a mutant blastocysts do not proliferate in vitro, and therefore Bmpr1a mutant mESC
lines cannot be established (Di-Gregorio et al, 2007). Thus, the Bmp pathway must
function to some extent in BS69 KO embryos. Another interesting clue from our BS69 KO
mESC RNA-sequencing is the presence of Lefty2 on the list of down-regulated genes.
75

Lefty2 is a member of the TGFB family and is a feedback inhibitor of Nodal. Lefty2
knockdown in mESCs results in an increased ability to self-renew under differentiation-
promoting conditions (Kim et al, 2014). However, we did not see any difference in the
ability of BS69 KO mESCs to self-renew. They appeared to differentiate at a similar rate
to controls during the EB formation assay.

Our data suggests that BS69 would most likely be involved upstream of the Wnt and Fgf
pathways and downstream of the Bmp and Nodal pathways. We have found support for
this hypothesis in the literature. The Smad4 null cell line generates EBs with a high
proportion of similarities to BS69 KO EBs (Figure 3.10, (Costello et al, 2009). We found
that more genes were represented both in the differentially expressed gene lists from our
BS69 KO EBs and the Smad4 null EBs than from other lists we compared, such as the
Bmp target gene data (Fei et al, 2010). Notably, the Smad4 null was the only cell line in
which Fgf8 was down-regulated and Fgf4 upregulated upon differentiation, just as in our
BS69 KO EBs. Fgf4 is implicated to function in early neuroectoderm development in
concert with Bmp inhibition in Xenopus (Linker & Stern, 2004). However, in all the studies
we examined except for the Smad4-null paper, it was down-regulated wherever Fgf8 was
down-regulated. Despite the support for BS69 involvement in Smad4-mediated signaling,
there are caveats. Smad4 is required for both Bmp pathway and Nodal pathway
transduction, and we have shown that the Bmp pathway is likely to be active in BS69 KO
embryos. Therefore, Smad4 would have to be at least partially able to carry out its normal
function. As previously discussed, Nodal null embryos share many commonalities with
BS69 KO embryos, but they also have distinct differences. With the additional data
76

provided by our RNA-sequencing study, we see that there are also differences in gene
expression between Nodal and BS69 mutants. Nodal null embryos express goosecoid (Gsc)
and HNF3β/ Foxa2, two anterior primitive streak markers that are down-regulated in BS69
KO day 3 EBs (Figure 3.9A, (Camus et al, 2006). These similarities and differences to the
BS69 KO suggest that further studies are prudent to determine whether BS69 interacts with
Nodal signaling or if it functions through a different mechanism.

Our development of BS69 KO mESCs and EBs as a tool with which to study BS69 function
has allowed us to confirm our in vivo results: BS69 is required for the development of
mesendoderm. Due to our success in maintaining pluripotent BS69 KO mESCs, we can
also surmise that BS69 is unlikely to abrogate Bmp signaling.

77

Figure 3.10 One quarter of BS69 KO day 3 EB DEGs are also differentially expressed in
SMAD4 null day 4 EBs. Comparison between the DEG lists from our BS69 day 3 EB RNA-
seq data and the microarray data provided by (Costello et al, 2009).

78

Chapter 4: Long-Term Consequences of BS69 Function
4.1 Abstract
Our in vitro system allows us to probe BS69 function over a longer time period than we
are able to in vivo. Therefore, we can ask whether BS69 affects organogenesis. Following
gastrulation, the embryo is patterned and begins to differentiate toward myriad tissues.
Using the EB formation assay, we find that neurogenesis is promoted in the BS69 KO,
resulting in an up-regulation of neuroectoderm markers at day 5. Interestingly, we see that
while more neurons are formed in BS69 KO EBs at days 8 and 12, neuroectoderm remains
up-regulated. Conversely, these mature EBs do not efficiently generate mesoderm-derived
tissues and cannot form contractile cardiomyocytes. The small amount of mesoderm
differentiation shows that BS69 KO embryos do specify some mesendoderm, but are
delayed. Although BS69 affects neuroectoderm and mesendoderm differentiation, it is not
required for the formation of endothelial cells and does not affect the rate of proliferation
or apoptosis in vitro. Finally, although the Bmp pathway has been implicated in blocking
neuroectoderm, here we show that it is not abrogated in BS69 KO cells.

79

4.2 Introduction
After gastrulation, the embryo begins the process of organogenesis. Here, the progenitor
cells differentiate, multiply, and—in some cases—migrate, to organize each respective
organ and tissue. The signaling factors that were required during gastrulation play roles
here too: Stringent regulation of Bmp signaling is required to promote anterior surface
ectoderm rather than anterior neural differentiation of the ectoderm and Wnt signaling must
be timed in a specific manner in order to form the heart (Davis et al, 2004; Ueno et al,
2007). The gradients that are established for the purpose of gastrulation determine not only
the patterning, but also the differentiation of precursors.

One of the advantages of the EB formation assay is that in the absence of maternal cues,
cells may survive in vitro longer than an embryo of the same genotype would survive in
utero. This is true for the BS69 KO. Whereas BS69 KO embryos die at E7.5, we can
propagate BS69 KO EBs until at least day 12, which corresponds to approximately E15.5.
Therefore, we can determine whether BS69 is required for the differentiation of more
mature tissues. Because our results in Chapter 3 showed that mesendoderm was not formed
in the BS69 KO, but neuroectoderm was over-specified, we hypothesized that this would
be reflected in older EBs. Therefore, we would expect to see a down-regulation of genes
that are involved in mesoderm and endoderm development and an up-regulation of genes
that function in neuroectoderm. To study this, we again used RNA-seq to acquire a global
picture of gene regulation in day 5 EBs, which corresponds to approximately E8.5.

80

During and after gastrulation, regions of mesoderm become specified and different genes
are required for each multipotent precursor. For example, Mesp1 is required for the
migration of mesoderm cells to the future region where heart development takes place
(Kitajima et al, 2000; Saga et al, 1999). Later, Isl1 specifies the second heart field (SHF)
(Cai et al, 2003). In neural development, the gene Sox2 is a pan-neural precursor marker
that demarcates prospective neural tissue from the epiblast onward (Avilion et al, 2003;
Wood & Episkopou, 1999). Six3 marks specifically the anterior neuroectoderm, which
contributes later to the forebrain and eyes (Oliver et al, 1995). In progressively more mature
EBs, we examined the down-regulation of mesoderm and up-regulation of neuroectoderm
differentiation, and show that BS69 promotes neuroectoderm specification as development
progresses. Additionally, we tested the possible requirement for BS69 in the Bmp pathway,
and we show that Bmp signaling is unobstructed in BS69 KO cells.

81

4.3 Results
4.3.1 BS69 KO EBs Continue to Lack Mesoderm and Over-specify Neuroectoderm as
They Mature

In Chapter 2, we showed that BS69 is expressed throughout embryonic tissue through
E9.25. Thus far, we have only examined BS69 KO EBs up to a stage equivalent to E6.5.
To determine the long-term consequences of BS69 function and how they correlate to
BS69’s role at day 3, we performed RNA-sequencing on day 5 control and KO EBs. We
used the same experimental conditions and analysis as for our mESC and day 3 EB
experiments. Importantly, we saw that BS69 expression was decreased in day 3 and day 5
EBs. In comparison, day 5 EBs have a higher number of DEGs, with 364 in total. Also,
more DEGs are up-regulated than down-regulated (Figure 4.1). Of the down-regulated
genes, the top ten gene ontology categories based on DAVID analysis are all Wnt or
mesendoderm-related (Figure 4.1A). The top term, Wnt receptor signaling pathway-
calcium modulating pathway, has a much higher fold-enrichment than the other terms. The
7 genes in this category are all Wnt ligands, which is consistent with the down-regulation
of Wnt pathway genes seen in the day 3 RNA-sequencing results. The mesendoderm
related gene ontology terms include development of glands, skeleton, and heart. For
example, the gene Mesp1 is the earliest marker of cardiac precursor cells and is required
for normal heart morphogenesis (Saga, 1998)(Saga, 1998)(Saga, 1998)(Saga, 1998). In
addition, Isl1 is also required for cardiogenesis, specifically to form the second heart field
(SHF) (Cai et al, 2003). Both of these genes are down-regulated in BS69 KO day 5 EBs
(Figure 4.1A). This suggests that the BS69 KO EBs might not correctly specify
mesendoderm derived precursors of early heart development as they mature.

82

Although the up-regulated DEGs in BS69 KO day 5 EBs are more numerous than the
down-regulated genes at 188 out of 364, they cannot be classified into gene ontology
categories that reveal an obvious and consistent pattern. Rather, they include terms related
to the senses, circulation, intracellular organization, and behavior. The terms are also
relatively less enriched than the categories related to the down-regulated genes (Figure
4.1B). Notably, Sox2 and Six3 are up-regulated in the KO EBs at this stage. Sox2 marks
pluripotent cells in the blastocyst and epiblast, but it is also expressed throughout early
neuroectoderm. Six3 is also an early neuroectoderm marker that is expressed in the anterior
of the embryo starting at E6.5 (Oliver et al, 1995). This suggests that BS69 KO EBs could
preferentially specify neuroectoderm lineage cells that are capable of differentiating into
mature cell types, in which case we would expect to find neurons in older EBs. Another
possibility is that the immature neuroectoderm cells that we detect in BS69 KO day 5 EBs
could continue to self-renew as immature neural progenitors without differentiating. To
determine which scenario occurs in the BS69 KO, we analyzed older EBs to discover the
long-term consequences of BS69 function.

83

Figure 4.1 Day 5 DEGs confirm trends from mESC and day 3 RNA-sequencing. A. Venn
diagram showing the total number of DEGs determined by RNA sequencing analysis of
BS69 KO and control mESCs and day 3 EBs. B. Tabulated DEGs per sample with numbers
down-regulated versus up-regulated. C, D. GO_BP_FAT terms from DAVID Functional
Annotation of Day 5 EB differentially expressed genes. C. Top 10 of 54 total down-
regulated gene categories (Count: 5, EASE: 0.01). D. Top 10 of 24 total up-regulated gene
categories (Count: 5, EASE: 0.1). E. Up-regulation of two early neural genes. (Ashburner
et al, 2000; Dennis et al, 2003).

84

4.3.2 Absence of BS69 Promotes Neural Precursor Specification and Differentiation
To determine whether neural differentiation was indeed occurring preferentially in the
BS69 KO EBs, we analyzed day 8 and day 12 EBs for the presence of mature neurons. We
demonstrated highly increased expression of the mature neuronal marker Tuj1 in the BS69
KO EBs (Figure 4.2A, B). Tuj1 is present in more KO than control EBs at day 8 and day
12, and appears to also be present in a higher proportion of cells per EB in the KO. (Figure
4.2A, B). Consistently, Neurofilament (NF) is also expanded at day 12 in the KO (Figure
4.2C). Together, these results suggest that without BS69 expression, mouse embryonic
stem cells preferentially specify neuroectoderm lineage cells which are able to differentiate
into mature neurons.

85

Figure 4.2 Precocious BS69 KO EBs differentiation into mature neurons in vitro.
Immunofluorescent staining of day 8 and day 12 control and BS69 KO embryoid bodies.
A. Tuj1 immunofluorescence on day 8 EB cryosections. B. Tuj1 immunofluorescence on
day 12 EB cryosections. C. Neurofilament immunofluorescence on day 12 EB
cryosections. All sections are 10um. All images are at 200x magnification.

86

The higher numbers of mature neurons in BS69 KO EBs does not preclude the possibility
that these EBs also retain populations of immature neuroectoderm lineage cells. Moreover,
in the EB formation assay, groups have reported finding immature, and even pluripotent
cells, as many as seven days after removal of pluripotency supplements, which suggests
that even our control cell lines may contain some early neuroectoderm cells (Toumadje et
al, 2003). To determine whether there are more early neuroectoderm progenitors in the
BS69 KO day 12 EBs relative to controls, we performed WMISH with probes specific for
early neuroectoderm markers. Our results show that all three genes tested—Sox1, Hesx1,
and Six3—were expressed more highly in BS69 KO EBs than in controls (Figure 4.3).
Sox1, an early pan-neural marker, is seen in the wild-type embryo starting at E7.5 in the
neural plate, which forms at the proximal-anterior side of the embryo (Pevny et al, 1998).
In EBs, we see that Sox1 is highly expressed in the BS69 KO EBs at day 12, but in contrast
the control EBs have little to no Sox1 expression (Figure 4.3A). Furthermore, expression
of Sox1 in the KO appears to be symmetrical, suggesting a lack of axis specification. In
contrast, Sox1 expression in the control EBs is asymmetric and restricted, possibly
indicative of in vitro anterior-posterior axis formation. Hesx1 is a transcription regulator
that is required for anterior neural development (Martinez-Barbera et al, 2000). It is first
expressed at E6.5 at the onset of gastrulation, with expression continuing in the rostral
ectoderm through E10.5. In the BS69 KO EB, Hesx1 expression is increased in comparison
to the control and lacks regional restriction (Figure 4.3B). Six3, as described previously, is
also an anterior neural marker and is up-regulated symmetrically in BS69 KO (Figure 4.3C,
(Oliver et al, 1995). These experiments suggest that the population of early neuroectoderm
87

cells persists for as long as 12 days in the BS69 KO while a fraction differentiates into
mature neurons.

88

Figure 4.3 BS69 KO embryoid bodies retain higher expression of early neuroectoderm
markers than controls. Control and BS69 KO mESCs were grown in hanging drops without
LIF or 2i for 12 days and then subjected to WMISH using antisense RNA probes. A. Sox1
probe. B. Hesx1 probe. C. Six3 probe. All images are to the same scale.

89

Because BS69 KO EBs contain both increased expression of mature neural markers and
early neuroectoderm markers, we asked whether they also harbor more immature neurons.
To answer this question, we first performed immunofluorescent staining on day 5 EB
cryosections using an antibody against Nestin, a marker of central nervous system neural
precursors. We did not see an increase in Nestin positive cells in the BS69 KO day 5 EBs
(Figure 4.4). Both the control and KO EBs had limited staining that was predominantly
found at EB perimeters. This suggests that BS69 KO EBs do not prematurely differentiate
into neural progenitors (Figure 4.4A). We next examined day 8 EBs for Nestin and Sox2,
which is also expressed in neural precursors. At this stage, BS69 KO EBs did contain more
cells that were Sox2 positive, but Nestin staining appeared relatively equal to controls
(Figure 4.4B).

90

Figure 4.4 BS69 KO EBs do not show precocious specification of immature neurons. A.
Nestin immunofluorescence of day 5 control and KO EBs. B. Sox2 and Nestin
immunofluorescence of day 8 EBs. Control and BS69 KO mESCs were grown in hanging
drops without LIF or 2i for eight days followed by cryo-sectioning and staining with
antibodies as labeled. Arrowheads indicate regions positive for Nestin. DAPI is used to
mark nuclei. All sections are 10µm. All images are at 200x magnification.

91

4.3.3 BS69 is Required for Efficient Mesoderm Differentiation and Cardiomyocyte
Contraction

Our in vivo and in vitro results consistently suggest that we should expect BS69 KO EBs
to have a limited ability to differentiate into mesendoderm-lineage cells. In our RNA-seq
data, mesendoderm genes were consistently down-regulated in BS69 KO EBs at both day
3 and day 5, so we should expect to find less mature mesendoderm lineage cell types in
older EBs. To check this prediction, we looked at mesoderm derived tissues, specifically
skeletal muscle, smooth muscle, and cardiac muscle. We used an antibody against mMF-
20, which specifically marks skeletal muscle to stain day 8 EB sections. Surprisingly, we
found that some BS69 KO EBs do contain areas with significant MF-20 protein, though
less than controls (Figure 4.5A). In addition, our cardiac muscle marker, cardiac troponin-
t (cTnT), was also greatly reduced in KO EBs (Figure 4.5B). Similarly, using an antibody
against smooth muscle actin (SMA) we observed reduced staining for smooth muscle in
BS69 KO EBs (Figure 4.5C). Moreover, when we differentiated control and KO cells under
culture conditions that promote cardiomyogenesis, BS69 KO EBs were unable to form the
beating patches of cells that indicate cardiomyocytes, while controls formed numerous
beating patches (Figure 4.5D). Day 8 EBs are also known to form cardiomyocytes, but
none of the BS69 KO EBs that we examined showed any signs of movement, whereas most
of our control EBs did (Data not shown). Therefore, it appears that BS69 KO EBs can
differentiate into some cTnT positive cells, but they are unable to contract. Though these
results consistent with our previous data, they are surprising since we did not find evidence
of mesendoderm marker expression in BS69 KO day 3 EBs. Thus, the reduced expression
of these mesendoderm markers may suggest that lineage specification and differentiation
are delayed rather than fully blocked in absence of BS69.
92

Figure 4.5 Decreased efficiency of mesoderm differentiation in BS69 KO EBs. A-C.
Immunofluorescent staining of day 8 and day 12 control and BS69 KO embryoid bodies.
All sections are 10µm. A. MF20 immunofluorescence of day 8 EB cryosections. B. cTnT
immunofluorescence of day 12 EB cryosections. C. SMA immunofluorescence of day 12
EB cryosections. D. Phase-contrast images of control and BS69 KO cell lines grown for 8
days in cardiomyocyte-promoting conditions. All images are at 200x magnification.

93

To test this further, we analyzed day 12 EBs using WMISH with probes for Bmp4 and
Nodal, two genes that are expressed in mesendoderm and which were decreased in BS69
KO E7.5 embryos. We saw that Bmp4 and Nodal were expressed in BS69 KO day 12 EBs
at a comparable level to controls, and although this is in contrast to our in vivo data, it
agrees with our later discovery of mesendoderm-lineage tissues in day 12 EBs and
emphasizes that some mesendoderm tissues may not be completely blocked, but only
delayed (Figure 4.6).

94

Figure 4.6 BS69 KO embryoid bodies recover Bmp and Tgfβ pathway ligand expression.
Control and BS69 KO mESCs were grown in hanging drops without LIF or 2i for twelve
days and then subjected to whole mount in situ hybridization using antisense RNA probes.
A. Bmp4 probe. B. Nodal probe.

95

Since Bmp4 and Nodal expression was observed in BS69 KO day 12 EBs, we were curious
at what timepoint the expression of mesendoderm genes began. Therefore, we used
WMISH to determine whether BS69 KO EBs began to differentiate toward mesendoderm
by day 5. For Bmp4, although we can see some expression at day 5 in BS69 KO EBs, the
level is reduced in comparison to controls (Figure 4.7A). Interestingly, Brachyury, another
marker of mesendoderm formation whose expression we showed to be absent at day 3 and
in E7.5 embryos, was expressed at day 5 in BS69 KO EBs at slightly lower levels than
controls (Figure 4.7B). Although prolonged culture of EBs showed that some markers of
mesendoderm differentiation appear to be recovered in BS69 KO EBs at day 5, we also
wanted to determine the expression status of the anterior neural gene Hesx1, which was
expanded in the BS69 KO embryo (Figure 2.6F). Surprisingly, Hesx1 WMISH showed
that expression levels appear similar in the control and BS69 KO (Figure 4.7C).

96

Figure 4.7 BS69 KO EBs show delayed expression of primitive streak markers. Control
and BS69 KO mESCs were grown in hanging drops without LIF or 2i for five days and
then subjected to whole mount in situ hybridization using antisense RNA probes. A. Bmp4
probe. B. Brachyury probe. C. Hesx1 probe.

97

Although reduced, the presence of Brachyury expression in BS69 KO day 5 EBs suggests
that canonical Wnt signaling occurs in these EBs, so we stained for β-catenin in day 5 EBs
(Figure 4.8). The control EBs show a higher level of β-catenin staining on the interior of
the EBs, and overlap with DAPI suggests that some of the staining may be nuclear. There
appears to be less β-catenin staining on the interior of BS69 KO EBs, but it is still present.
Some overlap with DAPI may also be present in the KO, but to a lesser degree than the
control. Due to weak staining, this data is inconclusive and needs to be repeated using a
more sensitive method of β-catenin detection.

98

Figure 4.8 β-catenin is expressed in BS69 KO day 5 EBs. Immunofluorescent staining
using an antibody against β-catenin. Boxes indicate magnified regions. Arrowheads
indicate possible overlap of β-catenin and DAPI. All sections are 10µm. All images are at
200x magnification.

99

4.3.4 BS69 is not Required for Endothelial Cell Formation, the Rate of Apoptosis, or
Bmp Signaling

So far, in our study we have examined the consequences of BS69 function in older EBs for
differences in neural and muscle differentiation, but we have not yet looked at other cell
types. To address this, we analyzed EBs for the presence of endothelial cells, which are
present in many tissues. platelet/endothelial cell adhesion molecule 1 (Pecam1) is widely
expressed in vascular endothelial cells as well as in extraembryonic mesoderm (Redick &
Bautch, 1999). Immunofluorescent antibody staining shows that BS69 KO EBs are positive
for Pecam1 and contain comparable amounts to control EBs (Figure 4.9A). Another
endothelial marker, cadherin 2 (N-cadherin) is expressed in a variety of cell types including
in the brain, heart, pancreas, and vasculature (Esni et al, 2001; Redies & Takeichi, 1993;
Volk & Geiger, 1984). We used an antibody against N-cadherin to examine its protein
levels in day 8 EBs by immunofluorescence. Our results were similar to those for Pecam1:
N-cadherin is expressed in BS69 KO EBs at similar levels to control EBs (Figure 4.9B).
Therefore, BS69 KO cell lines may be capable of differentiating into vascular endothelium.
These results suggest that in vitro, BS69 is not required for endothelial cell differentiation.

100

Figure 4.9 BS69 KO EBs differentiate into endothelium at a similar rate as controls. A.
Immunofluorescent staining of day 8 EBs using an antibody against Pecam1. B. N-cadherin
immunofluorescent staining of control and KO day 8 EB cryosections. DAPI is used to
mark all nuclei. All sections are 10µm. All images are at 200x magnification.

101

To complete our picture of the long term consequences of BS69 function in vitro, we have
yet to address the question of whether BS69 KO EB cells proliferate and apoptose similarly
to controls. To answer this, we performed immunofluorescent staining with an antibody
against Ki67 and we performed TUNEL staining (Figure 4.10). The majority of cells in
both the control and KO lines showed widespread positive Ki67 staining (Figure 4.10A).
This supports our in vivo Ki67 staining in which we saw the same result (Figure 2.5A).
Surprisingly, in vitro we observed no difference in the number of TUNEL positive cells
between the control and KO EBs, which is in contrast to our in vivo data (Figure 2.5C, D;
Figure 4.10B).

As a whole, our data on the long-term consequences of BS69 function shows that it is
required for efficient mesendoderm formation, to inhibit the expansion of early
neuroectoderm, and to promote neural differentiation. In prolonged culture of BS69 KO
EBs, mesendoderm is not completely blocked, rather it can form but appears to be delayed.
In contrast, and neuroectoderm can differentiate, but a large proportion of undifferentiated
cells remain.

102

Figure 4.10 BS69 KO EBs proliferation and apoptosis rates are similar to controls. A.
Ki67 immunofluorescent staining of day 8 EB cryosections. B. TUNEL assay of day 8 EB
cryosections. Control and BS69 KO mESCs were grown in hanging drops without LIF or
2i for eight days followed by cryo-sectioning and staining with antibodies as labeled. All
sections are 10µm. All images are at 200x magnification.

103

In Chapter 2 we discussed the similarity of the BS69 KO phenotype to the Bmpr1a KO
phenotype. These parallels between the two mutations suggest that BS69 could be involved
in the canonical Bmp pathway and none of our data thus far has directly addressed this
point. Therefore, to determine this possibility is true, we investigated whether the Bmp
effector proteins SMAD family members 1, 5, and 8 (Smad1/5/8) are phosphorylated in
day 8 BS69 KO embryoid bodies (Figure 4.11A).

Using immunofluorescent staining, we found that Smad1/5/8 are phosphorylated in the
BS69 KO at a similar level to the controls (Figure 4.11A). To confirm Bmp pathway target
activity, we performed RTPCR using primers for Id2, which is a target of canonical Bmp
signaling with phosphorylated-Smad (pSmad) binding sites that are well-characterized
(find paper). Our results show that Id2 expression is present in the BS69 KO in mESCs,
day 3 EBs, and day 5 EBs at similar levels to the controls, which suggests that BS69 is not
required for canonical Bmp pathway activity (Figure 4.11B). Furthermore, it has been
shown that Bmpr1a knockout mESCs cannot be propagated. They do not proliferate, which
is in contrast to BS69 KO mESCs (Figure 3.2, (Di-Gregorio et al, 2007). This refutes the
hypothesis that BS69 is required for canonical Bmp signaling.
104

Figure 4.11 BS69 KO embryoid bodies contain phosphorylated Smad1/5/8 and express
Bmp target genes. A. Immunofluorescent staining of day 8 control and BS69 KO embryoid
bodies. Control and BS69 KO mESCs were grown in hanging drops without LIF or 2i for
eight days followed by cryo-sectioning and staining with antibodies as labeled. All sections
are 10µm. All images are at 200x magnification. B. RNA was isolated from mESCs, day
3, and day 5 control and BS69 KO EBs (grown as previously stated) and subjected to
RTPCR using primers against Id2. Gapdh serves as a loading control. NC = negative
control.

105

4.4 Discussion

4.4.1 Summary
Because BS69 expression continues as the embryo develops, we analyzed older BS69 KO
EBs. We performed RNA-sequencing on day 5 BS69 KO and control EBs and found that
Wnt signaling remains down-regulated in the KO, in addition to genes involved in gland,
skeleton, and heart development (Figure 4.1A). The up-regulated DEGs outnumbered the
down-regulated ones in day 5 EBs, but gene ontology analysis did not reveal an obvious
pattern (Figure 4.1B). However, two genes that are expressed in early anterior
neuroectoderm, Sox2 and Six3, were up-regulated (Figure4.1E, (Oliver et al, 1995; Wood
& Episkopou, 1999). This is consistent with our results from our embryonic studies where
we saw anterior neuroectoderm expanded throughout the epiblast (Figure 2.6F).

We followed the fate of the BS69 KO EBs and determined that the early up-regulation of
neuroectoderm led to an increase in neural tissue at later time points (Figure 4.2). We
examined day 12 EBs for the presence of early neuroectoderm markers and found that they
are strongly up-regulated, suggesting that much of the neuroectoderm remains immature
(Figure 4.3). Additionally, neural precursors did not develop prematurely in the BS69 KOs
(Figure 4.4). This suggests that BS69 potentially regulates neuron differentiation at
multiple stages, resulting in expanded neuroectoderm, reduced neural precursors, and
increased mature neurons.

Surprisingly, we found that development of skeletal muscle, heart muscle, and smooth
muscle does take place in the BS69 KO, but it is delayed and reduced (Figure 4.5). We
106

found that development of mesendoderm began as early as day 5 in BS69 KO EBs (Figure
4.7). Endothelial cells, conversely, are unaffected by the loss of BS69 (Figure 4.9).
Similarly, neither proliferation nor apoptosis are altered in the BS69 KO (Figure 4.10).
Although proliferation is likewise unaffected in vivo, BS69 KO E7.5 embryos had more
apoptotic cells than controls (Figure 2.5B, C).

Finally, we found that Bmp signaling is not affected in the BS69 KO as we had previously
hypothesized, as shown by the presence of phosphorylated SMAD1/5/8 and Id2 expression
in mESCs, day 3 EBs, and day 5 EBs (Figure 4.11). This is consistent with Bmp’s known
role in maintaining self-renewal and proliferation in mESCs (Ying et al, 2003). However,
it was surprising to find both active Bmp signaling and expanded neuroectoderm in the
BS69 KO because the Bmp pathway is known to block premature neuroectoderm
specification (Di-Gregorio et al, 2007).

107

4.4.2 Signaling Pathway Analysis
The RNA-sequencing results of day 5 EBs demonstrate that mesendoderm-derivative
genes are down-regulated in the BS69 KO (Figure 4.1A). Mesp1 and Isl1, as previously
mentioned, are required for heart development. Isl1 specifies the second heart field (SHF)
in a Wnt-dependent manner, and Mesp1 expression requires Wnt and Oct4 interaction
(Klaus et al, 2007; Li et al, 2013). Furthermore, a Wnt target gene and trophoblast
differentiation marker, Cdx2, was the most highly down-regulated gene in day 5 BS69 KO
EBs (He et al, 2008). Beyond its requirement in trophoblasts, Cdx2 is also necessary for
axial elongation in the post-gastrulation mouse embryo (Chawengsaksophak et al, 2004).
The posterior truncation phenotype of the Cdx2 mutant mouse embryo is similar to that of
the Wnt3a mutant, which is a target of Axin2 (Qian et al, 2011; Takada et al, 1994).
Incidentally, Axin2 is also a target of the canonical Wnt pathway and down-regulated in
the BS69 KO (Qian et al, 2011). This data suggests that the down-regulation of
mesendoderm-related genes in day 5 BS69 KO EBs may be secondary to Wnt pathway
abrogation. The most enriched gene ontology category in day 5 BS69 KO EBs was Wnt
signaling. Upon closer inspection, we saw that all of the DEGs in this category were Wnt
ligands, supporting our hypothesis that BS69 function is upstream of Wnt signaling.

Loss of Wnt signaling may also cause the down-regulation of EMT-related genes that we
observe in day 5 BS69 KO EBs. Snail expression, which is required to down-regulate E-
cadherin in EMT, is decreased in the absence of Wnt signaling (Hierholzer & Kemler,
2010). Fgf signaling is also a known requirement for Snail expression (Ciruna & Rossant,
2001). Evidence has suggested that Wnt3a—which is expressed throughout the developing
108

mesendoderm after gastrulation—and Fgf8 may regulate each other’s expression through
a feed-forward loop. Down-regulation of either the canonical Wnt pathway or Fgf signaling
results in decreased levels of both Fgf8 and Wnt3a, which supports this idea (Hierholzer &
Kemler, 2010). Because multiple Wnt ligands, Wnt targets, and Snail are down-regulated
in the BS69 KO, BS69 may indirectly affect Fgf signaling through the Wnt pathway.
Furthermore, of the 34 genes that are differentially expressed in both day 3 and day 5 EBs,
3 are Wnt ligands, specifically Wnt2b, Wnt3, and Wnt5b, suggesting that BS69’s putative
effect on the Wnt pathway is consistent at both time points.

Our previous data suggested that the Bmp and Nodal pathways were likely candidates for
direct interaction with BS69. However, based on our day 3 EB data and ability to propagate
BS69 KO mESCs, we have suggested that the Bmp pathway is likely to function normally
in BS69 KO EBs. Our day 5 EB data supports the hypothesis that Bmp signaling is not
directly affected by BS69. There are no down-regulated Bmp ligands in day 5 BS69 KO
EBs (Figure 4.1A). Additionally, the well-established Bmp target gene ID2 is not down-
regulated, as demonstrated by its absence from the differentially expressed gene lists and
by similar expression to controls in mESCs, day 3 EBs, and day 5 EBs as shown by RTPCR
(Figure 4.1A, 4.11B). Furthermore, phosphorylation of SMAD 1/5/8, which is required for
Bmp signal transduction, is present in day 8 BS69 KO EBs (Figure 4.11A). We have thus
shown that BS69 is not required for canonical Bmp signaling.

We have not proven that our last candidate pathway, Nodal, does not play a role in BS69
function, but the data that we gathered from day 5 EBs suggests that it is unlikely. By day
109

5, Nodal is no longer down-regulated in BS69 KO EBs (Figure 4.1A). Nodal stimulates its
own transcription, so normal expression levels of the ligand suggests that the Nodal
signaling pathway is intact (Brennan et al, 2001). Additionally, the known Nodal target
genes Lefty1 and Cerl are not down-regulated. This suggests that BS69 is not required for
Nodal signaling.

110

Chapter 5: Concluding Remarks
5.1 Conclusions

Until now, the role of the gene BS69 during development has not been examined. For the
first time, we show that BS69 is required for embryonic development beyond E7.5.
Molecular analysis reveals that BS69 KO embryos do not express gastrulation or early
mesendoderm markers at E7.5, nor do they specify the prospective location of the primitive
streak at E6.5. Additionally, they have increased levels of Hesx1 and decreased levels of
Oct4, which suggests that they prematurely differentiate into neuroectoderm.

To further study the functions of BS69 in pluripotency and differentiation, we derived
mESCs from BS69 KO blastocysts. We were able to successfully derive these cell lines
and establish a useful model system in vitro. This allowed us to demonstrate that BS69 is
not required to maintain mESC self-renewal and pluripotency. However, BS69 is required
for mesendoderm differentiation in day 3 EBs, as shown by the down-regulation of related
genes in BS69 KOs. This in vitro data supports our findings in BS69 KO E7.5 embryos.

Allowing the EBs to differentiate further revealed that BS69 KO EBs up-regulate the
expression of early neuroectoderm markers at day 5. Later, they are able to form more
neurons than controls, but they predominantly maintain prolonged expansion of early
neuroectoderm. Physiologically, this suggests that during gastrulation BS69 might inhibit
or restrict differentiation into mature neurons, but is dispensable for early neuroectoderm
specification. We also discovered that mesendoderm differentiation is not completely
blocked in BS69 KO EBs, but its formation is delayed and less efficient in the long term.
111

Specifically, BS69 KO EBs down-regulated cardiomyogenesis-related genes at day 5, and
were unable to form contractile cardiomyocytes at day 8. Additionally, although the BS69
KO embryo phenocopies the Bmpr1a mutant mouse, using BS69 KO mESCs and EBs we
have shown that Bmp signaling is surprisingly not abrogated.

Our data consistently support a role for BS69 in mesendoderm specification and
gastrulation. Of the 34 genes that are differentially expressed in both BS69 KO day 3 and
day 5 EBs, 7 are known to be expressed during gastrulation. They are all down-regulated
at both day 3 and day 5 in BS69 KO EBs. Of these genes, four are known Wnt pathway
genes: Wnt2b, Wnt3, Wnt5b, and Axin2. As previously discussed, Wnt3 and Axin2 are
involved in canonical Wnt pathway regulation of gastrulation and mesoderm formation
(Liu et al, 1999; Qian et al, 2011). Less is known about Wnt5b and Wnt2b, but both are
expressed at E7.5 and Wnt5b is important in regulating gastrulation movement in zebrafish
(Gavin et al, 1990; Kemp et al, 2005; Lin et al, 2010). Also on both day 3 and day 5 EB
DEG lists are Evx1, Mixl1, and Meis2. Although required starting at an earlier stage of
development, Evx1 is expressed in the primitive streak during gastrulation (Dush & Martin,
1992). A putative target of Wnt signaling, Mixl1 is also expressed in the primitive streak
during gastrulation and is required for axial mesoderm development (Choi et al, 2015; Hart
et al, 2002). Finally, Meis2 is expressed in the anterior primitive streak and is involved in
somite formation (Cecconi et al, 1997). The consistency with which mesendoderm-related
genes are down-regulated in the BS69 KO supports our hypothesis that BS69 is required
for gastrulation and germ layer specification.
112

Taken together, our data shows that BS69 is required for the formation of mesendoderm
and to prevent precocious differentiation toward neuroectoderm during mouse
embryogenesis and embryonic stem cell differentiation.

113

5.2 Proposed Model of BS69 Function in Germ Layer Specification

We have revealed an important role for the gene BS69 in the development of the embryo
and confirmed this finding in vitro. Though there are relatively few studies on BS69,
recently it has been implicated in chromatin modifications in cancer. Many parallels have
been found between cancer and embryonic development; genes that are oncogenic in adults
can have important functions in pluri/multipotency, differentiation, and morphogenesis.
Additionally, these same genes may be key to regenerative medicine. Just as the potent
oncogene and developmental regulator C-myc was used to reprogram somatic cells to an
embryonic state, other factors about which we know little could play essential roles in
driving pluripotent stem cells toward specific fates for clinical use. Therefore, a complete
understanding of embryonic development, including genes like BS69, is important for
progress in both regenerative medicine and cancer therapy.

Figure 5.1 Model of BS69 function in early embryogenesis.

114

5.3 New Hypotheses for BS69 Function in Embryonic Development
Instead of interacting with transcription factors, perhaps BS69 functions by interacting with
chromatin, as was suggested in 2006 and strongly confirmed in 2014 (Guo et al, 2014; Lan
& Shi, 2014; Velasco et al, 2006; Wen et al, 2014). Moreover, BS69 contains three known
chromatin-interacting domains, and it is known that chromatin modification plays a role in
germ layer formation. In 2006, a study found by immunoprecipitation that BS69 binds the
chromatin modifying proteins BRG1, HDAC1, and EZH2 through its C-terminus in quail
fibroblast cells (Velasco et al, 2006). Therefore, we will discuss the likelihood of each
protein’s interaction with BS69 in mouse embryogenesis by examining its known function
in this context. BRG1 is an ATP-dependent helicase that is part of the SWI/SNF chromatin
remodeling complex (Wang et al, 1996). In the mouse, it is required for embryonic survival
beyond the peri-implantation stage (Bultman et al, 2000). This suggests that BRG1 binding
may not be required for BS69 function in the embryo, since BS69 KO embryos survive
longer than BRG1 mutants. HDAC1 mutants, on the other hand, survive longer than BS69
KO embryos and die by E9.5 (Montgomery et al, 2007). Furthermore, when HDAC1 is
knocked out in mESCs, cardiomyocyte differentiation increases, whereas the BS69 KO
does not form contractile cardiomyocytes (Dovey et al, 2010). This suggests that HDAC1
and BS69 may have opposing functions, but there is no additional data to reinforce this
supposition. Finally, EZH2 is the enzymatic component of polycomb repressive complex
2 (PRC2) and is required for embryonic development beyond gastrulation (O'Carroll et al,
2001). Besides EZH2, PRC2 contains two other proteins: embryonic ectoderm
development (EED), and suppressor of zeste 12 (SUZ12). Loss of any of these three
components renders the complex nonfunctional, and results in the same phenotype as loss
115

of EZH2 (Morin-Kensicki et al, 2001; Pasini et al, 2004). In cell lines that lack PRC2
components, there is an expansion of posterior embryonic and extraembryonic mesoderm
and a decrease in neuroectoderm upon differentiation (Shen et al, 2008). Because PRC2’s
function is to trimethylate histone 3 lysine 27, a repressive chromatin mark, this suggests
that one of the functions of trimethylated histone 3 lysine 27 (H3K27me3) is to restrict the
transcription of posterior genes. This is supported by ChIP data that shows H3K27me3
deposition at the promoters of important developmental regulators (Boyer et al, 2006). The
phenotype of expanded mesendoderm in PRC2 mutants is opposite of what we see in BS69
KO embryos or cells. This suggests that if an interaction between EZH2 and BS69 occurs
in early development, it is likely to be inhibitory.

Recent findings implicate BS69 in another epigenetic role, though not related to polycomb
complexes. A study in 2014 reported that BS69 binds to trimethylated histone 3 lysine 36
(H3K36me3) on the specific histone 3 variant H3.3 (Wen et al, 2014). Furthermore, the
authors found through ChIP-sequencing analysis that BS69 and trimethylated histone
variant H3.3 lysine 36 (H3.3K36me3) frequently colocalize in gene bodies. The crystal
structure of this interaction shows that BS69’s Bromodomain, a newly discovered zinc-
finger domain, and PWWP domain form a pocket in which the trimethyl group on H3K36
sits. Functionally, the authors suggest that BS69 regulates RNA polymerase II. Although
associated with highly expressed genes, they found that BS69 likely represses the
elongation step of RNA polymerase II transcription, thereby exerting control over the level
of transcript production (Wen et al, 2014). If this hypothesis applies to BS69 function in
embryonic development and mESC differentiation, BS69 might repress the expression of
116

early neuroectoderm genes. If increased neuroectoderm gene expression can override
posterior specification, then the lack of BS69 might result in epiblast differentiation toward
neuroectoderm. However, our data shows that BS69 KO cells have more down-regulated
DEGs than controls until day 5 of EB formation, which conflicts with the idea that BS69
functions mainly as a transcriptional repressor of RNA polymerase II-bound genes.
Furthermore, the authors of this 2014 study performed RNA-sequencing on BS69 knock-
down osteosarcoma (U2OS) cells and found that while the up-regulated DEGs were
associated with specific gene ontology terms, the down-regulated DEGs were not. This is
the opposite from what we have consistently seen from our RNA-sequencing data.
Therefore, although published data strongly supports an interaction between H3.3K36me3
and BS69, our data on BS69 function during embryogenesis does not.

Another study in 2014 also found that BS69 binds to H3.3K36me3, but suggests that it
could have a different function, namely, the regulation of intron retention, a poorly
understood type of pre-mRNA splicing (Guo et al, 2014). The authors found that BS69
associates with the EFTUD2 subunit of the U5 small nuclear ribonucleic protein (snRNP)
spliceosome and regulates intron retention in an H3.3K36me3 binding-dependent manner
(Guo et al, 2014). They found that among the splicing events that were affected by BS69
knockdown, intron retention was altered the most. Furthermore, the same phenotype was
observed in multiple cell types including HeLa, lung cancer (A549), and lung fibroblasts
(HFL1), leading the authors to suggest that this is a non-cell type dependent mechanism.
They also observed that instances of intron retention decreased in BS69 knockdown cells
and increased when EFTUD2 was lost. If this is true, then BS69’s function may be to
117

increase intron retention by inhibiting EFTUD2 and thereby preventing splicing.
Interestingly, the authors found that knockdown of SETD2, the enzyme that trimethylates
H3K36me3, yields the same changes in intron retention as BS69 knockdown. This supports
the model that BS69 promotes intron retention by inhibiting H3.3K36me3-dependent
spliceosome function. To determine whether BS69 functions the same way in early
embryonic development and mESCs, we would need to discover if the lack of BS69 leads
to increased intron retention. Fortuitously, we obtained transcript-level data from our RNA-
sequencing, which included information on whether transcripts were from retained introns.
There were no differentially expressed transcripts (DETs) that were categorized as retained
introns in mESCs. Day 3 EB DETs, however, were approximately 9% retained intron
transcripts and day 5 contained 8% (data not shown). The retained intron transcripts were
split evenly between up and down-regulation in day 3 EBs, but day 5 EBs had more than
twice as many that were up-regulated. Therefore, our data neither supports nor refutes the
hypothesis that BS69 regulates intron retention, but suggests that deeper biochemical and
bioinformatic analyses are necessary.

Our examination of BS69 function in EB differentiation has yielded important results and
interesting questions. We have shown that BS69 is required for normal mesendoderm
lineage development but is not necessary for neuroectoderm specification in vitro, which
correlates with our in vivo data. However, we have also shown that BS69 is unlikely to
function as a transcriptional co-repressor in this context. Instead, new studies will be
needed to determine whether BS69 interacts with chromatin in early embryonic
development and differentiation.
118

5.4 A Putative Mechanism of BS69 Function in Early Embryogenesis

Newly published evidence suggests that BS69 may interact with chromatin through the
proteins BRG1, HDAC1, or EZH2 (Velasco et al, 2006). Therefore, here we examine how
this data may help explain the role of BS69 during early embryogenesis. Based on our data
and the phenotype of BRG1, HDAC1, and EZH2 mutant mice, we believe that the PRC2
component, EZH2, is the most likely BS69 binding partner in early development, but
additional biochemical experiments would be required to confirm this. Recently published
data has also suggested that BS69 interacts with chromatin, but by reading the chromatin
modification H3.3K36me3 (Guo et al, 2014; Wen et al, 2014). Intriguingly, a connection
between H3.3 and PRC2 has been discovered, which suggests that both hypotheses
regarding BS69 chromatin interaction may be correct.

Histone variant H3.3 is inserted into gene bodies by the chaperone protein Hira. This
deposition occurs throughout the cell cycle, as opposed to the S-phase limited deposition
of canonical H3 isoforms, H3.1 and H3.2. In embryogenesis, Hira is required for
development past E9.5 (Roberts et al, 2002). The Hira mutant embryo also exhibits reduced
neuroepithelium and increased mesendoderm, presumably due to lack of H3.3 deposition
in gene bodies (Roberts et al, 2002). According to a 2013 paper, H3.3 is needed to silence
embryonic and trophectoderm development genes (Banaszynski et al, 2013). In this study,
the authors performed an EB formation assay in which they saw up-regulation of
trophectoderm, primitive endoderm, and mesoderm, but no change in ectoderm or
pluripotency markers. Moreover, the genes that become up-regulated in the H3.3 KO were
missing the normal H3K27me3 marks. Therefore, the authors suggest that H3.3 is required
119

for PRC2 binding of these developmental genes (Banaszynski et al, 2013). This illuminates
a surprising possible connection between the ability of BS69 to bind PRC2 and the recently
discovered interaction between H3.3K36me3 and BS69. Additionally, both PRC2 and
H3.3 mutants form expanded mesendoderm, while BS69 is required for mesendoderm
development. This suggests that BS69 may function to inhibit H3.3-dependent PRC2
binding. If BS69 competes with or otherwise inhibits PRC2 to prevent it from binding H3.3
in mesendoderm-regulating genes, the lack of BS69 could potentially result in the
phenotype we see in BS69 KO embryos and EBs (Figure 5.2).

120

Figure 5.2 Putative mechanism of BS69 function. A. BS69 binding to H3.3K36me3
prevents EZH2-mediated trimethylation of H3.3K27me3, which allows transcription of
mesendoderm genes and blocks neuroectoderm. B. Lack of BS69 allows EZH2 to
trimethylate H3.3K27me3, which shuts down mesendoderm gene expression and results in
the specification of neuroectoderm.
121

5.5 Future Directions

5.5.1 Rescue of BS69 Function in Germ Layer Specification
Studies have attributed multiple functions to BS69 based on experiments using different
BS69 isoforms and truncated constructs. For some functions, the N-terminal domains are
required, for others the C-terminal, and some require the full-length protein. We would like
to discern which domains are required for BS69’s role in mesendoderm formation. We
have already attempted to generate a tagged full-length BS69 lentiviral construct, but they
rendered transduced cells unviable upon induction of the gene (Data not shown). We
attempted this procedure with a wild-type full-length BS69 and a MYND-domain mutant,
each fused to eGFP and preceded by an inducible promoter. Both constructs caused cell
death in mESCs only upon induction with doxycycline, whereas control constructs lacking
BS69 survived. Previous groups have successfully expressed an N-terminal portion of
BS69 in various cell lines, so our next step is to generate BS69 constructs lacking different
functional domains (Guo et al, 2014; Wen et al, 2014). To test the function of each
construct, we will incorporate them into inducible lentiviral vectors with an eGFP fusion
and transduce them into BS69 KO mESCs. The ability to induce expression will allow us
to control the amount of construct that is expressed, and the eGFP fusion will simplify
verification of induction. As a rescue assay, we will attempt to differentiate the transduced
cells into mature cell types such as contractile cardiomyocytes. Additionally, we will use
the EB formation assay to compare the ability of each construct to down-regulate neuron
formation.

122

5.5.2 Detection of BS69 Interaction with Chromatin during Pluripotency and Germ
Layer Specification by ChIP-seq Analysis

We know that BS69 is expressed in mESCs and is not required to maintain a self-renewing
state. However, it appears to play an early role since BS69 KO mESCs had 106 genes that
were differentially expressed from controls. To discern what this role may be and to test
the recent finding that BS69 is a chromatin reader, we plan to perform ChIP-sequencing
on BS69 and compare its localization with H3.3 and H3K36me3 in mESCs. Because we
can expand the mESCs rapidly, we will be able to obtain ample amounts of chromatin.
Although the binding of BS69 to H3.3K36me3 is already established in the literature, an
antibody that can successfully pull down full-length BS69 is not currently available on the
market. To surmount this obstacle, we will use an eGFP-tagged N-terminal BS69 lentiviral
construct from our rescue experiment (see above). Once we transduce our KO mESCs with
the lentivirus and select for positive cells, we will induce expression and isolate nuclear
protein. We will use the resulting isolate for ChIP sequencing with antibodies against
eGFP, H3.3, and H3K36me3. We expect to see significant overlap between the regions
bound by all three antigens. Additionally, we expect to find that BS69 associates with the
genes that were differentially expressed in our RNA-sequencing experiments. To
determine whether BS69’s interaction with chromatin changes during lineage
specification, we will repeat this experiment using chromatin from day 3 EBs.

123

5.3.3 Identification of BS69 Interactome in Pluripotency and Early Germ Layer
Specification by Mass Spectrometry

Ideally, we would like to determine by mass spectrometry whether BS69 binds any proteins
at its MYND domain in mESCs and day 3 EBs. One approach is to express a tagged C-
terminal BS69 construct for pulldown. However, an obstacle to proceeding with this
experiment is the potential existence of a functional short isoform of BS69 comprised of
only the C-terminal exons, BRAM1 (Kurozumi et al, 1998). If we confirm its existence, it
should be localized to the cytoplasm due to its lack of an NLS. Therefore, we can avoid
cross-contamination with BRAM1 binding partners by purifying the nuclear fraction from
the cytoplasmic fraction. Alternatively, we could generate an antibody against an epitope
in the N-terminal region of BS69 that would be capable of pulling down the full-length
protein. Once we complete the pull-down, we will use mass spectrometry to determine the
proteins that interact with BS69.

124

5.3.4 CRISPR/Cas-9 knockout of BS69 in hESCs
BS69 is highly conserved between mouse and human, so we propose to study its function
in hESCs. We have already attempted to use lentiviral shRNA technology, but because
BS69 is haplosufficient we have been unable to achieve significant knockdown with any
of the four shRNA constructs that we tested (Data not shown). Recently, CRISPR/Cas-9
technology has become a new tool to efficiently ablate both alleles of a targetd gene.
Therefore, we propose to use this technology to create BS69 KO hESCs. After ablating the
gene with CRISPR/Cas-9 vectors and specific guide RNA, we will culture and analyze
pluripotency gene expression in the KO cell lines. We will then utilize the embryoid body
formation assay to differentiate the BS69 KO hESCs. To determine if BS69 serves the
same function in hESCs as in mESCs, we will use RNA-sequencing to analyze gene
expression in BS69 KO hESCs and EBs. We will then analyze the tissues from each germ
layer that are up or down-regulated in the human BS69 KO lines using
immunofluorescence, RTPCR, and WMISH.

125

5.3.5 Chimera Formation Assay
We would like to test whether cells that lack BS69 are able to differentiate into
mesendoderm in an environment containing wild-type cells. To address this, we plan to
generate a chimera using BS69 KO mESCs injected into a wild-type blastocyst. Because
our BS69 KO cells contain β-gal, we will collect the chimeric embryos between E10.5 and
E13.5, section them, and stain the sections with X-gal to visualize the extent of
incorporation. If the KO cells are restricted to neuroectoderm lineages, that would suggest
that BS69 is required for mesendoderm specification in a cell autonomous manner.
Conversely, if KO cells are present in the developing mesoderm and endoderm tissues, that
would support the hypothesis that BS69 function is non-cell autonomous, allowing wild-
type cells to rescue the ability of KO cells to form mesendoderm.

126

Chapter 6: Materials and Methods

6.1 Animals
BS69 KO embryonic stem cell lines were generated through gene-trap knock-in by Bay
Genomics Gene Trap Consortium. Mouse lines were generated by injecting cells into
C57BL/6 blastocysts, which were then implanted into pseudo-pregnant females. The
resulting chimeras were crossed to each other and resulting F1 generation BS69
heterozygous mice were selected for founder breeding. BS69 heterozygous mice were
crossed with wild-type CD1 mice to increase litter size. BS69 heterozygous mice were then
bred and females checked for plugs each morning and evening for three days post-mating.
The time at which the plug was seen was designated embryonic day 0.5 (E0.5) for all
embryo isolation protocols. Mice were housed at the University of Southern California
under Institutional Animal Care and Use Committee (IACUC) protocol number 11526.

6.2 Genotyping
To propagate the BS69 heterozygous mouse line, both β-gal detection and polymerase
chain reaction (PCR) were used as genotyping tools. Between postnatal days 18 and 20,
mouse pups were ear-tagged and tail-clipped, removing approximately 1/8 inch from the
tip of the tail. Tail clips were collected and incubated overnight at 37°C in X-gal staining
solution (5mM EGTA, pH 8, 2mM MgCl2, 0.2% NP-40, 0.1% sodium deoxycholate, 2mM
CaCl2, 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 1mg/mL X-gal
(Sigma-Aldrich)). The following day, tail clips were visually inspected for blue staining,
indicative of β-gal activity. The X-gal staining solution was then removed and replaced
127

with 20u of tail lysis buffer (50mM Tris pH 8.0, 2mM NaCl, 10mM EDTA, and 0.1% SDS)
plus 1mg/ml proteinase K. Tail clips were then incubated overnight at 56°C. The following
day, 230ul of sterile water was added and samples were centrifuged for 1 minute at 12,000
rotations per minute (rpm). Samples were then analyzed by PCR using a standardized
master mix (Standard Taq Buffer, MgCl2, deoxyribonucleotides (dNTPs), and Taq
polymerase) (New England Biolabs) plus primers for the LacZ gene that encodes β-gal
(Table 6.1).

6.3 Embryo Isolation
Pregnant female mice were humanely euthanized per USC IACUC protocol 11526 at 6.5
and 7.5 days post-conception. After confirming cessation of heartbeat, a lateral incision
was made using dissecting scissors through the epidermis, dermis, and abdominal muscle
layer over the uterus. The incision was widened to form a flap, exposing the abdominal
organs. Beneath the visceral adipose tissue, the uterine horns were located and severed
from the body cavity just below the ovaries and above the cervix. The uterine horn was
then removed using forceps (Dumont) and deciduae were separated with scissors into a
petri dish filled with diethyl pyrocarbonate (DEPC)-treated phosphate-buffered saline
(PBS). The uterine muscle was removed from each decidua using two pairs of forceps
while working under a dissection microscope (Leica). Each decidua was then removed to
a new petri dish with fresh DEPC-treated PBS and dissected using two pairs of forceps to
expose the embryo. Embryos were collected in DEPC-treated PBS at E6.5 and E7.5 (Shea
& Geijsen, 2007).
128

6.4 RNA Probe Preparation
RNA antisense probes for in situ hybridization were prepared using the DIG RNA Labeling
Kit (SP6/T7) (Roche). The provided standard RNA labeling reaction protocol was used as
written except where T3 RNA polymerase was substituted for T7 or Sp6 according to the
template being used. The optional 15 minute DNase I incubation was performed (Roche).
Templates for Nodal, Bmp4, Brachyury, Wnt3, Oct4, Sox1, Six3, Hesx1, and HNF3β were
generously provided by the F. Mariani laboratory. Successful probe synthesis was
determined by denaturing gel electrophoresis. A 1% agarose gel was made with 1M 3-(N-
morpholino)propan sulfonic acid (MOPS) buffer and 2% formaldehyde. RNA probes were
mixed with loading dye, denatured at 70°C for 5 minutes, cooled on ice, and run with
MOPS buffer at 80 volts for 20 minutes.

Probe Citation
Wnt3 (Liu et al, 1999)
Hesx1 (Thomas et al, 1995)
Six3 (Oliver et al, 1995)
HNF3B (Sasaki & Hogan, 1994)
Brachyury (Wilkinson et al, 1990)
Nodal (Conlon et al, 1994)
Sox1 F. Mariani laboratory
Nodal F. Mariani laboratory
Oct4 F. Mariani laboratory

Table 6.1 RNA antisense probe list. Probes used in WMISH are listed with citations.

129

6.5 Whole Mount In Situ Hybridization

Post isolation, E6.5 and E7.5 embryos were fixed in 4% paraformaldehyde/PBS overnight
at 4°C and then dehydrated through a graded methanol/PBS series to 100% methanol and
stored at -80°C for at least one day. Before hybridization, embryos were rehydrated through
the methanol/PBS series and washed in PBS with 0.1% Tween-20 (PBT). Briefly, embryos
were equilibrated in pre-hybridization buffer and then incubated at 70°C for 1-2 hours.
RNA probes were added to hybridization buffer at 1ng/ml and incubated with embryos
overnight at 70°C. The following day, embryos were washed in formamide-based buffers
of decreasing stringency and finally in maleic acid buffer with 0.1% Tween-20 (MABT).
Embryos were blocked for one hour with 10% normal goat serum (Sigma-Aldrich) and 1%
Boehringer’s Blocking Reagent (Roche) in MABT. Anti-digoxygenin (DIG) alkaline
phosphatase antibody (Roche) was diluted to 1:2000 and incubated with the embryos
overnight at 4°C. Embryos were washed extensively with MABT and then developed in
BM Purple (Roche) for 2-48 hours. Samples were washed with PBT and equilibrated
gradually to 80% glycerol in PBS for photographing with a dissection microscope (Leica).

6.6 BS69 KO mESC Line Establishment

BS69 KO and control blastocysts were used to establish mESC lines. At E3.5, blastocysts
were flushed from the uterus into warmed mESC medium with 20mM 4-(2-hydroxyethyl)-
1-piperazineethanesulfonic acid (HEPES) and immediately plated onto 12-well cell culture
dishes (Nagy & Gossler, 2002). Blastocysts and mESCs were cultured in mESC medium
containing KO Dulbecco’s Modified Eagle’s Medium (DMEM) Optimized for ES Cells
130

(Life Technologies), 15% mESC-certified fetal bovine serum (FBS) (HyClone, Thermo
Scientific), 100U/mL LIF (ESGRO®, Millipore), 2i: 1µM PDO3 and 3µM CHIR (Ying et
al, 2008), 100μM non-essential amino acids (NEAA) (Life Technologies), 2mM L-
glutamine (Life Technologies), and 100μM β-mercaptoethanol (Life Technologies),
50µg/ml penicillin/streptomycin (Life Technologies). The ICM of each blastocyst was
manually removed using a glass capillary tube, trypsinized, and plated onto mitotically-
inactivated mouse embryonic fibroblasts (MEFs) after four to five days. mESCs were
subsequently passaged every 2-4 days with 0.25% trypsin-EDTA. To deplete mESCs of
MEFs for genotype analysis, RTPCR, and EB formation, mESCs were passaged onto
0.01% gelatin-coated 10cm cell culture plates for 30-60 minutes and then transferred to
fresh 0.01% gelatin-coated 10cm cell culture plates. This procedure was repeated during
passaging until no MEFs remained (2-4 passages). RNA was extracted as described
elsewhere in this section and mESCs were genotyped using BS69 primers (Table 6.1). All
cell culture was carried out under sterile conditions and all cells were incubated in a 37°C
humidified chamber with 5% CO2.

6.7 EB Formation
Established mESCs growing on 0.01% gelatin-coated plates were trypsinized to a single-
cell suspension with 0.25% Trypsin-EDTA (Life Technologies). The trypsin was then
quenched with differentiation media (mESC media sans LIF, PD03, and CHIR) and cells
were counted using a hemacytometer. Cells were diluted to a concentration of 25,000
cells/ml, yielding 1000 cells per 40ul droplet (Mansergh et al, 2009). On the underside of
the lid of a petri dish, 40ul droplets of cell suspension were placed in rows using an 8-
131

channel multipipette. Approximately 60-70 of these hanging drops were placed per lid.
Finally, 2ml of sterile PBS was added to the bottom of the petri dishes and the lids were
carefully replaced. EBs were grown in a humidified 37°C incubator with 5%CO2.

6.8 Flow Cytometry
mESCs on 0.01% gelatin-coated plates were trypsinized with 0.25% trypsin-EDTA,
neutralized with media, and spun-down for 3 minutes at 300g. Cells were then washed in
5ml sterile PBS and spun down for 3 minutes at 300g. Then, in 0.5ml sterile PBS, cells
were re-suspended and gently vortexed as 4.5ml chilled 70% ethanol was added drop-wise.
Cells were fixed for 2 hours at 4C and spun down for 3 minutes at 300g. Next, cells were
re-suspended in 0.5ml propidium iodide (PI) staining solution (0.01mg/ml PI and 0.5U
RNase in 0.1% Triton X-100 (in PBS)) and incubated in the dark at 37C for 30 minutes.
Cells were spun down for 3 minutes at 300g, re-suspended in 1ml PBS, and analyzed by
flow cytometry on an ARIA II (BD Biosciences) in the USC Flow Cytometry Core. The
single cell population was gated and applied to the cell count versus PI histogram. The
analysis was quantified using FACSDiva software (BD Biosciences).

6.9 RNA Isolation and RTPCR
RNA isolation from mESCs and EBs was carried out according to the Qiagen RNeasy
Micro Kit handbook instructions for cells, with the exception that day 12 EBs were
homogenized using a hand-held homogenizer (Qiagen). RNA concentrations were
determined using a Nanodrop (Thermo Scientific). Reverse transcription was carried out
using the SuperScript First-Strand Synthesis System for RTPCR using provided Oligo(dT)
132

primers (Life Technologies). Resulting cDNA was then analyzed by PCR (as described
previously) using specific primers (Table 6.1). Primers were designed using NCBI
PrimerBLAST and purchased from Eurofins Genomics.

Table 6.2 PCR primer list. Primers used for genotyping PCR and RTPCR are listed.

6.10 β-galactosidase detection

Whole embryos were collected in PBS (as previously described) and immediately fixed in
cold 0.2% glutaraldehyde (Electron Microscopy Sciences) for 2 minutes at 4°C. Samples
were then washed well in cold PBS and incubated overnight at 37°C in X-gal staining
solution (5mM EGTA, pH 8, 2mM MgCl2, 0.2% NP-40, 0.1% sodium deoxycholate, 2mM
CaCl2, 5mM potassium ferricyanide, 5mM potassium ferrocyanide, 1mg/mL X-gal
(Sigma-Aldrich)). The reaction was halted by washing samples three times with PBS.
Embryos were then equilibrated gradually to 80% glycerol and photographed on a
dissection microscope (Leica). Embryos were stored in 80% glycerol at -20°C. For
133

sections, whole embryos that were already X-gal stained and photographed were cleared
of glycerol by 3-5 washes in PBS for 10 minutes each. Embryos were then equilibrated
overnight in 15% sucrose, 7.5% gelatin in PBS. The following day, samples were frozen
in cryomolds in 15% sucrose, 7.5% gelatin in PBS and subsequently cryosectioned at a
thickness of 10um and placed on slides. Samples were mounted in glycerol and sealed with
coverslips and clear nail polish before imaging (Zeiss).
6.11 Immunohistochemistry

Embryos and embryoid bodies were equilibrated overnight in 15% sucrose, 7.5% gelatin
in PBS. The next day, samples were embedded in 15% sucrose, 7.5% gelatin in PBS and
frozen at -80°C until sectioning. Samples were cryosectioned at a thickness of 10um and
placed on slides. Until staining, slides were stored at -20°C. Once thawed, samples were
fixed in 4% paraformaldehyde for 10 minutes at room temperature and then washed twice
with PBS. Samples were permeabilized with 0.1% TritonX-100 in PBS for 10 minutes
followed by two washes in PBS. Samples were then blocked in 5% heat inactivated goat
serum with 0.1% bovine serum albumin (BSA) in 0.1% TritonX-100 for 1 hour at room
temperature. Primary antibody was diluted per Table 6.2 in blocking solution, applied to
samples, and incubated at 4°C overnight in a humidified staining tray. Samples were
washed three times in PBS and subsequently incubated in secondary antibody, which was
diluted in 0.1% BSA in PBS per Table 6.3 for 1 hour at room temperature. Slides were
washed three times with PBS and incubated with 0.05ug/ml DAPI for 5 minutes. Samples
were washed twice with PBS and mounted in fluorescent mounting medium. Finally, each
slide was covered with a coverslip and sealed with nail polish.
134

Table 6.3 Immunofluorescence primary antibody list. Primary antibodies, working
dilutions, and companies with catalog numbers are listed.

Table 6.4 Immunofluorescence secondary antibody list. Secondary antibodies, working
dilutions, and companies with catalog numbers are listed.

135

6.12 RNA-sequencing
RNA was isolated as described above and transferred to the USC Epigenome Center for
further preparation and sequencing. There, mRNA was reverse-transcribed and
multiplexed (Illumina). cDNA was then run for 75 cycles on a NextSeq 500 for paired-end
sequencing (Illumina).

6.13 Bioinformatics
The USC Norris Medical Library Bioinformatics Services Program provided bioinformatic
analysis for all RNA-seq data. Sequences were analyzed using Partek Flow software with
upper quartile normalization and multi-model statistical analysis (Partek Inc.). Subsequent
gene ontology analysis was carried out using DAVID Functional Annotation (Ashburner
et al, 2000; Dennis et al, 2003).

136

References

Adjaye J, Huntriss J, Herwig R, BenKahla A, Brink TC, Wierling C, Hultschig C, Groth
D, Yaspo ML, Picton HM, Gosden RG, Lehrach H (2005) Primary differentiation in the
human blastocyst: comparative molecular portraits of inner cell mass and trophectoderm
cells. Stem Cells 23: 1514-1525

Ang SL, Wierda A, Wong D, Stevens KA, Cascio S, Rossant J, Zaret KS (1993) The
formation and maintenance of the definitive endoderm lineage in the mouse: involvement
of HNF3/forkhead proteins. Development 119: 1301-1315

Ansieau S, Leutz A (2002) The conserved Mynd domain of BS69 binds cellular and
oncoviral proteins through a common PXLXP motif. J Biol Chem 277: 4906-4910

Arkell RM, Tam PP (2012) Initiating head development in mouse embryos: integrating
signalling and transcriptional activity. Open Biol 2: 120030

Arnold SJ, Hofmann UK, Bikoff EK, Robertson EJ (2008) Pivotal roles for
eomesodermin during axis formation, epithelium-to-mesenchyme transition and
endoderm specification in the mouse. Development 135: 501-511

Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski
K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S,
Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G (2000) Gene ontology:
tool for the unification of biology. The Gene Ontology Consortium. Nat Genet 25: 25-29

Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003)
Multipotent cell lineages in early mouse development depend on SOX2 function. Genes
Dev 17: 126-140

Banaszynski LA, Wen D, Dewell S, Whitcomb SJ, Lin M, Diaz N, Elsässer SJ, Chapgier
A, Goldberg AD, Canaani E, Rafii S, Zheng D, Allis CD (2013) Hira-dependent histone
H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells. Cell 155:
107-120

Beck S, Le Good JA, Guzman M, Ben Haim N, Roy K, Beermann F, Constam DB (2002)
Extraembryonic proteases regulate Nodal signalling during gastrulation. Nat Cell Biol 4:
981-985

Ben-Haim N, Lu C, Guzman-Ayala M, Pescatore L, Mesnard D, Bischofberger M, Naef
F, Robertson EJ, Constam DB (2006) The nodal precursor acting via activin receptors
induces mesoderm by maintaining a source of its convertases and BMP4. Dev Cell 11:
313-323

Boyer LA, Plath K, Zeitlinger J, Brambrink T, Medeiros LA, Lee TI, Levine SS, Wernig
M, Tajonar A, Ray MK, Bell GW, Otte AP, Vidal M, Gifford DK, Young RA, Jaenisch R
137

(2006) Polycomb complexes repress developmental regulators in murine embryonic stem
cells. Nature 441: 349-353

Braam SR, Mummery CL (2010) Human stem cell models for predictive cardiac safety
pharmacology. Stem Cell Res 4: 155-156

Branford WW, Yost HJ (2002) Lefty-dependent inhibition of Nodal- and Wnt-responsive
organizer gene expression is essential for normal gastrulation. Curr Biol 12: 2136-2141

Brennan J, Lu CC, Norris DP, Rodriguez TA, Beddington RS, Robertson EJ (2001)
Nodal signalling in the epiblast patterns the early mouse embryo. Nature 411: 965-969

Bultman S, Gebuhr T, Yee D, La Mantia C, Nicholson J, Gilliam A, Randazzo F,
Metzger D, Chambon P, Crabtree G, Magnuson T (2000) A Brg1 null mutation in the
mouse reveals functional differences among mammalian SWI/SNF complexes. Mol Cell
6: 1287-1295

Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J, Evans S (2003) Isl1 identifies a
cardiac progenitor population that proliferates prior to differentiation and contributes a
majority of cells to the heart. Dev Cell 5: 877-889

Camus A, Perea-Gomez A, Moreau A, Collignon J (2006) Absence of Nodal signaling
promotes precocious neural differentiation in the mouse embryo. Dev Biol 295: 743-755

Cano A, Pérez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo
F, Nieto MA (2000) The transcription factor snail controls epithelial-mesenchymal
transitions by repressing E-cadherin expression. Nat Cell Biol 2: 76-83

Cecconi F, Proetzel G, Alvarez-Bolado G, Jay D, Gruss P (1997) Expression of Meis2, a
Knotted-related murine homeobox gene, indicates a role in the differentiation of the
forebrain and the somitic mesoderm. Dev Dyn 210: 184-190

Chawengsaksophak K, de Graaff W, Rossant J, Deschamps J, Beck F (2004) Cdx2 is
essential for axial elongation in mouse development. Proc Natl Acad Sci U S A 101:
7641-7645

Chen T, Dent SY (2014) Chromatin modifiers and remodellers: regulators of cellular
differentiation. Nat Rev Genet 15: 93-106

Cheng SK, Olale F, Bennett JT, Brivanlou AH, Schier AF (2003) EGF-CFC proteins are
essential coreceptors for the TGF-beta signals Vg1 and GDF1. Genes Dev 17: 31-36

Choi SC, Choi JH, Cui LH, Seo HR, Kim JH, Park CY, Joo HJ, Park JH, Hong SJ, Yu
CW, Lim DS (2015) Mixl1 and Flk1 Are Key Players of Wnt/TGF-β Signaling During
DMSO-Induced Mesodermal Specification in P19 cells. J Cell Physiol 230: 1807-1821

138

Chong JJ, Yang X, Don CW, Minami E, Liu YW, Weyers JJ, Mahoney WM, Van Biber
B, Cook SM, Palpant NJ, Gantz JA, Fugate JA, Muskheli V, Gough GM, Vogel KW,
Astley CA, Hotchkiss CE, Baldessari A, Pabon L, Reinecke H, Gill EA, Nelson V, Kiem
HP, Laflamme MA, Murry CE (2014) Human embryonic-stem-cell-derived
cardiomyocytes regenerate non-human primate hearts. Nature 510: 273-277

Ciruna B, Rossant J (2001) FGF signaling regulates mesoderm cell fate specification and
morphogenetic movement at the primitive streak. Dev Cell 1: 37-49

Ciruna BG, Rossant J (1999) Expression of the T-box gene Eomesodermin during early
mouse development. Mech Dev 81: 199-203

Cobben JM, Weiss MM, van Dijk FS, De Reuver R, de Kruiff C, Pondaag W, Hennekam
RC, Yntema HG (2014) A de novo mutation in ZMYND11, a candidate gene for 10p15.3
deletion syndrome, is associated with syndromic intellectual disability. Eur J Med Genet
57: 636-638

Conlon FL, Lyons KM, Takaesu N, Barth KS, Kispert A, Herrmann B, Robertson EJ
(1994) A primary requirement for nodal in the formation and maintenance of the
primitive streak in the mouse. Development 120: 1919-1928

Costello I, Biondi CA, Taylor JM, Bikoff EK, Robertson EJ (2009) Smad4-dependent
pathways control basement membrane deposition and endodermal cell migration at early
stages of mouse development. BMC Dev Biol 9: 54

Davis S, Miura S, Hill C, Mishina Y, Klingensmith J (2004) BMP receptor IA is required
in the mammalian embryo for endodermal morphogenesis and ectodermal patterning.
Dev Biol 270: 47-63

Dennis G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA (2003)
DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol
4: P3

DeScipio C, Conlin L, Rosenfeld J, Tepperberg J, Pasion R, Patel A, McDonald MT,
Aradhya S, Ho D, Goldstein J, McGuire M, Mulchandani S, Medne L, Rupps R, Serrano
AH, Thorland EC, Tsai AC, Hilhorst-Hofstee Y, Ruivenkamp CA, Van Esch H, Addor
MC, Martinet D, Mason TB, Clark D, Spinner NB, Krantz ID (2012) Subtelomeric
deletion of chromosome 10p15.3: clinical findings and molecular cytogenetic
characterization. Am J Med Genet A 158A: 2152-2161

Di-Gregorio A, Sancho M, Stuckey DW, Crompton LA, Godwin J, Mishina Y,
Rodriguez TA (2007) BMP signalling inhibits premature neural differentiation in the
mouse embryo. Development 134: 3359-3369

Diczfalusy E (1969) Steps in the reproductive process susceptible to regulation. Bull
World Health Organ 40: 479-491
139

Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF,
Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) Induced
pluripotent stem cells generated from patients with ALS can be differentiated into motor
neurons. Science 321: 1218-1221

Ding J, Yang L, Yan YT, Chen A, Desai N, Wynshaw-Boris A, Shen MM (1998) Cripto
is required for correct orientation of the anterior-posterior axis in the mouse embryo.
Nature 395: 702-707

Dovey OM, Foster CT, Cowley SM (2010) Histone deacetylase 1 (HDAC1), but not
HDAC2, controls embryonic stem cell differentiation. Proc Natl Acad Sci U S A 107:
8242-8247

Downs KM, Davies T (1993) Staging of gastrulating mouse embryos by morphological
landmarks in the dissecting microscope. Development 118: 1255-1266

Dufort D, Schwartz L, Harpal K, Rossant J (1998) The transcription factor HNF3beta is
required in visceral endoderm for normal primitive streak morphogenesis. Development
125: 3015-3025

Dunn NR, Vincent SD, Oxburgh L, Robertson EJ, Bikoff EK (2004) Combinatorial
activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse
embryo. Development 131: 1717-1728

Dush MK, Martin GR (1992) Analysis of mouse Evx genes: Evx-1 displays graded
expression in the primitive streak. Dev Biol 151: 273-287

Elsaesser SJ, Goldberg AD, Allis CD (2010) New functions for an old variant: no
substitute for histone H3.3. Curr Opin Genet Dev 20: 110-117

Esni F, Johansson BR, Radice GL, Semb H (2001) Dorsal pancreas agenesis in N-
cadherin- deficient mice. Dev Biol 238: 202-212

Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from
mouse embryos. Nature 292: 154-156

Fei T, Xia K, Li Z, Zhou B, Zhu S, Chen H, Zhang J, Chen Z, Xiao H, Han JD, Chen YG
(2010) Genome-wide mapping of SMAD target genes reveals the role of BMP signaling
in embryonic stem cell fate determination. Genome Res 20: 36-44

Feinstein PG, Kornfeld K, Hogness DS, Mann RS (1995) Identification of homeotic
target genes in Drosophila melanogaster including nervy, a proto-oncogene homologue.
Genetics 140: 573-586

140

Fossat N, Jones V, Garcia-Garcia MJ, Tam PP (2012) Modulation of WNT signaling
activity is key to the formation of the embryonic head. Cell Cycle 11: 26-32

Gallico GG, O'Connor NE, Compton CC, Kehinde O, Green H (1984) Permanent
coverage of large burn wounds with autologous cultured human epithelium. N Engl J
Med 311: 448-451

Gavin BJ, McMahon JA, McMahon AP (1990) Expression of multiple novel Wnt-1/int-
1-related genes during fetal and adult mouse development. Genes Dev 4: 2319-2332

Giefing M, Zemke N, Brauze D, Kostrzewska-Poczekaj M, Luczak M, Szaumkessel M,
Pelinska K, Kiwerska K, Tönnies H, Grenman R, Figlerowicz M, Siebert R, Szyfter K,
Jarmuz M (2011) High resolution ArrayCGH and expression profiling identifies PTPRD
and PCDH17/PCH68 as tumor suppressor gene candidates in laryngeal squamous cell
carcinoma. Genes Chromosomes Cancer 50: 154-166

Gilbert S (2003) Developmental Biology, Seventh edn.: Sinauer Associates, Inc.

Guo R, Zheng L, Park JW, Lv R, Chen H, Jiao F, Xu W, Mu S, Wen H, Qiu J, Wang Z,
Yang P, Wu F, Hui J, Fu X, Shi X, Shi YG, Xing Y, Lan F, Shi Y (2014)
BS69/ZMYND11 reads and connects histone H3.3 lysine 36 trimethylation-decorated
chromatin to regulated pre-mRNA processing. Mol Cell 56: 298-310

Haegel H, Larue L, Ohsugi M, Fedorov L, Herrenknecht K, Kemler R (1995) Lack of
beta-catenin affects mouse development at gastrulation. Development 121: 3529-3537

Hart AH, Hartley L, Sourris K, Stadler ES, Li R, Stanley EG, Tam PP, Elefanty AG,
Robb L (2002) Mixl1 is required for axial mesendoderm morphogenesis and patterning in
the murine embryo. Development 129: 3597-3608

Hateboer G, Gennissen A, Ramos YF, Kerkhoven RM, Sonntag-Buck V, Stunnenberg
HG, Bernards R (1995) BS69, a novel adenovirus E1A-associated protein that inhibits
E1A transactivation. EMBO J 14: 3159-3169

He S, Pant D, Schiffmacher A, Meece A, Keefer CL (2008) Lymphoid enhancer factor 1-
mediated Wnt signaling promotes the initiation of trophoblast lineage differentiation in
mouse embryonic stem cells. Stem Cells 26: 842-849

Hemmati-Brivanlou A, Melton D (1997) Vertebrate neural induction. Annu Rev Neurosci
20: 43-60

Hierholzer A, Kemler R (2010) Beta-catenin-mediated signaling and cell adhesion in
postgastrulation mouse embryos. Dev Dyn 239: 191-199

141

Huelsken J, Vogel R, Brinkmann V, Erdmann B, Birchmeier C, Birchmeier W (2000)
Requirement for beta-catenin in anterior-posterior axis formation in mice. J Cell Biol
148: 567-578

Hughes-Davies L, Huntsman D, Ruas M, Fuks F, Bye J, Chin SF, Milner J, Brown LA,
Hsu F, Gilks B, Nielsen T, Schulzer M, Chia S, Ragaz J, Cahn A, Linger L, Ozdag H,
Cattaneo E, Jordanova ES, Schuuring E, Yu DS, Venkitaraman A, Ponder B, Doherty A,
Aparicio S, Bentley D, Theillet C, Ponting CP, Caldas C, Kouzarides T (2003) EMSY
links the BRCA2 pathway to sporadic breast and ovarian cancer. Cell 115: 523-535

Ikeda O, Sekine Y, Mizushima A, Oritani K, Yasui T, Fujimuro M, Muromoto R, Nanbo
A, Matsuda T (2009) BS69 negatively regulates the canonical NF-kappaB activation
induced by Epstein-Barr virus-derived LMP1. FEBS Lett 583: 1567-1574

Kemp C, Willems E, Abdo S, Lambiv L, Leyns L (2005) Expression of all Wnt genes
and their secreted antagonists during mouse blastocyst and postimplantation
development. Dev Dyn 233: 1064-1075

Kim D-K, Cha Y, Ahn H-J, Kim G, Park K-S (2014) Lefty1 and lefty2 control the
balance between self-renewal and pluripotent differentiation of mouse embryonic stem
cells. Stem Cells Dev 23: 457-466

Kiskinis E, Eggan K (2010) Progress toward the clinical application of patient-specific
pluripotent stem cells. J Clin Invest 120: 51-59

Kitajima S, Takagi A, Inoue T, Saga Y (2000) MesP1 and MesP2 are essential for the
development of cardiac mesoderm. Development 127: 3215-3226

Klaus A, Saga Y, Taketo MM, Tzahor E, Birchmeier W (2007) Distinct roles of
Wnt/beta-catenin and Bmp signaling during early cardiogenesis. Proc Natl Acad Sci U S
A 104: 18531-18536

Kooistra SM, van den Boom V, Thummer RP, Johannes F, Wardenaar R, Tesson BM,
Veenhoff LM, Fusetti F, O'Neill LP, Turner BM, de Haan G, Eggen BJL (2010)
Undifferentiated embryonic cell transcription factor 1 regulates ESC chromatin
organization and gene expression. Stem Cells 28: 1703-1714

Kurozumi K, Nishita M, Yamaguchi K, Fujita T, Ueno N, Shibuya H (1998) BRAM1, a
BMP receptor-associated molecule involved in BMP signalling. Genes Cells 3: 257-264

Ladendorff NE, Wu S, Lipsick JS (2001) BS69, an adenovirus E1A-associated protein,
inhibits the transcriptional activity of c-Myb. Oncogene 20: 125-132

Lagutin OV, Zhu CC, Kobayashi D, Topczewski J, Shimamura K, Puelles L, Russell
HRC, McKinnon PJ, Solnica-Krezel L, Oliver G (2003) Six3 repression of Wnt signaling
142

in the anterior neuroectoderm is essential for vertebrate forebrain development. Genes
Dev 17: 368-379

Lan F, Shi Y (2014) Histone H3.3 and cancer: A potential reader connection. Proc Natl
Acad Sci U S A

Lawson KA, Dunn NR, Roelen BA, Zeinstra LM, Davis AM, Wright CV, Korving JP,
Hogan BL (1999) Bmp4 is required for the generation of primordial germ cells in the
mouse embryo. Genes Dev 13: 424-436

Leahy A, Xiong JW, Kuhnert F, Stuhlmann H (1999) Use of developmental marker genes
to define temporal and spatial patterns of differentiation during embryoid body formation.
J Exp Zool 284: 67-81

Lee B, Song H, Rizzoti K, Son Y, Yoon J, Baek K, Jeong Y (2013) Genomic code for
Sox2 binding uncovers its regulatory role in Six3 activation in the forebrain. Dev Biol
381: 491-501

Li Y, Yu W, Cooney AJ, Schwartz RJ, Liu Y (2013) Brief report: Oct4 and canonical
Wnt signaling regulate the cardiac lineage factor Mesp1 through a Tcf/Lef-Oct4
composite element. Stem Cells 31: 1213-1217

Lin S, Baye LM, Westfall TA, Slusarski DC (2010) Wnt5b-Ryk pathway provides
directional signals to regulate gastrulation movement. J Cell Biol 190: 263-278

Linker C, Stern CD (2004) Neural induction requires BMP inhibition only as a late step,
and involves signals other than FGF and Wnt antagonists. Development 131: 5671-5681

Liu P, Wakamiya M, Shea MJ, Albrecht U, Behringer RR, Bradley A (1999)
Requirement for Wnt3 in vertebrate axis formation. Nat Genet 22: 361-365

Lutterbach B, Sun D, Schuetz J, Hiebert SW (1998) The MYND motif is required for
repression of basal transcription from the multidrug resistance 1 promoter by the t(8;21)
fusion protein. Mol Cell Biol 18: 3604-3611

Mansergh FC, Daly CS, Hurley AL, Wride MA, Hunter SM, Evans MJ (2009) Gene
expression profiles during early differentiation of mouse embryonic stem cells. BMC Dev
Biol 9: 5

Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured
in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A 78:
7634-7638

Martinez-Barbera JP, Rodriguez TA, Beddington RS (2000) The homeobox gene Hesx1
is required in the anterior neural ectoderm for normal forebrain formation. Dev Biol 223:
422-430
143

Masselink H, Bernards R (2000) The adenovirus E1A binding protein BS69 is a
corepressor of transcription through recruitment of N-CoR. Oncogene 19: 1538-1546

Masselink H, Vastenhouw N, Bernards R (2001) B-myb rescues ras-induced premature
senescence, which requires its transactivation domain. Cancer Lett 171: 87-101

Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda
A, Matoba R, Sharov AA, Ko MSH, Niwa H (2007) Pluripotency governed by Sox2 via
regulation of Oct3/4 expression in mouse embryonic stem cells. Nature Cell Biology 9:
625-U626

MATHE G, AMIEL JL, SCHWARZENBERG L, CATTAN A, SCHNEIDER M,
DEVRIES MJ, TUBIANA M, LALANNE C, BINET JL, PAPIERNIK M, SEMAN G,
MATSUKURA M, MERY AM, SCHWARZMANN V, FLAISLER A (1965)
SUCCESSFUL ALLOGENIC BONE MARROW TRANSPLANTATION IN MAN:
CHIMERISM, INDUCED SPECIFIC TOLERANCE AND POSSIBLE ANTI-
LEUKEMIC EFFECTS. Blood 25: 179-196

Matthews JM, Bhati M, Lehtomaki E, Mansfield RE, Cubeddu L, Mackay JP (2009) It
takes two to tango: the structure and function of LIM, RING, PHD and MYND domains.
Curr Pharm Des 15: 3681-3696

Megaw R, Dhillon B (2014) Stem cell therapies in the management of diabetic
retinopathy. Curr Diab Rep 14: 498

Meno C, Gritsman K, Ohishi S, Ohfuji Y, Heckscher E, Mochida K, Shimono A, Kondoh
H, Talbot WS, Robertson EJ, Schier AF, Hamada H (1999) Mouse Lefty2 and zebrafish
antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation. Mol Cell
4: 287-298

Merrill BJ, Pasolli HA, Polak L, Rendl M, Garcia-Garcia MJ, Anderson KV, Fuchs E
(2004) Tcf3: a transcriptional regulator of axis induction in the early embryo.
Development 131: 263-274

Mesnard D, Donnison M, Fuerer C, Pfeffer PL, Constam DB (2011) The
microenvironment patterns the pluripotent mouse epiblast through paracrine Furin and
Pace4 proteolytic activities. Genes Dev 25: 1871-1880

Mesnard D, Guzman-Ayala M, Constam DB (2006) Nodal specifies embryonic visceral
endoderm and sustains pluripotent cells in the epiblast before overt axial patterning.
Development 133: 2497-2505

Mishina Y, Suzuki A, Ueno N, Behringer RR (1995) Bmpr encodes a type I bone
morphogenetic protein receptor that is essential for gastrulation during mouse
embryogenesis. Genes Dev 9: 3027-3037
144

Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M,
Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of
pluripotency in mouse epiblast and ES cells. Cell 113: 631-642

Miura S, Singh AP, Mishina Y (2010) Bmpr1a is required for proper migration of the
AVE through regulation of Dkk1 expression in the pre-streak mouse embryo. Dev Biol
341: 246-254

Molenaar M, van de Wetering M, Oosterwegel M, Peterson-Maduro J, Godsave S,
Korinek V, Roose J, Destrée O, Clevers H (1996) XTcf-3 transcription factor mediates
beta-catenin-induced axis formation in Xenopus embryos. Cell 86: 391-399

Montgomery RL, Davis CA, Potthoff MJ, Haberland M, Fielitz J, Qi X, Hill JA,
Richardson JA, Olson EN (2007) Histone deacetylases 1 and 2 redundantly regulate
cardiac morphogenesis, growth, and contractility. Genes Dev 21: 1790-1802

Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Goedel A,
Höhnke C, Hofmann F, Seyfarth M, Sinnecker D, Schömig A, Laugwitz KL (2010)
Patient-specific induced pluripotent stem-cell models for long-QT syndrome. N Engl J
Med 363: 1397-1409

Morin-Kensicki EM, Faust C, LaMantia C, Magnuson T (2001) Cell and tissue
requirements for the gene eed during mouse gastrulation and organogenesis. Genesis 31:
142-146

Mummery CL, Zhang J, Ng ES, Elliott DA, Elefanty AG, Kamp TJ (2012)
Differentiation of human embryonic stem cells and induced pluripotent stem cells to
cardiomyocytes: a methods overview. Circ Res 111: 344-358

Nagy A, Gossler A (2002) ES Cell Protocols. In Molecular Embryology of the Mouse, pp
26-33. Cold Spring Harbor Laboratories

O'Carroll D, Erhardt S, Pagani M, Barton SC, Surani MA, Jenuwein T (2001) The
polycomb-group gene Ezh2 is required for early mouse development. Mol Cell Biol 21:
4330-4336

Ogata-Kawata H, Yamada K, Uesaka-Yoshino M, Kagawa N, Miyamoto K (2007) BS69,
a corepressor interacting with ZHX1, is a bifunctional transcription factor. Front Biosci
12: 1911-1926

Oliver G, Mailhos A, Wehr R, Copeland NG, Jenkins NA, Gruss P (1995) Six3, a murine
homologue of the sine oculis gene, demarcates the most anterior border of the developing
neural plate and is expressed during eye development. Development 121: 4045-4055

145

Pasini D, Bracken AP, Jensen MR, Lazzerini Denchi E, Helin K (2004) Suz12 is essential
for mouse development and for EZH2 histone methyltransferase activity. EMBO J 23:
4061-4071

Pelton TA, Sharma S, Schulz TC, Rathjen J, Rathjen PD (2002) Transient pluripotent cell
populations during primitive ectoderm formation: correlation of in vivo and in vitro
pluripotent cell development. J Cell Sci 115: 329-339

Perea-Gomez A, Vella FD, Shawlot W, Oulad-Abdelghani M, Chazaud C, Meno C,
Pfister V, Chen L, Robertson E, Hamada H, Behringer RR, Ang SL (2002) Nodal
antagonists in the anterior visceral endoderm prevent the formation of multiple primitive
streaks. Dev Cell 3: 745-756

Pevny LH, Sockanathan S, Placzek M, Lovell-Badge R (1998) A role for SOX1 in neural
determination. Development 125: 1967-1978

Pruitt K, Brown G, Hiatt S, Thibaud-Nissen F, A, O E, CM F, J H, MJ L, KM M, MR M,
NA OL, S P, B R, SH R, LD R, A S, H S, P T, RE T, C W, D W, J W, W W, M D, P K,
DR M, TD M, JM O (2013) RefSeq: an update on mammalian reference sequences.
Nucleic Acids Research: 7

Qian L, Mahaffey JP, Alcorn HL, Anderson KV (2011) Tissue-specific roles of Axin2 in
the inhibition and activation of Wnt signaling in the mouse embryo. Proc Natl Acad Sci
U S A 108: 8692-8697

Redick SD, Bautch VL (1999) Developmental platelet endothelial cell adhesion molecule
expression suggests multiple roles for a vascular adhesion molecule. Am J Pathol 154:
1137-1147

Redies C, Takeichi M (1993) Expression of N-cadherin mRNA during development of
the mouse brain. Dev Dyn 197: 26-39

Rivera-Perez JA (2007) Axial specification in mice: ten years of advances and
controversies. J Cell Physiol 213: 654-660

Rivera-Pérez JA, Hadjantonakis AK (2014) The Dynamics of Morphogenesis in the Early
Mouse Embryo. Cold Spring Harb Perspect Biol

Rivera-Pérez JA, Mager J, Magnuson T (2003) Dynamic morphogenetic events
characterize the mouse visceral endoderm. Dev Biol 261: 470-487

Roberts C, Sutherland HF, Farmer H, Kimber W, Halford S, Carey A, Brickman JM,
Wynshaw-Boris A, Scambler PJ (2002) Targeted mutagenesis of the Hira gene results in
gastrulation defects and patterning abnormalities of mesoendodermal derivatives prior to
early embryonic lethality. Mol Cell Biol 22: 2318-2328

146

Robertson EJ (2014) Dose-dependent Nodal/Smad signals pattern the early mouse
embryo. Semin Cell Dev Biol 32: 73-79

Rogers MB, Hosler BA, Gudas LJ (1991) Specific expression of a retinoic acid-
regulated, zinc-finger gene, Rex-1, in preimplantation embryos, trophoblast and
spermatocytes. Development 113: 815-824

Römer AM, Lühr I, Klein A, Friedl A, Sebens S, Rösel F, Arnold N, Strauss A, Jonat W,
Bauer M (2013) Normal mammary fibroblasts induce reversion of the malignant
phenotype in human primary breast cancer. Anticancer Res 33: 1525-1536

Saga Y (1998) Genetic rescue of segmentation defect in MesP2-deficient mice by MesP1
gene replacement. Mech Dev 75: 53-66

Saga Y, Miyagawa-Tomita S, Takagi A, Kitajima S, Miyazaki J, Inoue T (1999) MesP1
is expressed in the heart precursor cells and required for the formation of a single heart
tube. Development 126: 3437-3447

Sahara M, Santoro F, Chien KR (2015) Programming and reprogramming a human heart
cell. EMBO J 34: 710-738

Sasaki H, Hogan BL (1994) HNF-3 beta as a regulator of floor plate development. Cell
76: 103-115

Schwartz SD, Regillo CD, Lam BL, Eliott D, Rosenfeld PJ, Gregori NZ, Hubschman JP,
Davis JL, Heilwell G, Spirn M, Maguire J, Gay R, Bateman J, Ostrick RM, Morris D,
Vincent M, Anglade E, Del Priore LV, Lanza R (2015) Human embryonic stem cell-
derived retinal pigment epithelium in patients with age-related macular degeneration and
Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet
385: 509-516

Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, Yuan GC, Orkin SH (2008) EZH1
mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining
stem cell identity and executing pluripotency. Mol Cell 32: 491-502

Sirard C, de la Pompa JL, Elia A, Itie A, Mirtsos C, Cheung A, Hahn S, Wakeham A,
Schwartz L, Kern SE, Rossant J, Mak TW (1998) The tumor suppressor gene
Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse
embryo. Genes Dev 12: 107-119

Song J, Oh SP, Schrewe H, Nomura M, Lei H, Okano M, Gridley T, Li E (1999) The
type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral
head development in mice. Dev Biol 213: 157-169

147

Srinivas S, Rodriguez T, Clements M, Smith JC, Beddington RS (2004) Active cell
migration drives the unilateral movements of the anterior visceral endoderm.
Development 131: 1157-1164

Stover D. (2000) Growing Hearts from Scratch. Popular Science, Vol. 256, pp. 46-50.

Sun X, Meyers EN, Lewandoski M, Martin GR (1999) Targeted disruption of Fgf8
causes failure of cell migration in the gastrulating mouse embryo. Genes Dev 13: 1834-
1846

Takada S, Stark KL, Shea MJ, Vassileva G, McMahon JA, McMahon AP (1994) Wnt-3a
regulates somite and tailbud formation in the mouse embryo. Genes Dev 8: 174-189

Takaki H, Oshiumi H, Sasai M, Kawanishi T, Matsumoto M, Seya T (2009)
Oligomerized TICAM-1 (TRIF) in the cytoplasm recruits nuclear BS69 to enhance NF-
kappaB activation and type I IFN induction. Eur J Immunol 39: 3469-3476

Takaoka K, Yamamoto M, Hamada H (2011) Origin and role of distal visceral endoderm,
a group of cells that determines anterior-posterior polarity of the mouse embryo. Nat Cell
Biol 13: 743-752

Tam PP, Behringer RR (1997) Mouse gastrulation: the formation of a mammalian body
plan. Mech Dev 68: 3-25

Tam PPL, Loebel DAF (2007) Gene function in mouse embryogenesis: get set for
gastrulation. Nat Rev Genet 8: 368-381

Tam PPL, Loebel DAF, Tanaka SS (2006) Building the mouse gastrula: signals,
asymmetry and lineages. Curr Opin Genet Dev 16: 419-425

ten Berge D, Koole W, Fuerer C, Fish M, Eroglu E, Nusse R (2008) Wnt signaling
mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3: 508-
518

Teo AK, Arnold SJ, Trotter MW, Brown S, Ang LT, Chng Z, Robertson EJ, Dunn NR,
Vallier L (2011) Pluripotency factors regulate definitive endoderm specification through
eomesodermin. Genes Dev 25: 238-250

Thomas PQ, Johnson BV, Rathjen J, Rathjen PD (1995) Sequence, genomic organization,
and expression of the novel homeobox gene Hesx1. J Biol Chem 270: 3869-3875

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS,
Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science
282: 1145-1147

148

Tortelote GG, Hernandez-Hernandez JM, Quaresma AJ, Nickerson JA, Imbalzano AN,
Rivera-Perez JA (2013) Wnt3 function in the epiblast is required for the maintenance but
not the initiation of gastrulation in mice. Dev Biol 374: 164-173

Toumadje A, Kusumoto K, Parton A, Mericko P, Dowell L, Ma G, Chen L, Barnes DW,
Sato JD (2003) Pluripotent differentiation in vitro of murine ES-D3 embryonic stem
cells. In Vitro Cell Dev Biol Anim 39: 449-453

Ueno S, Weidinger G, Osugi T, Kohn AD, Golob JL, Pabon L, Reinecke H, Moon RT,
Murry CE (2007) Biphasic role for Wnt/beta-catenin signaling in cardiac specification in
zebrafish and embryonic stem cells. Proc Natl Acad Sci U S A 104: 9685-9690

Umehara H, Kimura T, Ohtsuka S, Nakamura T, Kitajima K, Ikawa M, Okabe M, Niwa
H, Nakano T (2007) Efficient derivation of embryonic stem cells by inhibition of
glycogen synthase kinase-3. Stem Cells 25: 2705-2711

Velasco G, Grkovic S, Ansieau S (2006) New insights into BS69 functions. J Biol Chem
281: 16546-16550

Volk T, Geiger B (1984) A 135-kd membrane protein of intercellular adherens junctions.
EMBO J 3: 2249-2260

Wan J, Zhang W, Wu L, Bai T, Zhang M, Lo KW, Chui YL, Cui Y, Tao Q, Yamamoto
M, Akira S, Wu Z (2006) BS69, a specific adaptor in the latent membrane protein 1-
mediated c-Jun N-terminal kinase pathway. Mol Cell Biol 26: 448-456

Wang W, Côté J, Xue Y, Zhou S, Khavari PA, Biggar SR, Muchardt C, Kalpana GV,
Goff SP, Yaniv M, Workman JL, Crabtree GR (1996) Purification and biochemical
heterogeneity of the mammalian SWI-SNF complex. EMBO J 15: 5370-5382

Weinstein DC, Ruiz i Altaba A, Chen WS, Hoodless P, Prezioso VR, Jessell TM, Darnell
JE (1994) The winged-helix transcription factor HNF-3 beta is required for notochord
development in the mouse embryo. Cell 78: 575-588

Wen H, Li Y, Xi Y, Jiang S, Stratton S, Peng D, Tanaka K, Ren Y, Xia Z, Wu J, Li B,
Barton MC, Li W, Li H, Shi X (2014) ZMYND11 links histone H3.3K36me3 to
transcription elongation and tumour suppression. Nature 508: 263-268

Wilkinson DG, Bhatt S, Herrmann BG (1990) Expression pattern of the mouse T gene
and its role in mesoderm formation. Nature 343: 657-659

Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF,
Metcalf D, Nicola NA, Gough NM (1988) Myeloid leukaemia inhibitory factor maintains
the developmental potential of embryonic stem cells. Nature 336: 684-687

149

Wilson PA, Hemmati-Brivanlou A (1995) Induction of epidermis and inhibition of neural
fate by Bmp-4. Nature 376: 331-333

Wolpert L (1992) Gastrulation and the evolution of development. Dev Suppl: 7-13

Wood HB, Episkopou V (1999) Comparative expression of the mouse Sox1, Sox2 and
Sox3 genes from pre-gastrulation to early somite stages. Mech Dev 86: 197-201

Xiao J, Yang R, Biswas S, Qin X, Zhang M, Deng W (2015) Mesenchymal Stem Cells
and Induced Pluripotent Stem Cells as Therapies for Multiple Sclerosis. Int J Mol Sci 16:
9283-9302

Yamaguchi TP, Harpal K, Henkemeyer M, Rossant J (1994) fgfr-1 is required for
embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev 8:
3032-3044

Yamaguchi TP, Takada S, Yoshikawa Y, Wu N, McMahon AP (1999) T (Brachyury) is a
direct target of Wnt3a during paraxial mesoderm specification. Genes Dev 13: 3185-3190

Yeo C, Whitman M (2001) Nodal signals to Smads through Cripto-dependent and Cripto-
independent mechanisms. Mol Cell 7: 949-957

Ying QL, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins
suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration
with STAT3. Cell 115: 281-292

Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A
(2008) The ground state of embryonic stem cell self-renewal. Nature 453: 519-523

Yu B, Shao Y, Zhang C, Chen Y, Zhong Q, Zhang J, Yang H, Zhang W, Wan J (2009)
BS69 undergoes SUMO modification and plays an inhibitory role in muscle and neuronal
differentiation. Exp Cell Res 315: 3543-3553

Yuasa S, Itabashi Y, Koshimizu U, Tanaka T, Sugimura K, Kinoshita M, Hattori F,
Fukami S-i, Shimazaki T, Ogawa S, Okano H, Fukuda K (2005) Transient inhibition of
BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic
stem cells. Nat Biotechnol 23: 607-611

Zhang W, Chan HM, Gao Y, Poon R, Wu Z (2007) BS69 is involved in cellular
senescence through the p53-p21Cip1 pathway. EMBO Rep 8: 952-958

Zhou X, Sasaki H, Lowe L, Hogan BL, Kuehn MR (1993) Nodal is a novel TGF-beta-
like gene expressed in the mouse node during gastrulation. Nature 361: 543-547

150

Zohn IE, Li Y, Skolnik EY, Anderson KV, Han J, Niswander L (2006) p38 and a p38-
interacting protein are critical for downregulation of E-cadherin during mouse
gastrulation. Cell 125: 957-969

151

Appendices

Appendix 1: RNA-sequencing analysis data: BS69 KO mESCs vs
Control mESCs

Chromosome gene_status gene_symbol
MultiModel
Fold-Change
MultiModel
p-value
6 KNOWN Gm8038 -5.36911 0.00075616
4 KNOWN Gm11237 -3.78318 0.000458552
10 NOVEL Gm6763 -3.1739 0.000127577
10 NOVEL Gm4340 -3.17177 0.0010444
7 KNOWN Gm3989 -3.16857 0.00013392
5 KNOWN Gm7982 -3.08645 4.58E-06
7 KNOWN Zscan4b -3.06346 1.91E-07
10 NOVEL Gm6763 -3.03089 0.00421054
11 KNOWN Tmem92 -3.01572 8.72E-11
7 KNOWN Gm3994 -3.00738 0.000321073
13 KNOWN Gm21818 -2.99918 0.00248772
4 KNOWN Gm11239 -2.96639 0.000318247
1 KNOWN Lefty2 -2.96433 0.00359635
13 KNOWN Tcstv3 -2.93471 0.00257113
10 NOVEL Gm8764 -2.92941 0.000612805
10 NOVEL Gm20765 -2.91914 0.000399357
10 NOVEL Gm21304 -2.8851 7.90E-06
5 KNOWN Gm16522 -2.83496 0.00945774
12 KNOWN Gm8332 -2.83036 0.000250943
13 NOVEL B020031M17Rik -2.81233 0.000439786
13 PUTATIVE Gm21761 -2.80092 0.000115105
5 KNOWN Gm6502 -2.78114 1.11E-07
12 KNOWN Gm5662 -2.78103 9.72E-05
6 KNOWN Gm5580 -2.77265 0.00693435
10 NOVEL Gm4301 -2.76974 0.00184233
5 KNOWN Gm16429 -2.76832 0.000755705
10 NOVEL Gm21312 -2.76408 1.70E-05
12 NOVEL Gm2016 -2.75421 4.28E-08
10 KNOWN Gm9046 -2.74821 0.000640588
4 KNOWN BC080695 -2.73288 2.36E-06
7 KNOWN Zscan4e -2.71724 4.99E-06
12 NOVEL Gm8300 -2.71526 1.88E-05
13 KNOWN Actn2 -2.69742 0.00778686
10 KNOWN Gm4303 -2.68598 0.000828293
152

5 KNOWN AA792892 -2.68252 0.00326315
8 NOVEL Gm27167 -2.66968 7.15E-05
4 KNOWN Gm11236 -2.66 0.00571787
13 KNOWN Gm20767 -2.64846 0.000629178
11 KNOWN Gm12183 -2.62131 1.43E-07
13 NOVEL Tcstv1 -2.602 0.0026835
8 KNOWN Inpp4b -2.5817 0.000319475
6 KNOWN Gm8994 -2.56076 0.00198608
5 KNOWN BC061212 -2.54672 0.0032534
4 KNOWN Gm13119 -2.52343 0.000107389
10 KNOWN Gm9048 -2.51061 0.00414036
13 KNOWN Gm21762 -2.50903 0.000159129
4 KNOWN Gm13057 -2.49979 0.00439486
5 KNOWN Gm7942 -2.49153 0.00177143
2 NOVEL Gm13693 -2.47081 0.000529492
4 NOVEL Pramef25 -2.44112 0.00350584
11 KNOWN Gm11543 -2.39493 0.000456905
4 NOVEL Gm11757 -2.3877 0.00290358
7 KNOWN Usp17ld -2.38568 0.00011085
4 NOVEL Gm13040 -2.37181 0.000662349
12 NOVEL Gm4027 -2.36427 9.92E-06
2 NOVEL Gm13691 -2.36167 4.27E-05
8 KNOWN Gm5117 -2.33451 0.000343689
4 KNOWN Gm13043 -2.32844 0.00228185
1 KNOWN Il1r2 -2.32389 0.00143395
7 KNOWN Gm3984 -2.31891 0.00700541
2 NOVEL Gm13694 -2.31329 0.000100563
2 NOVEL Gm13698 -2.30793 3.53E-05
6 KNOWN Cml2 -2.28244 0.000125509
16 KNOWN Hunk -2.27438 0.00441933
2 NOVEL Gm13697 -2.27075 0.00837659
X KNOWN Gm5939 -2.26831 0.00930176
4 NOVEL Gm11756 -2.26819 0.0054793
12 KNOWN Gm2022 -2.26597 0.00334536
X KNOWN Xpnpep2 -2.24759 0.00181568
4 NOVEL Gm12794 -2.23709 0.00750074
7 KNOWN Gm10678 -2.23672 0.00223074
5 KNOWN Gm6351 -2.2203 0.00433057
5 KNOWN Gm10424 -2.21971 0.00564473
5 KNOWN Gm7682 -2.21464 0.000813877
4 NOVEL Gm13871 -2.21136 0.000942542
153

14 KNOWN Trac -2.19769 0.00155396
7 NOVEL Gm5891 -2.18881 0.00782539
12 KNOWN Gm2046 -2.18573 0.00802834
10 NOVEL Gm4308 -2.1225 0.00111633
11 NOVEL Gm15698 -2.11476 0.0017142
10 KNOWN Gm8711 -2.11078 0.000646994
12 NOVEL RP23-291K4.3 -2.11005 0.0052287
10 KNOWN Gm9044 -2.08567 0.00274474
16 KNOWN Robo1 -2.04537 0.00651046
9 KNOWN Gm27205 -2.02897 0.00359225
18 KNOWN Mep1b -2.02377 0.00699465
7 KNOWN Gm4003 -2.02278 0.00160922
6 KNOWN Ccdc136 2.00532 0.00400688
2 KNOWN Optn 2.00872 0.00820658
7 KNOWN Atf5 2.12034 0.00573357
1 KNOWN Unc80 2.33706 0.00020342
4 KNOWN Sdc3 2.33923 0.00402662
9 KNOWN Fxyd6 2.36184 2.22E-05
2 KNOWN Ckmt1 2.43823 0.00073139
4 KNOWN Fblim1 2.44653 0.00732489
12 KNOWN Meg3 2.49114 0.000153785
2 KNOWN Chac1 2.89051 0.00437638
5 KNOWN Galnt11 3.10859 0.002393
3 KNOWN Trim2 3.27759 0.00274192
1 KNOWN Col6a3 3.28901 0.0070049
19 KNOWN Prune2 3.30287 0.00242647
1 KNOWN Cyb5r1 3.49919 2.12E-06
2 KNOWN Grin1 3.59225 5.27E-07
8 KNOWN Cpe 4.9852 0.00115727
3 KNOWN Frem2 9.40955 0.00586902
1 KNOWN Chit1 10.4697 3.95E-15

154

Appendix 2: RNA-sequencing analysis data: BS69 KO day 3 EBs vs
Control day 3 EBs

Chromosome gene_status gene_symbol
MultiModel
Fold-Change
MultiModel
p-value
17 KNOWN T -24.4321 3.32E-10
2 KNOWN Sp5 -19.5409 2.38E-08
18 KNOWN Wnt8a -14.3803 2.77E-21
2 KNOWN Foxa2 -10.5924 1.02E-06
9 KNOWN Eomes -8.8459 2.99E-06
6 KNOWN Evx1 -8.66996 5.46E-09
14 KNOWN RP23-421C24.1 -8.26929 0.00069544
1 KNOWN Mixl1 -7.22995 8.58E-07
2 KNOWN Meis2 -6.95701 8.15E-05
11 KNOWN Wnt3 -6.57671 5.65E-09
9 KNOWN Foxb1 -6.32194 0.00157147
12 KNOWN Gsc -5.68586 0.0043028
3 KNOWN Gbp2 -5.32716 3.02E-05
19 KNOWN Fgf8 -5.16005 0.00072656
6 KNOWN Ccnd2 -5.10636 0.00218421
5 KNOWN Tgfbr3 -4.85132 0.0003611
7 KNOWN Wnt11 -4.83324 0.00534139
2 KNOWN Frzb -4.75321 0.00017158
13 KNOWN Fst -4.69317 0.00919045
9 KNOWN Nt5e -4.6856 0.00089805
X KNOWN Porcn -4.65436 5.02E-07
12 KNOWN Nrcam -4.26556 0.00150876
17 KNOWN Pla2g7 -4.21521 0.00543396
12 KNOWN Ism2 -4.21063 1.66E-05
X KNOWN Cxx1c -4.12418 0.00012417
7 KNOWN Mogat2 -4.00257 0.00089886
11 KNOWN Slit3 -3.99359 0.00352762
6 KNOWN Adamts9 -3.99344 0.00035895
11 KNOWN Axin2 -3.9801 3.36E-11
6 NOVEL 5730457N03Rik -3.89701 0.00695829
14 KNOWN Tnfrsf19 -3.79613 0.00144349
X KNOWN Efnb1 -3.71545 0.00558697
12 KNOWN Dact1 -3.46927 3.41E-06
X KNOWN Zfp36l3 -3.39997 2.80E-05
5 KNOWN Fzd10 -3.34985 1.62E-06
155

11 KNOWN Grm6 -3.33312 5.12E-05
6 KNOWN Wnt5b -3.24598 0.0063519
14 KNOWN Phf11d -3.16549 0.00387172
X KNOWN Ebp -3.07589 0.00839565
3 KNOWN Pdzk1 -3.06742 0.00915043
3 KNOWN Lphn2 -3.05351 0.00484135
3 KNOWN Vangl1 -2.96343 0.00272051
10 KNOWN Nodal -2.94797 0.00030479
2 KNOWN Bmp7 -2.9087 0.00094941
X KNOWN Gria3 -2.88459 0.00683828
3 KNOWN Dennd2c -2.87157 8.19E-07
11 KNOWN Krt19 -2.85903 0.00059984
3 KNOWN Txnip -2.85673 0.00308107
8 KNOWN Lzts1 -2.84542 2.50E-05
X KNOWN Cxx1a -2.82191 1.29E-05
3 KNOWN Flg -2.77277 0.0073842
5 KNOWN Pcdh7 -2.75261 0.0035049
11 KNOWN Cabp7 -2.7486 0.00010756
6 KNOWN Slc2a3 -2.71666 1.64E-08
9 KNOWN Rbp1 -2.65212 0.00108027
X KNOWN Nrk -2.61573 4.39E-06
7 KNOWN Relt -2.60785 0.00103178
X KNOWN BC023829 -2.60686 3.97E-06
X KNOWN Sat1 -2.6006 3.58E-09
6 KNOWN Slc4a5 -2.58457 0.00013815
2 NOVEL Gm8898 -2.56932 0.00015092
4 NOVEL Gm17167 -2.54652 0.00465678
2 KNOWN Cyct -2.54569 0.00315144
11 KNOWN Pecam1 -2.52688 0.00120127
11 KNOWN Upp1 -2.5178 0.00032353
14 KNOWN Il17rd -2.50694 0.00465265
18 KNOWN Sall3 -2.50557 0.00165546
4 KNOWN Epha2 -2.49716 0.00518411
11 KNOWN Cep112 -2.48063 1.52E-08
14 KNOWN Ltb4r2 -2.40859 0.00053844
2 KNOWN Slc4a11 -2.38855 0.00475913
X KNOWN Zic3 -2.38179 1.47E-08
5 KNOWN Pdgfa -2.37582 0.00226827
5 KNOWN Gm17132 -2.33422 2.51E-05
13 KNOWN Zmynd11 -2.32915 0.00242743
3 KNOWN Gm21962 -2.30886 0.00585219
156

7 KNOWN Gm3998 -2.3003 0.00385078
4 KNOWN Trp53inp1 -2.25513 0.00112031
7 KNOWN Peg3 -2.25197 0.00136074
1 KNOWN Acsl3 -2.24644 1.67E-09
17 KNOWN Cdkn1a -2.24107 1.54E-10
17 KNOWN Ptk7 -2.23585 3.14E-28
X NOVEL RP23-99K18.3 -2.20234 7.07E-06
11 KNOWN Cdc42ep4 -2.18606 2.94E-08
18 KNOWN Prob1 -2.17685 0.00234144
2 KNOWN Il15ra -2.16757 0.00047816
X KNOWN Cxx1b -2.16532 0.00036955
18 KNOWN Nme5 -2.15235 0.00182115
X KNOWN Cask -2.1368 0.00049665
1 NOVEL RP24-529L13.2 -2.13216 0.0098907
5 KNOWN Sdk1 -2.13032 0.00053721
7 KNOWN Sct -2.12878 0.00133615
10 KNOWN Perp -2.12514 0.0003446
11 KNOWN Cpd -2.12421 0.00124904
18 KNOWN Dnd1 -2.11612 0.00213523
15 KNOWN Krt7 -2.11327 0.0099201
18 KNOWN Pou4f3 -2.11306 0.00163508
7 KNOWN Cd81 -2.10684 0.00018565
17 KNOWN Scube3 -2.10386 0.00306452
2 KNOWN Sfmbt2 -2.09914 0.00143418
8 KNOWN Slc5a5 -2.09809 0.00065135
11 KNOWN Tex19.1 -2.09739 0.00604401
2 KNOWN Cercam -2.09251 0.00532115
X KNOWN Itm2a -2.07729 0.00404106
3 KNOWN Wnt2b -2.07683 0.00250876
14 KNOWN Ltb4r1 -2.07293 0.00173341
3 KNOWN Npr1 -2.04757 0.00540632
1 KNOWN Btg2 -2.04009 0.00739342
10 KNOWN Gm26543 -2.03963 0.0022826
2 KNOWN Ptprt -2.02269 0.00020504
11 KNOWN Tcn2 -2.02079 0.00172273
2 NOVEL Gm14420 -2.02063 0.00516928
X KNOWN Lamp2 -2.0124 6.37E-05
15 KNOWN Fbln1 -2.00414 0.0066605
14 KNOWN Tmem254c 2.00503 0.00045877
2 KNOWN Lsm14b 2.01497 2.32E-07
19 KNOWN Rps6ka4 2.02575 0.00397173
157

4 KNOWN Arhgef19 2.04163 0.00039474
7 KNOWN Sephs2 2.06258 4.12E-09
3 KNOWN Dnttip2 2.06827 0.00725194
17 KNOWN C430042M11Rik 2.06932 0.00083923
19 KNOWN Rasgrp2 2.0731 0.00105831
2 KNOWN Cbln4 2.07623 0.00364668
17 KNOWN Tubb4a 2.09431 0.00030976
8 KNOWN Rab20 2.14145 0.00928934
3 KNOWN Ap1ar 2.14222 0.00666465
5 KNOWN Ptpn13 2.14896 0.00193027
17 KNOWN Ttbk1 2.15513 0.00130152
10 KNOWN Dcn 2.15553 0.00229748
12 KNOWN Rapgef5 2.17118 0.00367701
3 KNOWN Mettl14 2.1764 0.00979759
3 KNOWN Rab25 2.17902 0.00153008
18 KNOWN 4930426D05Rik 2.20076 0.00865997
2 NOVEL E530001K10Rik 2.21818 0.00097224
4 KNOWN Tmem54 2.22368 0.00653851
8 KNOWN Tbc1d9 2.22825 0.00296687
3 KNOWN Pla2g12a 2.23235 3.66E-08
14 KNOWN Duxbl2 2.23438 0.0067821
4 KNOWN Foxd3 2.24232 8.71E-07
11 KNOWN 2810408A11Rik 2.3093 0.0059873
3 KNOWN Camk2d 2.30951 1.27E-05
3 KNOWN Trmt10a 2.33092 5.11E-08
19 KNOWN Ccdc85b 2.3324 0.00169253
7 KNOWN Cend1 2.4702 3.20E-05
19 KNOWN Dtx4 2.47184 0.00464726
7 KNOWN Coro1a 2.48926 0.0066548
7 KNOWN Fgf4 2.48996 7.54E-05
7 KNOWN Fam57b 2.51926 0.00117086
3 KNOWN 1810037I17Rik 2.52651 0.00445689
3 KNOWN Gclm 2.5326 5.63E-06
15 KNOWN Cpt1b 2.54877 0.00297089
3 KNOWN Gar1 2.59143 0.00232331
3 KNOWN Ank2 2.61058 6.23E-06
4 KNOWN Fbxo44 2.64836 0.00809927
3 KNOWN Synpo2 2.66922 1.47E-05
3 KNOWN Ugt8a 2.69961 0.00015376
10 KNOWN Atcay 2.70031 0.00015307
6 KNOWN Calcr 2.73695 0.00012596
158

2 KNOWN Insm1 2.75801 2.18E-06
9 KNOWN Rgl3 2.8324 0.00033416
1 KNOWN Cdk5r2 2.89527 0.0093431
15 KNOWN AI848285 3.20646 0.00061606
7 KNOWN Spon1 3.23132 0.0013868
2 KNOWN Wt1 3.31349 0.00029785
1 KNOWN Chit1 3.68814 1.65E-06
14 KNOWN Tmem254a 3.79582 0.00187994
14 KNOWN Tmem254b 3.79582 0.00187994
19 KNOWN Plce1 3.83785 0.00213708
17 KNOWN Park2 3.87231 3.12E-11
1 KNOWN Cyb5r1 4.43471 5.07E-08
10 KNOWN Slc35d3 4.55901 3.94E-06

159

Appendix 3: RNA-sequencing analysis data: BS69 KO day 5 EBs vs
Control day 5 EBs

Chromosome gene_status gene_symbol
MultiModel
Fold-Change
MultiModel
p-value
5 KNOWN Cdx2 -12.2803 0.000241278
2 NOVEL CTNND1 -11.7314 1.68E-06
7 KNOWN Fgf3 -8.72567 0.000513255
14 KNOWN Wnt5a -8.44002 3.78E-05
1 KNOWN Nrp2 -7.9022 0.000762576
2 KNOWN Hoxd9 -7.55538 0.00432767
6 KNOWN Ccnd2 -6.26258 0.000586433
11 KNOWN Hoxb1 -6.08738 1.91E-05
3 KNOWN Syt6 -5.58589 0.0073452
6 KNOWN Plxna4 -5.58068 2.10E-05
1 KNOWN Mixl1 -5.24268 1.27E-05
2 NOVEL Gm28036 -5.22905 3.22E-10
17 KNOWN Six2 -5.05434 4.34E-14
2 KNOWN Adra2b -5.00252 7.65E-07
8 KNOWN Cdh11 -4.96053 0.000152147
1 KNOWN Wnt6 -4.79764 0.00467207
5 KNOWN Tbx3 -4.76916 0.00572765
11 KNOWN Hoxb2 -4.52014 2.24E-06
9 KNOWN Bmper -4.42789 0.00112434
5 KNOWN Limch1 -4.40764 0.00030315
7 KNOWN Kcnc1 -4.01303 0.00258039
2 KNOWN Ccdc3 -4.00466 0.00417966
7 KNOWN Peg3 -3.97953 1.04E-07
4 KNOWN Glis1 -3.94749 0.000287451
17 KNOWN Tiam2 -3.92876 1.56E-07
6 KNOWN Adcyap1r1 -3.84955 0.000507419
13 KNOWN Zmynd11 -3.83001 5.60E-09
14 KNOWN Nynrin -3.8075 2.46E-06
9 KNOWN Cbl -3.7782 3.00E-07
5 KNOWN Msx1 -3.75223 0.00172519
11 KNOWN Hs3st3b1 -3.69041 1.56E-10
2 KNOWN Hoxd1 -3.68701 0.00396676
6 KNOWN Wnt2 -3.6703 0.00433161
7 KNOWN Lmo1 -3.65663 4.68E-17
160

5 KNOWN Pdgfra -3.6406 0.00216544
4 KNOWN Palm2Akap2 -3.60897 1.47E-05
2 KNOWN Dusp2 -3.60603 5.23E-06
7 KNOWN Mesp1 -3.59287 0.00185622
6 KNOWN Gata2 -3.58163 0.000976331
11 KNOWN Hoxb3 -3.56431 0.000560141
1 KNOWN ZBED6 -3.44017 3.61E-05
6 NOVEL 5730457N03Rik -3.41043 1.89E-05
6 KNOWN Wnt5b -3.40509 1.25E-06
6 KNOWN Hoxa13 -3.37787 1.13E-05
5 KNOWN Rimbp2 -3.35179 0.00156829
17 KNOWN Rftn1 -3.34742 0.00343568
6 KNOWN Evx1 -3.3277 0.000178488
8 KNOWN Tnks -3.25501 1.15E-17
16 KNOWN Klhl6 -3.2427 0.00305205
3 KNOWN Wnt2b -3.19281 6.82E-07
16 KNOWN Bace2 -3.19253 0.000252163
11 KNOWN Ksr1 -3.15064 0.00116641
6 KNOWN Plxnd1 -3.07038 0.000167973
8 KNOWN Lzts1 -3.01776 2.92E-06
7 KNOWN Etv2 -3.00817 0.00620916
13 KNOWN Isl1 -2.99849 0.00540486
14 KNOWN Xpo4 -2.97403 6.56E-10
2 KNOWN Fbn1 -2.96792 0.00952093
5 KNOWN Htra3 -2.91211 0.00362796
9 KNOWN Tirap -2.91179 3.36E-05
5 KNOWN Tmem119 -2.90746 1.25E-06
3 KNOWN Dennd2c -2.89271 1.99E-06
11 KNOWN Axin2 -2.89012 1.93E-15
12 KNOWN Nin -2.87593 4.78E-08
17 KNOWN Zfp871 -2.84888 0.000836215
11 KNOWN Wnt3 -2.82696 4.25E-08
18 KNOWN Nfatc1 -2.8266 0.00042647
17 KNOWN Pde10a -2.817 0.00745532
7 KNOWN Cyp2s1 -2.81285 0.00699202
2 NOVEL Gm2026 -2.80316 2.96E-05
9 KNOWN Pxylp1 -2.80107 0.0087623
13 KNOWN Pde4d -2.77956 0.00252332
2 KNOWN St6galnac4 -2.77237 0.00226402
X KNOWN 6330419J24Rik -2.74675 9.10E-05
10 KNOWN Nuak1 -2.70903 2.53E-06
161

2 KNOWN Gm9864 -2.6946 0.00899114
7 KNOWN Pwwp2b -2.67833 0.000337235
5 KNOWN Fzd10 -2.65005 2.02E-06
19 KNOWN Vldlr -2.64546 0.000309742
4 KNOWN Prkaa2 -2.64446 0.00828903
19 KNOWN Gna14 -2.63967 0.00502077
8 KNOWN Dlc1 -2.63651 0.0065688
8 KNOWN Pdpr -2.63267 0.000431641
12 KNOWN Trim9 -2.63089 0.000448434
2 KNOWN Snai1 -2.62474 3.67E-08
3 KNOWN Lor -2.61436 0.00318927
11 KNOWN Hs3st3a1 -2.60846 7.61E-05
15 KNOWN Pdgfb -2.6063 0.0053179
11 KNOWN Cyb5d1 -2.5933 0.0072671
16 KNOWN Boc -2.59015 0.00025202
X KNOWN Chst7 -2.58576 3.56E-05
16 KNOWN B3gnt5 -2.5601 1.81E-06
2 KNOWN Itga8 -2.5522 0.00892614
1 KNOWN Ptgs2 -2.53467 0.00540198
17 KNOWN Lnpep -2.53043 0.00357601
2 KNOWN Mmp9 -2.52982 1.77E-08
15 KNOWN Shank3 -2.52288 0.000639695
6 KNOWN Zc3hav1l -2.521 0.000113366
1 KNOWN Hsd17b7 -2.51895 8.20E-05
14 KNOWN Gm2670 -2.47932 0.00187258
4 KNOWN Gabbr2 -2.4748 0.000298466
3 KNOWN Hey1 -2.46687 0.000160152
7 KNOWN Capn5 -2.46558 0.00231396
11 KNOWN Rel -2.46095 3.06E-05
11 KNOWN Wnt9b -2.45345 0.000674541
11 KNOWN Klhl11 -2.45318 0.000907191
6 KNOWN Tead4 -2.44809 0.00390838
X KNOWN Fam122b -2.44554 0.000123961
5 KNOWN Pcdh7 -2.44494 0.00319239
X KNOWN Zcchc12 -2.44379 0.00241147
1 KNOWN Slc39a10 -2.44036 6.41E-21
3 KNOWN Trpc3 -2.43372 0.000290311
14 KNOWN Dlg5 -2.43063 0.00598969
10 KNOWN Col13a1 -2.41978 0.00375268
9 KNOWN Clmp -2.40003 0.00749462
14 KNOWN Wdfy2 -2.3996 0.00995767
162

17 KNOWN Rasgrp3 -2.37533 0.00229373
9 KNOWN Prkar2a -2.37141 0.000624325
2 KNOWN Lrrn4 -2.3651 1.48E-13
16 KNOWN Hunk -2.34629 3.79E-05
16 KNOWN Phldb2 -2.3445 1.05E-06
9 KNOWN Chst2 -2.34358 0.00114601
4 KNOWN Mllt3 -2.34084 0.00346173
17 KNOWN Emilin2 -2.33814 0.00861564
18 KNOWN Gm26672 -2.33743 0.00144526
17 KNOWN Lama1 -2.33701 0.00378317
1 KNOWN Kif21b -2.33678 0.00935237
3 KNOWN Siah2 -2.32584 0.00961779
15 KNOWN Lmbrd2 -2.32431 0.00746793
X KNOWN Mum1l1 -2.31724 0.00248111
1 KNOWN Irs1 -2.28153 0.00136943
7 KNOWN Ube3a -2.25211 9.60E-07
13 KNOWN Ptch1 -2.22437 5.72E-05
2 KNOWN Adnp -2.22429 4.44E-08
6 KNOWN Itpr1 -2.21665 3.77E-08
2 KNOWN Pfkfb3 -2.21413 0.00233377
7 KNOWN Xylt1 -2.21114 0.000610157
1 KNOWN Gm10171 -2.20819 0.00807604
12 KNOWN Atad2b -2.19664 0.00392577
3 KNOWN SEMA6C -2.16532 0.000818085
15 KNOWN Zhx1 -2.16277 0.00230456
15 KNOWN Myc -2.16104 2.97E-06
16 KNOWN Dscam -2.14693 0.00430572
X KNOWN Gm8822 -2.13455 1.18E-14
1 KNOWN Syt14 -2.12538 0.000902761
4 KNOWN Ttc39a -2.12303 0.00279825
6 KNOWN Fbxl14 -2.10838 7.34E-13
7 KNOWN Tgfb1i1 -2.10048 0.00621395
12 KNOWN Btbd7 -2.0946 0.000390188
9 KNOWN Susd5 -2.09416 0.000304877
15 KNOWN Rnf19a -2.09406 3.85E-06
10 KNOWN Nt5dc3 -2.08942 0.0046886
2 KNOWN Nrarp -2.08793 3.94E-06
4 KNOWN Man1c1 -2.08752 0.000782528
11 KNOWN Arl4d -2.08349 0.000241271
14 KNOWN Dock9 -2.08322 0.000397389
7 KNOWN Dock1 -2.08198 1.53E-09
163

8 KNOWN Maf -2.08181 0.00429959
10 KNOWN Zfp365 -2.07699 0.00502742
7 KNOWN Anpep -2.07431 0.00200423
14 KNOWN Klf12 -2.06517 0.00265912
9 KNOWN Rab8b -2.06261 0.000535994
2 KNOWN Olfm1 -2.05626 2.16E-05
2 KNOWN Meis2 -2.0526 0.00100511
3 KNOWN Magi3 -2.04839 0.000191098
2 KNOWN Fam171a1 -2.04816 0.00100089
13 KNOWN Gas1 -2.03882 6.02E-09
7 KNOWN Cemip -2.03689 0.00577144
11 KNOWN Stat3 -2.0349 0.000515133
1 KNOWN Itpkb -2.0344 0.000481995
7 KNOWN Zfp568 -2.03092 1.86E-13
2 KNOWN Slc32a1 -2.01965 0.00496399
X KNOWN Sowahd -2.01411 0.00399987
6 KNOWN Nr2c2 -2.01374 0.00242639
2 KNOWN Cdh4 -2.00786 0.00136665
1 KNOWN Disp1 -2.00041 0.000271091
1 KNOWN Gm9747 2.00383 0.000946416
7 KNOWN Car11 2.00522 0.0006279
4 KNOWN Pnrc2 2.00745 1.84E-06
2 KNOWN Npdc1 2.01605 0.000675503
6 KNOWN Gm16042 2.01627 0.00206702
12 KNOWN 2410004P03Rik 2.01937 0.00207093
7 KNOWN Sbk1 2.02148 7.95E-07
3 KNOWN Dclk2 2.0218 0.00563461
4 KNOWN Enho 2.02287 0.00239007
12 NOVEL RP24-69I6.1 2.02298 0.0022114
2 KNOWN B2m 2.02438 0.00520629
4 KNOWN Slc2a5 2.02771 0.000343064
4 KNOWN Rpl22 2.03052 0.000268553
11 KNOWN Selm 2.03485 1.87E-06
4 KNOWN Tekt2 2.03572 0.000890172
10 KNOWN Tjp3 2.04249 0.00470613
13 KNOWN Marveld2 2.04403 0.00964921
5 KNOWN Hvcn1 2.04641 0.00277681
4 KNOWN Rpl11 2.05748 1.42E-06
19 KNOWN Gng3 2.06032 0.00887004
5 KNOWN Ppp1r35 2.06129 2.62E-19
19 KNOWN Tcf7l2 2.06399 0.00247602
164

5 KNOWN Hfm1 2.06529 0.000989118
18 KNOWN Rnf165 2.0721 0.00964993
19 KNOWN Ankrd13d 2.07725 0.000655401
X KNOWN Pls3 2.08036 2.15E-11
9 KNOWN Angptl6 2.08206 0.00268771
8 KNOWN Cotl1 2.08827 1.37E-05
4 KNOWN Gpn2 2.09485 0.00584111
1 KNOWN Rgs20 2.09526 0.000688109
6 KNOWN Col1a2 2.0966 0.00289883
4 KNOWN Hmgn2 2.09847 8.80E-17
19 KNOWN Hps1 2.1033 0.000326383
3 KNOWN Tpd52 2.10611 0.00638633
X KNOWN Gm21887 2.10952 0.00964914
17 KNOWN Dynlt1b 2.11941 1.08E-08
6 KNOWN Calcr 2.12051 0.000893495
15 KNOWN Mapk15 2.12114 0.00431333
15 KNOWN Cbx7 2.12284 0.00768574
17 KNOWN Epcam 2.12499 0.00482386
4 KNOWN Ahdc1 2.12549 1.56E-06
1 KNOWN 2810025M15Rik 2.12694 0.00971619
3 KNOWN Abcd3 2.1542 0.000711033
6 KNOWN Eno2 2.1548 0.000976327
4 KNOWN Gm13248 2.15587 5.08E-08
10 KNOWN Mar9. 2.16101 0.000185541
7 KNOWN Sepw1 2.16472 2.73E-09
4 KNOWN Gm2564 2.16878 0.0020556
5 KNOWN Sdsl 2.18296 0.00216669
18 KNOWN C330018D20Rik 2.19247 0.000521274
4 KNOWN Atpif1 2.19594 1.27E-10
4 NOVEL Gm20707 2.20118 3.65E-06
4 KNOWN Necap2 2.20333 0.000671459
11 KNOWN Ybx2 2.20549 0.000260558
15 KNOWN Slc1a3 2.20691 0.00417214
14 KNOWN Zfhx2 2.20961 0.00319042
14 KNOWN Slain1 2.21371 0.00188674
4 KNOWN Slc25a33 2.21386 0.00258106
7 KNOWN Klc3 2.21522 0.000100091
4 KNOWN Cda 2.22106 0.00698181
4 KNOWN Tprgl 2.22186 2.07E-12
4 KNOWN Atp13a2 2.22313 4.46E-06
5 KNOWN Prom1 2.22392 0.000155638
165

4 NOVEL Gm17300 2.22562 0.00710801
19 KNOWN Rps6ka4 2.23183 0.0038391
7 KNOWN Spint2 2.23321 0.000500912
4 KNOWN Park7 2.24058 3.29E-12
17 NOVEL Gm26917 2.24584 5.32E-05
15 KNOWN Sepp1 2.25043 0.00201889
9 KNOWN Lars2 2.2511 0.000138854
19 KNOWN Nrxn2 2.25116 0.00999708
2 KNOWN Trp53i11 2.25148 1.03E-08
19 KNOWN Vwa2 2.25267 0.0059441
X KNOWN Tspan7 2.25489 7.13E-05
1 KNOWN Pax3 2.25755 0.00109968
11 KNOWN Btbd17 2.25856 1.80E-05
X KNOWN Kctd12b 2.25971 5.71E-05
1 KNOWN Nfasc 2.26627 0.0035016
4 KNOWN Prkcz 2.26991 0.00417681
10 KNOWN Aes 2.27291 7.37E-05
14 KNOWN Shisa2 2.27309 0.00128219
4 KNOWN Crocc 2.2787 1.31E-06
8 KNOWN BC068157 2.28776 0.00406024
7 KNOWN Art5 2.29459 3.95E-05
17 KNOWN Crb3 2.29625 0.000229867
19 KNOWN Sptbn2 2.29731 7.37E-06
4 KNOWN Rnf207 2.29961 2.67E-05
16 KNOWN Cpped1 2.30944 0.0021757
5 KNOWN Ldb2 2.31162 0.000793874
4 KNOWN Tal2 2.31284 0.00749277
1 KNOWN Pgap1 2.32124 0.00014526
4 KNOWN Gm2163 2.32389 0.00123987
X KNOWN Hdac6 2.32446 0.0011099
3 KNOWN Pde5a 2.32542 4.39E-06
17 KNOWN AY036118 2.33149 0.00171888
3 KNOWN Ank2 2.33603 7.08E-05
17 KNOWN 0610011F06Rik 2.35108 0.00120153
4 KNOWN Rcan3 2.3578 0.00874351
15 KNOWN Panx2 2.36166 6.51E-05
3 KNOWN Gclm 2.38795 6.93E-05
X KNOWN Gpc4 2.40937 9.87E-06
X KNOWN Irs4 2.44258 0.00175946
15 KNOWN Pde1b 2.46616 0.00208889
4 KNOWN Tcea3 2.46682 0.000274849
166

11 KNOWN Hap1 2.47199 0.00815268
9 KNOWN Myl3 2.488 0.00770474
4 KNOWN Cnksr1 2.49234 1.57E-06
17 KNOWN Mapk13 2.49507 0.000726299
3 KNOWN Celsr2 2.49782 0.000291521
4 KNOWN Zdhhc18 2.50049 1.92E-05
17 KNOWN Fsd1 2.50299 0.00114171
4 KNOWN Stmn1 2.50475 0.000344531
1 KNOWN Fzd5 2.51468 1.26E-05
9 KNOWN Cmtm8 2.52263 1.68E-08
4 KNOWN Nphp4 2.5359 9.07E-09
7 KNOWN Cacng7 2.54084 2.32E-05
4 NOVEL Lrp8os2 2.54518 0.00199676
7 KNOWN Qprt 2.54716 0.00471613
2 KNOWN Rprm 2.54719 0.000260215
5 KNOWN Cldn3 2.55761 0.000143449
4 KNOWN Snhg3 2.56328 0.00452547
19 KNOWN Myrf 2.56448 2.04E-06
4 KNOWN Miip 2.56753 3.43E-05
X KNOWN Klhl4 2.60044 0.00711779
4 KNOWN Nipal3 2.60814 0.00658001
4 KNOWN Trnp1 2.60957 3.93E-09
19 KNOWN Malat1 2.61905 0.000133637
2 KNOWN Vstm2l 2.63104 0.00829632
4 KNOWN Camta1 2.6332 0.000117427
4 KNOWN Cntfr 2.66831 1.67E-05
5 KNOWN Nptx2 2.67327 0.00328201
5 KNOWN Crmp1 2.68171 0.00181099
X KNOWN Pcsk1n 2.69513 0.00174163
2 KNOWN Slc27a2 2.71595 0.000865392
9 KNOWN Adamts15 2.71603 7.74E-05
11 KNOWN Hist3h2ba 2.74113 1.09E-05
7 KNOWN Klk8 2.74392 0.0040887
1 KNOWN Cyb5r1 2.7466 0.000140317
7 NOVEL Fam160a2 2.75834 4.47E-11
15 KNOWN Col2a1 2.76901 3.01E-09
3 KNOWN Camk2d 2.78581 3.29E-07
4 KNOWN Megf6 2.8437 0.00431767
4 KNOWN Alpl 2.85318 0.00601326
1 KNOWN Gm26649 2.86179 0.00453046
4 KNOWN Arhgef19 2.89569 0.00474979
167

4 KNOWN Pdpn 2.90362 8.74E-05
6 KNOWN Gadd45a 2.9306 5.33E-05
9 KNOWN Rgl3 2.95257 0.000328543
7 KNOWN Spon1 2.962 0.00683883
4 KNOWN Espn 2.9963 0.00388315
19 KNOWN Dtx4 3.00433 0.00459369
4 KNOWN Snhg12 3.01796 3.85E-06
1 KNOWN Igfbp2 3.02573 0.00359192
7 KNOWN Fam57b 3.03524 0.00302586
4 KNOWN Arhgef16 3.06333 1.78E-05
18 KNOWN Sema6a 3.08633 0.0020556
16 KNOWN B4galt4 3.10379 0.00984834
11 KNOWN Foxj1 3.19497 0.00157747
4 KNOWN Acot7 3.2083 2.11E-05
4 KNOWN Penk 3.25517 0.0029439
14 KNOWN Fam213a 3.25702 0.000100152
17 KNOWN Ltb 3.31395 1.92E-05
3 KNOWN Pla2g12a 3.31493 2.51E-09
18 KNOWN Fzd8 3.32813 3.43E-06
2 KNOWN Wfdc2 3.33467 8.48E-05
X KNOWN Eras 3.45321 6.66E-06
11 KNOWN Ramp3 3.45508 0.00287591
11 KNOWN Pipox 3.45743 0.00491225
3 KNOWN 1810037I17Rik 3.47855 0.000626444
19 KNOWN Foxd4 3.49175 6.20E-07
17 NOVEL Yam1 3.51718 1.71E-05
8 PUTATIVE Gm20388 3.58193 0.000370361
4 KNOWN Mfap2 3.61764 5.82E-06
17 KNOWN Six3os1 3.6635 0.000314687
5 KNOWN Uchl1 3.67432 6.32E-07
3 KNOWN Rab25 3.73029 4.63E-05
4 KNOWN Gm13251 3.78902 0.000857607
7 KNOWN Coro1a 3.80277 0.00753711
4 KNOWN Fbxo44 3.88905 0.00694281
7 KNOWN Calca 3.89651 0.00460407
3 KNOWN Sox2 4.14277 0.00222643
17 KNOWN Tubb4a 4.38539 2.95E-07
4 KNOWN Foxd3 4.4507 2.10E-05
4 KNOWN 2610305D13Rik 4.49865 0.00340057
3 KNOWN Ugt8a 4.92999 0.00148497
10 KNOWN Enpp3 5.0205 0.00504393
168

17 KNOWN Six3 5.55088 1.05E-06
7 KNOWN Utf1 6.22199 0.000381312

Abstract (if available)

Abstract The ultimate goal of pluripotent stem cell research is to devise new and better treatments for disease. Specifically, the ability to regenerate tissues and organs to replace those that have been damaged has been the hope of many researchers and patients alike. Although there has been significant progress toward this goal, the efficient and safe generation of many tissues from pluripotent stem cells is still beyond our ability. The embryo is the blueprint for the derivation of every tissue, so the more accurately we can imitate this in vitro, the better the future patient outcomes will be. In this dissertation, I aim to increase the understanding of early embryogenesis and illuminate a previously undiscovered role for the gene BS69, which has never before been implicated in development. To accomplish this objective, I first examine the role of BS69 in early mouse embryogenesis. Next, I utilize the robustness of mouse embryonic stem cell technology to determine whether BS69 functions correspondingly in vitro and to uncover its global effects on gene transcription. Finally, by studying the differentiation capacity of cells that lack BS69, I determine its long-term effects on development. Here, I show that BS69 is required for embryonic development. BS69 facilitates the differentiation of epiblast into mesendoderm during gastrulation by preventing the premature differentiation of neuroectoderm. Although this phenotype shares striking similarity to the effects of Bmp pathway abrogation, I have been able to derive mouse embryonic stem cells that lack BS69, suggesting that Bmp signaling is intact. I find that BS69 is not required for mouse embryonic stem cell pluripotency or differentiation into epiblast, but it is necessary for the in vitro differentiation of mesendoderm. Furthermore, BS69 is required for the efficient differentiation of mesoderm-derived tissues and prevents the expansion and persistence of early neuroectoderm. Upon direct analysis of Bmp signaling in the absence of BS69, I find that the Bmp pathway is active. Although more work remains to clarify the mechanism by which BS69 functions, my findings suggest that it is required for gastrulation. Through revealing a previously unknown function of BS69, this dissertation has added to the understanding of early embryonic development. Future studies that investigate the effect of BS69 on the derivation efficiency of specific tissues in vitro may uncover its usefulness in stem cell research and regenerative medicine.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Pleotropic potential of Stat3 in determining self-renewal, apoptosis, and differentiation in mouse embryonic stem cells

PDF

The role of Prkci in stem cell maintenance and cell polarity using a 3-D culture system

PDF

Role of STAT3 phosphorylation in mouse embryonic stem cell self-renewal and differentiation

PDF

Role of beta-catenin in mouse epiblast stem cell, embryonic stem cell self-renewal and differentiation

PDF

Molecular basis of mouse epiblast stem cell and human embryonic stem cell self‐renewal

PDF

The role of ERK1/2 in mouse embryonic stem cell fate control

PDF

Novel roles for Maf1 in embryonic stem cell differentiation and adipogenesis

PDF

Investigating the role of STAT3 in mouse and rat embryonic stem cell self-renewal and differentiation

PDF

The role of α-catlulin during tumor progression and early mouse development

PDF

Characterization of new stem/progenitor cells in skin appendages

PDF

Hepatic differentiation in human naïve stem cells compared to human embryonic stem cells

PDF

Characterization of human embryonic stem cell derived retinal pigment epithelial cells for age-related macular degeneration

PDF

The calcium-sensing receptor in the specification of normal and malignant hematopoietic cell localization in the bone marrow microenvironment

PDF

Identification and characterization of adult stem cells in the oral cavity

PDF

Utilizing zebrafish and mouse models to uncover the underlying genetics of human craniofacial anomalies

PDF

Roles of Klf4 in embryonic stem cells

PDF

Tissue-specific action of Msx genes in the regulation of skull vault development

PDF

Role of the bone marrow niche components in B cell malignancies

PDF

The histone H2A deubiquitinase MYSM1 regulates CCR9 expression on CD4⁺ T cells and thymocytes

PDF

The cancer stem-like phenotype: therapeutics, phenotypic plasticity and mechanistic studies

Asset Metadata

Creator Rippen, Marie (author)

Core Title The function of BS69 in mouse embryogenesis and embryonic stem cell differentiation

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 07/24/2017

Defense Date 06/18/2015

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag BS69,differentiation,embryogenesis,gastrulation,OAI-PMH Harvest

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Maxson, Robert E., Jr. (committee chair), Kobielak, Krzysztof (committee member), Warburton, David (committee member)

Creator Email marie.rippen@gmail.com,rippen@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-607259

Unique identifier UC11299785

Identifier etd-RippenMari-3706.pdf (filename),usctheses-c3-607259 (legacy record id)

Legacy Identifier etd-RippenMari-3706.pdf

Dmrecord 607259

Document Type Dissertation

Format application/pdf (imt)

Rights Rippen, Marie

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA