University of Southern California Dissertations and Theses

Retinoic acid and TGFβ signaling regulate cardiovascular development

(USC Thesis Other)

Retinoic acid and TGFβ signaling regulate cardiovascular development

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content

Retinoic acid and TGFβ signaling regulate cardiovascular development

by

Man Cheong Ma
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
August 2013

1

ACKNOWLEDGEMENTS

I would like to use this opportunity to thank my mentor, Dr. Henry Sucov, for his guidance and
support throughout my two years of graduate work. His passion and skeptical scientific attitude
have guided me towards critical thinking and independent scientific research. I would also like to
thank my committee members, Dr. Ching-Ling Lien and Dr. Mohammad Pashmforoush, for their
helpful suggestions and the former and current lab members from Dr. Sucov’s lab for their
discussion, encouragement, and contribution to the studies on TGFβ in mediating outflow tract
septation, especially Dr. Peng Li, Dr. Jingjing Zhou, and Dr. Bibha Choudhary. Last but not least,
I would like to express my deepest sincerity in thanking my parents. Without their full support
and love, I could not have pursued my dream freely.

2

TABLE OF CONTENTS
ACKNOWLEDGEMENTS 1
LIST OF TABLES 3
LIST OF FIGURES 4
ABSTRACT 5

CHAPTER 1: GENERAL INTRODUCTION 7
1.1 Morphogenesis and cell lineage of the heart 7
1.2 Heart outflow tract development 8
1.3 The role of retinoid signaling in cardiogenesis 9
1.4 TGFβ signaling and congenital heart disease 10
1.5 Retinoic Acid regulates differentiation of the SHF and TGFβ - 11
mediated OFT septation
1.6 Cardiovascular malformations in neural crest-specific type 2 11
TGFβ receptor mutant mice
CHAPTER 2: EXPERIMENTAL DESIGN AND PREPARATION 13
2.1 Mouse lines 13
2.2 Phenotype analysis 13
2.3 LacZ staining 13
CHAPTER 3: ECTOPIC ENDOCARDIAL EMT IS CAUSATIVE 15
FOR OFT SEPTATION FAILURE IN RA MUTANTS
3.1 Abstract 15
3.2 Introduction 16
3.3 Results 18
3.3.1 Altered endocardium specific TGFβ signaling accounts for 18
septation but not arch artery defects in RAR mutants
3.3.2 Ectopic mesenchymal transformation of the endocardium in the 22
distal OFT is causative specifically for compromised septation
3.4 Discussion 24
CHAPTER 4: TGFβ ACTIVATED KINASE 1 (TAK1) SIGNALING IN 26
NEURAL CREST CELLS MEDIATES OFT SEPTATION
4.1 Abstract 25
4.2 Introduction 26
4.3 Results: Tak1 Deficiency in the cNCC Lineage Leads to 27
Septation failure
4.4 Discussion 31
BIBLIOGRAPHY 33

3

LIST OF TABLES
Table1: 19
Rescue of outflow tract septation in RARα
1/RARβ mutants by specific reduction of
endothelial Tgfbr2 gene dosage
20
Table 2:
Arch artery defects at E14.5 assessed by
histology analysis
Table 3: 29
Deletion of Tak1 in cNCC lineage leads to
septation failure

4

LIST OF FIGURES
Fig1. Altered endothelial TGFβ signaling 21
rescued septation defect in RAR mutants
Fig 2. Rescue for ectopic EMT in RAR 23
mutants
Fig3. Altered Tak1 signaling pathway in cNCC 30
results in CAT and cleft palate
5

ABSTRACT

Congenital heart disease (CHD) occurs in approximately twelve out of 1000 live births
(Hoffman et al, 2004), and is the main source of infant mortality and morbidity. Additionally,
about three per 1000 live births will require some intervention during the first year of life (Gruber
and Epstein 2004). Thus, it is very important to maintain a tight regulation of the various
signaling pathways and different interactions between cell lineages in order to develop a healthy
and properly functional four chambered heart. With a better understanding of genetic regulation,
we will be able to identify the mechanisms behind this complex morphogenesis and ultimately
provide better insight into cardiovascular diseases. We used genetically modified mouse models
to primarily study the malformation called common arterial trunk (CAT) throughout this thesis.
Retinoic acid (RA), the biologically active derivative of vitamin A, is an important
morphogen during development. Deficiency in nutritional vitamin A and mutation in the nuclear
receptor for RA will result in CHD. Nearly one third of the CHD involves outflow tract (OFT)
malformations. For example, in retinoic acid receptor (RAR) mutants, SHF defects (elongation
and misalignment defects) occurred due to the deficiency in recruitment surrounding progenitor
cells into the OFT . Consequently, the OFT of RAR mutants has a misspecified proximal-distal
axis, and the proximal markers MLC2v and TGFβ2 are both ectopically expressed in the distal
OFT. In addition, the RAR mutants also experience a variety of pharyngeal arch artery and great
vessel defects. Using various tissue-specific genetic rescue approaches, we show that the
endothelial specific reduction of TGFβ receptor 2 (Tgfbr2) gene dosage in the RAR mutant
background restores proper septation in the OFT, but does not rescue arch artery defects and
alignment defects, demonstrating that the endocardium is the responsive tissue to excess TGFβ2,
and that arch artery formation and alignment defects are both independent of TGFβ signaling.
In addition to the migration of the SHF into the OFT, cardiac neural crest cells also
6

migrate towards the OFT at about E9.5-E10.0. These cells are required to initiate the formation
of the aorticopulmonary (A/P) septum (Jiang et al, 2000), which divides the aortic sac into aorta
and pulmonary vessels. Upon deletion of Tgfbr2 in neural crest cells (NCC) in our mouse model,
OFT septation fails and results in CAT, despite the normal migration pattern of NCC into the
OFT. Although this model also involves altered TGFβ signaling, the cellular basis of the CAT
phenotype in this model is quite different from the retinoic acid mutant. CAT in this mutant
background may be mediated by the non-canonical TGFβ signaling pathway. Using a neural
crest tissue specific mutant approach, we were able to recapitulate the CAT phenotype in the
Wnt1Cre;Tgfbr2 mutant by knocking out TAK1 protein in a tissue-specific way. This result
suggested that TGFβ response in the neural crest cell may be mediated by TAK signaling.

7

CHAPTER 1

GENERAL INTRODUCTION

1.1 Morphogenesis and cell lineage of the heart

Proper morphogenesis of the heart is essential because the heart is the first organ to be
developed and functionalized in the embryo. The formation of the mature vertebrate heart is
driven by several cell lineages: primary heart field, second heart field, cardiac neural crest, and
epicardial cells derived from the proepicardial organ (Srivastava 2006). The development of
the heart is a complicated process that requires the tight regulation of signaling pathways and
the integration of different cell lineages at unique domains as development continues.
During the early stage in heart development, the cardiac crescent (first heart field, aka-
FHF) moves anterior-laterally to form bilateral paired cardiogenic plates in the mouse embryo at
E7.5. Subjacent to these plates, endothelial cells differentiate and form the right and left
endocardial heart tubes (Kaufman and Bard 1999). These endothelial-lined vessels align in a
parallel fashion against each other and then fuse across the ventral midline to form a single
beating heart tube by E8.0. A regular heartbeat is established by E9.0. (Kaufman and Navaratnam
1981). At this stage, the linear heart tube consists of two layers of cells: the endocardium,
equivalent to the inner layer of the heart tube, and an exterior layer of myocardium. The FHF
mainly contributes to the left ventricle and the atria, but not the right ventricle or the outflow tract
(OFT).
When the heart tubes form, another cell lineage known as the second heart field (SHF),
which is a progenitor population of splanchnic and pharyngeal mesoderm that is located dorsal
to the pericardial cavity, migrates into the midline dorsally to the heart tube in the pharyngeal
mesoderm during E8-10.5 (Horsthuis et al, 2009; Kelly et al, 2001; Ward et al, 2005). Upon
rightward looping of the heart tube, SHF cells cross the pharyngeal mesoderm into the anterior
8

and posterior portions and populate a large portion of the OFT, the future right ventricle, and the
atria (Cai et al, 2003).
At approximately E9.5, cardiac neural crest cells (cNCC), originating from the lower
hindbrain between the optic placode and fourth somite (Creazzo et al, 1998;Martinsen et al,
1998), undergo epithelial mesenchymal transition (EMT) and migrate towards the heart through
the third, fourth, and sixth pharyngeal arches (Kirby et al, 2002). At E10.5, the cNCCs are
located between the endothelium and splanchnic mesoderm of the aortic sac. By E11, these cells
are required to initiate the formation of aorticopulmonary (A/P) septum (Jiang et al, 2000),
which divides the aortic sac into aorta and pulmonary vessels.
The fourth lineage of cardiac precursor cells is derived from the proepicardium (PE), which
migrates and covers the external surface of the entire heart, including the OFT, after the looping
in E9.0-11.0 (Komiyanma et al, 1987). Additionally, the epicardial cells undergo EMT and
contribute to cardiac fibroblasts, which are coronary vascular support cells (Cai et al, 2008). At
this stage, the heart consists of the following three layers: the outer epicardium layer for
protection, the inner epithelial layer as endocardium, and the middle cardiac muscle layer as
myocardium. With valve formation and septation between heart chambers, the heart has finally
become a four-chambered organ.
1.2 Heart outflow tract development

When the primitive heart tube forms in the midline through the coagulation of the FHF,
the SHF cells migrate to the heart tube and begin to be recruited from both ends. Myocardium
from the pharyngeal arch region adds to the lengthening OFT after the formation of the initial
heart tube (Kirby, 2002; Kirby, 2007). The addition of SHF-derived myocardium to the OFT
results in its elongation, which is necessary to allow the OFT to rotate sufficiently for the correct
alignment of the pulmonary and aortic trunks with their respective ventricles. In the outflow tract,
9

outflow tract cushions of the endocardium arise at E9.5 (Kisanuki et al, 2001; Mjaatvedt and
Markwald, 1989). The OFT cushions are located between the endocardium and myocardium, and
are formed by the infiltration of mesenchymal cells. The swelling of the cushions will contribute
to all septal and valvular structures that are essential to produce the four chambers of the heart as
well as the two separate outflow channels, the aorta and the pulmonary trunk (Fananapazir and
Kaufman, 1988). CNCCs also migrate through the anterior SHF into the outflow tract where they
also contribute to the endocardial cushions, while endodermal-derived mesenchymal cells are
located specifically in the proximal cushions. The migration and presence of the cNCCs are also
necessary for the septation of the OFT. In the absence of the neural crest cells, OFT septation and
arterial pole maturation are compromised (Hutson and Kirby, 2007).
1.3 The role of retinoid signaling in cardiogenesis

Retinoic Acid (RA) is a vitamin A derivative that is widely used as a signaling molecule
in vertebrate development (Li et al, 2012). Vitamin A is converted to retinol by retinol
acyltransferase and further converted to all-trans RA by retinal dehydrogenase (RALDH)
(Duester, 2000). Regulation of RA metabolism is mediated through specific RA receptors
inside the cell. Nuclear hormone receptors, RA receptors (RARs) and retinoid X receptors
(RXRs), will bind to each other to form an active receptor. There are three distinct RAR genes
and three distinct RXR genes, identified as α, β, and γ, each with 2 different isoforms expressed
in human and mice. The gene disruption of RA receptors or Raldh2 (Lee et al, 1997;
Mendelsohn et al, 1994) and vitamin A deficiency in rats (Wilson and Warkany, 1949) have
shown the importance of RA signaling in cardiovascular development, and in other organs as
well. Upon exposure of excess RA through maternal gavage around E9.0, the E10.0 embryos
showed underdeveloped atrioventricular cushions with fewer mesenchymal cells (Davis and
Sadler, 1981). RARα 1/RARβ and RARα 1/RARγ mutant embryos exhibit a single arterial
10

trunk (CAT), representing outflow tract septation defects. These mutants are also compromised
in the OFT alignment. Therefore, retinoid signaling plays an important role in regulating proper
formation and remodeling of the endocardial cushions during outflow tract septation as well as
chamber formation.
1.4 TGFβ signaling and congenital heart disease

TGFβ signaling regulates essential cell responses including proliferation, migration,
differentiation, and apoptosis. The precise control of the TGFβ signaling pathway is required for
the accurate development of the four-chambered human heart. To initiate signaling, the secreted
TGFβ ligands (TGFβ1, TGFβ2, and TGFβ3) must first bind to the TGFβ receptor 2 (TGFβR2)
on the cell surface. This binding further activates TGFβR1, which then phosphorylates SMAD2
and SMAD3 proteins. Phosphorylated SMAD2 and SMAD3 will then associate with SMAD4
and translocate into the nucleus to mediate gene expression. In mice, TGFβ1 is expressed in
endocardial-enothelial cells around E8.0 (Akhurst et al, 1990), while TGFβ2 is expressed in the
myocardium and outflow tract around E9.75 (Camenisch et al, 2002; Molin et al, 2003; Li et al,
2010). TGFβ3 is expressed in the cushion mesenchymal cells at a later stage, around E11.0
(Camenisch et al, 2002; Molin et al, 2003). Both Tgfbr1 and Tgfbr2 are expressed in the
primitive heart tube and in the myocardium, cushion mesenchyme, and endothelial cells (Lawler
et al, 1994; Mariano et al, 1998; Mummery 2001). It has been shown that TGFβ positively
promotes endocardial mesenchymal transformation in vitro (Brown et al, 1996; Eisenberg &
Markwald, 1995). In these studies (Dickson et al, 1995; Kaartinen et al, 1995), Tgfb1 -/- and
Tgfb3 -/- mutant embryos show no obvious cardiac defects. Since TGFβ2 is expressed in the
cushion myocardium and the cushion mesenchyme, and since Tgfb2-/- mutants have CHD with
major defects in the OFT cushion (Sanford et al, 1997), TGFβ2 seems to be a potential candidate
to regulate the EMT process for cushion formation. In addition to the SMAD-dependent
11

canonical signaling pathway, there is also non-SMAD signaling through Tak1/p38, which
signifies the importance of regulating these cellular response (Zhang, 2009).
1.5 Retinoic Acid regulates the differentiation of SHF and TGFβ-mediated OFT septation
Our lab has previously shown that the OFT phenotype of RAR mutants is a combination
of a deficiency in OFT lengthening and a failure in septation (Li et al, 2010). RA signaling is
required during E9.0-10.5 to promote the commitment and replenishment of Isl1
+
and Nkx2.5
+

progenitors to a Mef2c
+
fate. Due to the disruption of RA signaling, this recruitment process fails
in the RAR mutants. As a result, the OFT is shortened and ultimately misaligned. Since the
addition of Mef2c
+
cells to the end of the OFT is disrupted, the tissue that remains in the distal
OFT maintains a proximal identity, as evidenced by the expression of the proximal genes MLC2v
and Tgfb2. Excess TGFβ2 is the main cause for septation defects because reduction of
Tgfb2 gene dosage in the RAR mutants restores septation, but does not rescue the alignment
defects. Thus, the CAT septation defect results from altered TGFβ signaling, which in turn is due
to the misspecification of outflow tract. Furthermore, Li et al (2010) also showed that excess
TGFβ2 may be a potential candidate for explaining the ectopic location of endocardium-
mesenchymal transformation in the distal OFT. Although neural crest derived mesenchymal
cells also migrate into the cushions, endocardium-derived mesenchymal cells are only located
in the proximal cushions. With normal neural crest migration and differentiation in the RAR
mutants (Jiang et al, 2002), Li et al. (2010) used Tie2Cre/R26R mice to examine the
endocaridum-derived population, and showed ectopic enodcardium-derived mesenchymal
cells in the distal OFT where such cells are not normally located. Although our previous
results suggested that ectopic endocardial EMT correlates with the septation failure, it
remained possible that the neural crest cells in the OFT may respond to the excess TGFβ.

12

1.6 Cardiovascular malformations in neural crest-specific type 2 TGFβ receptor mutant
mice

In order to address the interaction between TGFβ and its receptors in the neural crest
population, our lab has previously crossed a conditional Tgfbr2 allele (Chytil et al, 2002) with the
Wnt1cre transgene (Danielian et al,, 1998). We observed cardiac defects and perinatal lethality in
neural crest-specific loss of Tgfbr2 mutants (Choudhary et al, 2006). In the study, NCC migration
into the OFT cushions and smooth muscle differentiation were normal in Wnt1cre/Tgfbr2
mutants, but there were defects in OFT cushion maturation and remodeling. Consequently, there
was a 100% penetrance of the CAT phenotype, which implied that neural crest cells require
TGFβ signaling to promote proper septation of the aorta and pulmonary trunk. Beside the CAT
phenotype, Choudhary et al. (2006) also report a highly penetrant interrupted aortic arch
phenotype due to improper regression of the left fourth pharyngeal arch artery with no
parathyroid gland defects. These results together confirm the importance of TGFβ signaling in
cNCCs for aortopulmonary septation (Arthur and Bamforth, 2011). However, the mechanism by
which the CAT phenotype arises in the Wnt1Cre/Tgfbr2 mutant is still not completely
understood.

13

CHAPTER 2

EXPERIMENTAL DESIGN AND PREPARATION

2.1 Mouse lines

All mouse lines used in this study have been previously mentioned: RARα 1 null (Li et al,
1993), RARβ null (Luo et al, 1995), R26R (Soriano, 1999), Tie2Cre (Kisanuki et al, 2001),
conditional Tgfbr2 (Chytil et al, 2002), conditional Tak1 (Sato et al, 2005), and Wnt1Cre
(Danielian et al, 1998). Our mating strategy in generating experimental embryos involves crosses
between RARα 1-/-, RARβ-/+ adults, often bearing heterozygous Cre, homozygous R26R alleles,
and homozygous conditional Tgfbr2 alleles. This is done to obtain a reasonable percentage of
experimental embryos per litter. Although the resulting littermate controls are
RARα 1-/- rather than wild type, as noted in the text, the absence of RARα 1 alone does not
result in any phenotype. All experiments with mice were performed in accordance with relevant
institutional and national guidelines as well as regulations that are approved by the USC
IACUC committee.
2.2 Phenotype Analysis

Genomic yolk sac DNA was isolated and genotyped by PCR. Embryos were isolated at
E14.5, fixed, embedded in paraffin, and sectioned 10 μm apart; serial sections were then
hematoxylin and eosin stained by standard procedures. CAT (common arterial trunk) was defined
by a single outflow vessel. In embryos where septation had occurred, the position of the aortic
outflow initiated from the right ventricle determined the diagnosis of DORV (double outlet right
ventricle). Serial sections were analyzed for arch arteries defects, and all embryos with CAT,
DORV, or arch arteries defects also had a ventricular septal defect, which is a hemodynamic
requirement of these phenotypes.

14

2.3 LacZ staining

Embryos were isolated in PBS, fixed in 0.2% glutaraldehyde for 5-20 minutes (stage-
dependent), and stained in X-gal staining solution as whole mount preparations. Embryos were
then embedded and paraffin sectioned. The staining solution contained potassium ferricyanide
(5mM), potassium ferrocyanide (5mM), magnesium chloride (2mM), and X-gal (1mg/ml) in
PBS.

15

CHAPTER 3

ECTOPIC ENDOCARDIAL EMT IS CAUSATIVE FOR OFT SEPTATION FAILURE
IN RA MUTANTS

3.1 Abstract

In RAR mutants, the SHF defects, including OFT shortening and misaligned outflow vessels,
are molecularly, genetically, and temporally distinguished from the septation defect in the OFT.
During E10.5, both cardiac neural crest cells and endothelial derived mesenchymal cells populate the
OFT. However, what remains unknown is which tissue(s) in the OFT is/are responsive to excess
TGFβ, which can thereby explain this phenotype. By using a genetic rescue approach in the retinoic
acid signaling mutant background, we found that the endocardium (the endothelium of the heart and
OFT) is the critical responsive tissue, and further confirmed that ectopic EMT in the distal segment
of the OFT is the main cause of the septation defect.

16

3.2 Introduction

During the early stages in heart development, there are two layers of epithelial cells in the
primitive heart tube: the inner endothelium (the future endocardium) and the outer myocardium.
These two layers are separated by the extracellular matrix termed cardiac jelly. During heart
looping, the myocardium induces the endothelium cells to undergo EMT – a process involving
the delamination of the epithelial cells and their transformation into mesenchymal cells, which
can migrate into the cushion matrix. This transition involves loss of apical-basal polarity and
disruption of cell-cell junctions while gaining a migratory phenotype and expression of
mesenchymal markers (Helen and Simon, 2011). The cardiac jelly will eventually develop into
the valves of the heart and facilitate the septation process of the OFT.
There are two origins for the mesenchymal cells in the OFT cushion: (1) the mesodermal
cells derived by endocardium EMT and (2) the migratory neural crest cells. Before the NCC
migrates into the cushion, the endocardial OFT cushions form as two opposing ridges, and can be
divided into proximal and distal components by an anatomical feature called the dogleg bend
(Webb et al, 2003). cNCCs migrate through the pharyngeal arches (third, fourth, and sixth) to the
aortic sac. By E11.5, the neural crest-derived cells have protruded into the dorsal wall of the
aortic sac, dividing the distal OFT into aortic and pulmonary channels (Saija et al, 2009), with the
cNCCs differentiating into smooth muscle cells in the arterial pole of the heart. The OFT is
initially a single tube without division, but the formation of the aorticopulmonary (A/P) septum
by the cCNCC divides the OFT into aortic and pulmonary vessels. The septation of the OFT is
completed by the A/P septum at around E11.5. A failure to form the A/P septum results in a heart
malformation known as CAT phenotype, in which the single outflow vessel persists.
Problems in the second heart field can also result in compromised outflow tract septation.
We previously mentioned that embryos lacking the α isoform of the RARα gene plus all isoforms
17

of the RARβ gene (RARα1/RARβ) have CAT and arch arteries defects with 100% penetrance. As
described in chapter 3.1, the OFT phenotype of RAR mutants results from two independent
processes. We showed that the CAT septation defect results specifically from altered TGFβ
signaling, which is an indirect consequence of outflow tract axial misspecification. Since Tgfb2 is
expressed by the myocardium in regions of EMT (proximal region of the OFT), and Tgfb2 null
mice have CHD with compromised OFT development, the excess TGFβ in the distal OFT (in the
RAR mutants) is a potential candidate for ectopic EMT in the distal segment, which does not
normally happen. Global reduction of Tgfb2 gene dosage in the RAR mutant background restored
proper septation in 50% of the embryos, though the alignment defect was not rescued. This result
implies that excess TGFβ is causative for septation defects but not alignment defects. During the
septation process, two different cell lineages, the endocardium-derived mesenchymal cells and
the neural crest-derived mesenchymal cells, are present in the OFT cushion. Both of the cell
lineages may respond to excess TGFβ and compromise the septation. In order to examine the
relative importance and role of endocardium-derived and neural crest-derived mesenchyme in the
overall process of outflow tract septation, we used a tissue-specific genetic rescue approach by
conditionally knocking out half of the Tgfbr2 gene dosage in either cell lineage. We showed that
endocardium-derived mesenchymal cells are the responsive lineage to excess TGFβ, and
conclude that excess TGFβ induces the endocardium to undergo ectopic epithelial-mesenchymal
transformation, which is the main cause of distortion of the OFT that is incompatible with normal
septation. Finally, the reduction of TGFβR2 in the endothelial cells (including the endocardium)
only restores septation, and does not rescue the alignment and arch artery defects, which implies
that excess TGFβ is only causative for compromised septation in the OFT.

18

3.3 Results:

3.3.1 Altered endocardium specific TGFβ signaling accounts for septation, but not arch
artery defects in RAR mutants

To address whether endocardium-derived or neural crest-derived mesenchyme would be
responsive to ectopic TGFβ expression and cause the OFT phenotypes, we decided to identify
which tissue in the OFT is responsive to excess TGFβ. We used a genetic rescue approach that
involved several different tissue-specific Cre lines (Tie2Cre-endothelial tissue specific and
Wnt1Cre-neural crest tissue specific) combined with a heterozygous Tgfbr2 (TGFβ-receptor 2)
conditional allele in the retinoic acid signaling mutant background. As previously stated, CAT is
a 100% penetrate phenotype in RAR mutants, and the single OFT originates from the right
ventricle. Additionally, the RAR mutants also have 100% penetrance of arch artery defects.
However, by specifically reducing the Tgfbr2 gene dosage by half in endothelial cells, including
the endocardium in the OFT, normal septation was restored in fifty percent of the embryos.
Reducing Tgfbr2 gene dosage specifically in the neural crest cells did not rescue the septation
defect in the RAR mutants (Table 1, Fig 1 A-D). In support of the previous results from our lab
(Li et al, 2010), the misalignment and lengthening of the OFT defects in RAR mutants are
molecularly distinct from septation defects. Consequently, for the rescued cases, the ascending
aorta always initiated from the right ventricle, resulting in the Double Outlet Right Ventricle
(DORV) phenotype (Table 1, Fig. 1C). Importantly, the arch artery defects were not rescued by
altering either the endothelial or neural crest Tgfbr2 gene dosage (Table 1, Table 2). This result
indicates that arch artery morphogenesis is independent of TGFβ signaling in the RAR mutants.
Taken together, these results show that the endocardium of the OFT is responding to the
excessive TGFβ in the proximal outflow tract and is the cause of the septation defects in the
RAR mutants.

19

Table1. Rescue of outflow tract septation in RARα 1/RARβ mutants by specific reduction
of endothelial TGFβR2 gene dosage

All embryos were isolated at E14.5, embedded in paraffin, and hematoxylin and eosin stained for
histology sections. Serial sections were collected and underwent cardiovascular defects analysis.
CAT (common arterial trunk) has a single outflow vessel. In DORV embryos, septation
occurred, but both outflow vessels initiated from the right ventricle. Arch artery defects result
from malformation and reorganization failure of the pharyngeal arch arteries into great vessels.
All abnormal phenotypes also included ventricular septal defect.

*The complete penetrance of CAT in germline RARα 1/RARβ embryos is based on 2 embryos
from the current study, plus many from earlier analyses, totaling over 100 embryos.

Genotype Mutants Litters CAT DORV VSD Arch Artery Normal
Defects

RARα 1-/-, RARβ -/- * * * 100% 0 0% * 100% * 100% 0 0%

Tie2Cre, 3 3 3 100% 0 0% 3 100% 3 100% 0 0%
RARα 1-/-, RARβ -/-

RARα 1-/-, RARβ -/-, 6 6 6 100% 0 0% 6 100% 6 100% 0 0%
Tgfbr2fl/+

Tie2Cre, 10 8 5 50% 5 50% 1 100% 10 100% 0 0%
RARα 1-/-, RARβ -/-, 0
Tgfbr2fl/+

Wnt1Cre, 7 6 7 100% 0 0% 7 100% 7 100% 0 0%
RARα 1-/-, RARβ -/-,
Tgfbr2fl/+

20

Table 2. Arch artery defects at E14.5 assessed by histology analysis

Embryos used in this analysis were the same embryos in Table 1, which all have arch artery
defects, and were further categorized into different arch artery abnormalities. For a given
phenotype, A-J designates different embryos.
Genotype Aortic arch abnormalities outflow

tract

E14.5 Right-sided Aberrant Missing Missing IAA-B R.retroes. L.retroes.

defects

embryos aorta origin of R/L L. pulm R. pulm

subcl. subcl.

pulm art. Art. Art

RARα 1-/-, A x x CAT

RARβ -/-

B x x CAT

Tie2Cre, A x CAT

RARα 1-/-,

RARβ -/-

B x x CAT

C x x CAT

RARα 1-/-, A x x x CAT

RARβ -/-,

Tgfbr2fl/+

B x x x x CAT

C x CAT

D x x CAT

E x CAT

F x x x CAT

Tie2Cre, A x x x CAT

RARα 1-/-,

RARβ -/-,

Tgfbr2fl/+

B x x CAT

C x CAT

D x CAT

E x CAT

F x DORV

G x DORV

H x x DORV

I x DORV

J x DORV

Wnt1Cre, A x x x x CAT

RARα1-/-,

RARβ -/-,

Tgfbr2fl/+

B x CAT

C x x x CAT

D x x x x CAT

E x x CAT

F x CAT

G x CAT

Embryos F-J with Tie2Cre, RARα 1-/-, RARβ -/-, Tgfbr2fl/+ genotype are the rescued embryos
with restored septation. Abbreviations: R. pulm. art. and L. pulm art., right and left pulmonary
artery, respectively; IAA-B, interrupted aortic arch; R. and L. retroes. subcl.,right and left
retroesophageal subclavian artery, respectively; CAT, common arterial trunk; DORV, double
outlet right ventricle.

21

Fig1. Altered endothelial -TGFβ signaling rescued septation defect in RAR mutants

H&E staining for E14.5 histology sections. A-D. Rescue of septation defects in E14.5 RAR
mutants by specifically reduced Tgfbr2 gene dosage in endocardium: Shown are (A) a normal
control embryo with normally septated ascending aorta and pulmonary trunk (B) a RARα
1/RARβ mutant with CAT (C) a Tie2Cre/ RARα 1/RARβ /Tgfbr2fl/+ mutant with rescued
septation, and (D) Wnt1Cre/RARα1/RARβ/Tgfbr2fl/+ mutant with CAT. Arrows show arch
artery defects (Right-sided aorta). Abbreviations: RV, right ventricle; Ao, ascending aorta; PT,
pulmonary trunk; CAT, common arterial trunk.

A

B

dAo

Ao

PT

RV

Control

dAo

RV

RARα 1-/-, RARβ -/-

CAT

C

D

dAo

dAo

PT

Ao

CAT

RV

Tie2Cre, RARα 1-/-, RARβ -/-,
Tgfbr2fl/+

Wnt1Cre, RARα 1-/-, RARβ -/-,
Tgfbr2fl/+

22

3.3.2 Ectopic mesenchymal transformation of the endocardium in the distal OFT is
causative specifically for compromised septation

Proper formation and remodeling of the endocardial cushion are significant for normal
OFT septation (Hoover et al, 2008). In normal embryos, endocardium-derived cells are
specifically located in proximal cushions, and neural crest derived cells migrate throughout the
cushions. Using the Wnt1Cre/R26R as a neural crest cell lineage marker, it was previously
demonstrated that the number, migration, distribution, spiral rotation, and ultimate differentiation
of neural crest cells were normal in RARα 1/RARβ mutant embryos (Jiang et al, 2002).
Therefore, we have hypothesized that excessive TGFβ in the mutants would induce the
endocardium to undergo ectopic epithelial-mesenchymal transformation, which causes a
distortion of the OFT, incompatible with normal septation. To address this issue, we performed
LacZ-staining on E10.5 mutant embryos with Tie2Cre –endothelial specific Cre lines combined
with a heterozygous Tgfbr2 (TGFβ receptor) conditional allele and R26R homozygous allele in
the retinoic acid signaling mutant background to specifically mark the endothelial-derived
mesenchymal cells (Fig 2 A-C). This approach indicated that reducing the endothelial- TGFβ
signaling in the OFT of RAR mutants was able to rescue the ectopic endocardium-derived
mesenchymal cells, which previously appeared in the distal segment of the outflow tract in
mutants (Li et al, 2010). This result further supports our hypothesis that the ectopic EMT of the
enodcardium in the distal OFT is the causative event for compromised septation.

23

Fig 2. Rescue for ectopic EMT in RAR mutants

The gray lines in panels A-C are positioned at the 90 degree bend that defines the boundary
between the proximal and distal segments of the outflow tract. A-C E10.5 control and
littermate mutant embryos all bearing Tie2Cre/R26R were isolated, sectioned and X-gal
stained; arrows point to endocardium-derived mesenchyme, which is mostly restricted in the
control and rescued embryos to the proximal OFT but present in mutant embryos in both
segments.

RARα 1-/- RARα 1-/- RARβ -/+ RARα 1-/- RARβ -/-

RARβ -/-
Tie2Cre/R26R
Tgfbr2fl/+
Tie2Cre/R26R
Tgfbr2fl/+
Tie2Cre/R26R

A

B

C

24

3.4 Discussion

In RAR mutants, the lack of replenishment of additional splanchnic mesoderm progenitors to
a Mef2c-positive fate is the cause of the SHF defect, which includes shortening and misalignment of
the OFT. Due to this shortening of the OFT, the cells normally fated to be the proximal OFT remain
at the distal segment. Moreover, these cells ectopically express the proximal markers MLC2v and
Tgfb2 in the distal segment of the OFT (Li et al, 2010). Although we have shown previously that this
ectopic expression of TGFβ in the outflow tract is causative for septation defects while the SHF
defect is independent to the TGFβ signaling, it is still relevant that the neural crest cells might also
respond to the ectopic TGFβ. Hence, the results in Chapter 3 demonstrated that the endocardium is
the critical responsive tissue to this ectopic TGFβ based on our ability to rescue 50% of the CAT
phenotype by altering the endothelial-Tgfbr2 gene dosage, while altering TGFβ signaling in the
neural crest cells; this did not rescue the CAT phenotype. In all the rescued cases, we have still
observed the DORV phenotype, which supports the previous analyses that the septation defect and
SHF defects are the results of two independent processes. Our results further suggest that the cause
for compromised OFT is the ectopic EMT in the distal segment of the OFT, as we have shown normal
EMT pattern in the rescued RAR mutants. Even though we still need to increase the number of
rescued EMT RAR mutants, we are assuming that the rescue percentage of the ectopic EMT will be
similar to the one in rescuing the septation defect. Finally, none of the RAR mutants can be rescued
from arch artery defects, implying that the morphogenesis of arch arteries is independent of TGFβ
signaling. In conclusion, our results explained in Chapter 3 have established the point that the
endocardium is the responsive tissue to the excessive TGFβ signaling in the RAR mutants, and
ecoptic EMT in the distal OFT is the cause for compromised septation.

25

CHAPTER 4

TGFΒ ACTIVATED KINASE 1 (TAK1) SIGNALING IN NEURAL CREST CELL
MEDIATES OFT SEPTATION

4.1 Abstract

While canonical (Smad-dependent) TGFβ signaling has been extensively studied in heart
development, there is very little known about the interaction of different cell lineages with the non-
canonical TGFβ signaling processes. A previous study (Choudhary et al, 2006) suggested that
altering TGFβ signaling in the neural crest cells can result in common arterial trunk (CAT).
However, the mechanism of how CAT arises still remains unclear. Hence we decided to delete

TGFβ -activated kinase 1 (TAK1) specifically in neural crest cells (NCC). The Wnt1Cre;Tak1
mutant embryos appeared to have the septation defects and result in the CAT phenotype.
Through a genetic synergy approach, we observed that while Wnt1Cre;Tak1fl/+;Tgbr2fl/+
mutants have normal heart development, some presented with a cleft palate. This result
suggested that TAK1 is thus required for proper palatogenesis through non-canonical TGFβ
signaling, while it is possibly TGFβ-independent for septation in the OFT. These results together
show that TAK1 is a critical component in cardiogenesis and palatogenesis.

26

4.2 Introduction

Cardiac neural crest cells (cNCCs) originate from the dorsal neural tube between the mid-
otic placode and the caudal boundary of the third somite (Hutson et al, 2007). These cells
delaminate from the neural tube and migrate past the pharyngeal endoderm into pharyngeal
arches 3, 4, and 6, and gain access to the heart as each arch extends to the ventral midline and
connects to the aortic sac (Jiang et al, 2002). The neural crest-derived mesenchymal cells migrate
into the distal part of the OFT and eventually form the A/P septum. Deficiencies in the formation
of the A/P septum result in a CAT phenotype, in which a single outflow tract structure persists
and is not transformed into distinct aortic and pulmonary vessels (Jiang et al, 2002). Many
signaling pathways are involved in the migration and condensation of cNCCs that are essential
for the development of the OFT and the aortic arch system (Waldo et al, 2005).
One of the signaling pathways that can function in the cNCCs is the TGFβ signaling
pathway. We described the TGFβ signaling pathway in Chapter 1.4 and its interaction with the
neural crest cell in Chapter 1.6. The Wnt1Cre;Tgfbr2 mutant has 100% penetrance of CAT and
interrupted aortic arch (IAA-B) penetrance through two independent processes. Inappropriate
apoptosis in the fourth arch artery results in IAA-B, while there is no associated apoptosis in
CAT (Choudhary et al, 2006). Therefore, TGFβ signaling controls the proper formation of the
A/P septum in the aortic sac and maintains the persistence of the 4th arch artery (Choudhary et
al, 2006). However, the mechanism by which the CAT phenotype arises in the Wnt1Cre/Tgfbr2
mutant remained unknown. In order to understand how the cNCCs are being organized and
directed to form the A/P septum, we have decided to look into non- canonical TGFβ signaling. In
addition to the canonical SMAD-dependent TGFβ pathway, TGFβ receptors can also activate the
members of the mitogen-activated protein kinase (MAPK) pathway, including TGFβ -activated
kinase 1 (TAK1) (Yamaguchi et al, 1995). Through a suppressor screen designed to identify
27

novel MAKP pathway members, TAK1 kinase activity was seen to be rapidly induced in
response to TGFβ (Yamaughi et al, 1995). At E10.5, TAK1 is ubiquitously expressed in the
whole embryo and by E12.5 the TAK1 expression starts to decline (Jadrich et al, 2003). Both in
vitro and in vivo studies have shown that TAK1 is able to elicit ligand-dependent TGFβ
responses (Ono et al, 2003; Yamaguchi et al, 1995; Zhang et al, 2000). Therefore, we decided to
conditionally knockout TAK1 in the neural crest populations and analyze heart development by
crossing the neural crest-specific Cre mouse line, Wnt1Cre, (Danielian et al, 1998) with a
transgenic mouse line with floxed TAK1 allele (Sato et al, 2005). By using a genetic synergetic
approach, we have crossed the Wnt1cre;Tgfbr2fl/+ mice with homozygous TAK1 conditional
transgenic in order to address whether the CAT phenotype in the Wnt1Cre;Tgfbr2 mutant is
mediated through the non-canonical TGFβ pathway.

28

4.3 Results

4.3.1 Tak1 Deficiency in the cNCC Lineage Leads to Septation Failure

Histological examinations of Wnt1Cre;TAK1 mutant mice have revealed that the
formation of the A/P septum failed and the OFT septation had been compromised. In all cases of
Wnt1Cre;TAK1 mutants, the single vessel OFT initiates from the middle of the ventricle (Table
2, Fig 3 A). These observations suggested a role for Tak1 during the septation process for the
OFT. Through a genetic mutant approach, we were able to recapitulate the CAT phenotype in
Wnt1Cre;Tgfbr2 mutants by knocking out the TAK1 gene conditionally in the neural crest cells.
This result suggests that TAK1 may be one of the potential TGFβ downstream signaling targets
which help to mediate proper A/P septum formation. In order to test whether TAK1 and TGFβ
work together in the cNCCs through the same pathway, which regulates OFT septation, we
decided to use a genetic synergetic approach to test. We conditionally reduced both the Tak1 and
Tgfbr2 genes by half, specifically in neural crest cells. No congenital heart defects have been
observed in the Wnt1Cre;Tak1fl/+;Tgfbr2fl/+ mutant mice, which all have normal heart
morphogenesis (Table2, Fig 3B). With this result, we cannot conclude whether TAK1 goes
through the TGFβ signaling pathway in OFT septation, because we might not have lowered the
Tak1 gene dosage enough in order to reproduce the CAT phenotype, and there might be
compensatory signaling pathways that maintain normal cardiogenesis. Subsequently, we
observed that two out of five Wnt1Cre;Tak1fl/+;Tgfbr2fl/+ mutants had cleft palate. This result
further supports that the existence of a role for non-canonical TGFβ signaling in the palatal
mesenchyme during palatogenesis. Nevertheless, the mechanism by which TAK1 mediates both
the cardiogenesis and palatogenesis needs to be clarified.
29

Table3. Deletion of Tak1 in the cNCC lineage leads to septation failure
All embryos were isolated at E14.5, embedded in paraffin, hematoxylin, and eosin stained for
histology sections. Serial sections were collected and for cardiovascular defects analysis. CAT
phenotype was scored as Table 1. Cleft palate is the malformation of the palatogenesis in which
the palatal shelves do not fuse together

Genotype Mutant Litter CAT Cleft palate Normal
cardiogensis
Wnt1Cre, 5 4 5 100% 5 100% 0 0%
Tak1fl/fl
Wnt1Cre, 5 3 0 0% 2 40% 5 100%
Tak1fl/+,
Tgfbr2fl/+

30

Fig3. Altered Tak1 signaling pathway in cNCCs results in CAT and cleft palate
H&E staining for E14.5 histology sections. (A) CAT phenotype in Wnt1Cre;Tak1fl/fl mutant (B)
Normal heart morphogenesis in Wnt1Cre;Tak1fl/+;Tgfbr2fl/+ (C) Cleft palate in
Wnt1Cre;Tak1fl/+;Tgfbr2fl/+. (B)-(C) histology sections are from same embryos. Abbreviations:
RV,right ventricle; Ao, ascending aorta; CAT, common arterial trunk; PT, pulmonary trunk; PS,
palatal shelf

A B

C

CAT
Ao

PT

RV

RV
PS

PS

Cleft palate

Wnt1Cre,Tak1fl/fl Wnt1Cre,Tak1fl/+,Tgfbr2fl/+ Wnt1Cre,Tak1fl/+,Tgfbr2fl/+

31

4.4 Discussion

The importance of TGFβ signaling in cardiogenesis has been studied extensively. For
example, TGFβ2 - null mutants are defective in outflow tract development (Sanford et al, 1997;
Bartram et al, 2001; Molin et al, 2004). Although TGFβ mainly signals through the Smad-
dependent canonical pathway, TGFβ can also signal through a Smad-independent pathway
(Massagué 2012). However, the mechanism by which the non-canonical pathway contributes to
TGFβ signaling in OFT septation remains poorly understood. In this chapter, we investigated
whether Tak1-mediated non-canonical TGFβ signaling plays a role in OFT septation and
palatogenesis.
We showed that the inactivation of the Tak1 gene in the neural crest lineage led to
septation failure. Wnt1Cre;Tak1fl/fl mutants died perinatally, and exhibited a CAT phenotype
and cleft palate defect with 100% penetrance (Song et al, 2013). A recent study, Song et al
(2003) has demonstrated that the cleft palate that exists in the Wnt1Cre;Tak1fl/fl mutants is a
secondary defect due to the malformation of the tongue, which obstructs palate elevation through
the regulation of Fgf10 expression in the tongue. However, how CAT exists in the mutants still
remains unknown, and we have hypothesized that TAK1 mediates OFT septation through non-
canonical TGFβ signaling. Nevertheless, the migration pattern of neural crest cells in the
Wnt1Cre;Tak1fl/fl mutants still need to be confirmed by lineage tracing analysis. In order to
examine the mechanism behind the CAT phenotype, we conditionally reduced Tak1and Tgfbr2
gene dosages by half, specifically in NCC, and none of the double heterozygous mutants
experienced any cardiovascular defects. Since TAK1 is activated by various signaling pathways
and stimuli including TLR ligands, TGFβ, BMPs, WNT, IL-1, and TNF (Ishitani et al, 2003;
Ninomiya-Tsuji et al, 1999; Shibuya et al, 1998), the proper cardiogenesis in
32

Wnt1Cre;Tak1fl/+;Tgfbr2fl/+ mutants may have resulted from compensatory mechanisms
between different signaling pathways. Contrarily, we did observe cleft palate in two out of five
Wnt1Cre;Tak1fl/+;Tgfbr2fl/+ mutants, which would suggest that there is a role for TAK1-
mediated non-canocial TGFβ signaling in palatogenesis. In conclusion, our results have
indicated that inactivation of Tak1 in the cNCC lineage does lead to a septation defect but the
mechanism by which TAK1 mediates OFT septation remains to be clarified.

33

BIBLIOGRAPHY

Arthur Helen M and Bamforth Simon D (2011) TGFβ signaling and congenital heart disease:
Insights from Mouse Studies. Birth Defects Research (Part A) 91:423-434.

Akhurst RJ, Lehnert SA, Faissner A (1990) TFG beta in murine morphogenetic processes: the
early embryo and cardiogenesis. Development 108:645-656.

BartramU, MolinDG, Wisse LJ, Mohamad A, Sanford LP, Doetschman T, Speer CP, Poelmann
RE, Gittenberger-DeGroot AC (2001) Double-outlet right ventricle and overriding tricuspid
valve reflect disturbances of looping, myocardialization, endocardial cushion differentiation,
and apoptosis in TGF-beta(2)-knockout mice.Circulation,2745–2752.

Brown CB, Boyer AS, Runyan RB, Barnett JV (1996) Antibodies to the Type II Tgfβeta
receptor block cell activation and migration during atrioventricular cushion transformation in the
heart. Developmental biology 174: 248-257.

Cai CL, Liang X, Shi Y, Chu PH, Pfaff S.L, Chen J (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.

Cai CL, Martin JC, Sun Y, Cui L, Wang L, Ouyang K, Yang L, Bu L, Liang X, Zhang X,
Stallcup WB, Denton CP, McCulloch A, Chen J, Evans SM (2008) A myocardial lineage derives
from Tbx18 epicardial cells. Nature 454: 104-108.

Camenish TD, Molin DG, Person A (2002) Temporal and distinct TGFbeta ligand requirements
during mouse and avian endocardial cushion morphogenesis. Dev Biol 248:170-181.

Choudhary B, Ito Y, Makita T, Sasaki T, Chai Y, Sucov HM (2006) Cardiovascular
malformations with normal smooth muscle differentiation in neural crest-specific type II Tgfβeta
receptor (Tgfβr2) mutant mice. Developmental biology 289: 420-429.

Chytil A, Magnuson MA, Wright CV (2002) Conditional inactivation of the TGF-beta type II
receptor using Cre: Lox. Genesis 32:73–75.

Creazzo TL, Godt RE, Leatherbury L, Conway SJ, and Kirby ML (1998) Role of cardiac
neural crest cells in cardiovascular development. Annu Rev Physiol 60, 267-286.

Davis LA, Sadler TW (1981) Effects of vitamin A on endocardial cushion development in the
mouse heart. Teratology 24, 139-148.

Duester G (2000) Families of retinoid dehydrogenases regulating vitamin A function:
production of visual pigment and retinoic acid. Eur. J. Biochem. 276, 4315-4324.

Eisenberg LM, Markwald RR (1995) Molecular regulation of atrioventricular
valvuloseptal morphogenesis. Circulation research 77: 1-6.

34

Fananapazir K, and Kaufman MH (1988) Observations on the development of the
aortico-pulmonary spiral septum in the mouse. J Anat 158, 157–72.

Gruber PJ, Epstein JA (2004) Development gone awry congenital heart disease. Circ Res
94:273–283.

Hierck BP, Molin DG, Boot MJ ,Poelmann RE ,Gittenberger-deGroot AC (2004) A chicken
model for DGCR6 as a modifier gene in the DiGeorge critical region. Pediatrics Research
56,440–448.

Hoffman JI, Kaplan S, Liberthson RR (2004). Prevalence of congenital heart disease. Am Heart J
147, 425–39.

Horsthuis T, Christoffels VM, Anderson RH, Moorman AF (2009) Can recent insights into
cardiac development improve our understanding of congenitally malformed hearts? Clinical
anatomy New York, NY 22: 4-20.

Jadrich JL, O'Connor MB, Coucouvanis E (2003) Expression of TAK1, a mediator of TGF-beta
and BMP signaling, during mouse embryonic development. Gene Expr Patterns 3(2):131-4.

Jiang X, Choudhary B, Merki E, Chien KR, Maxson RE, Sucov HM (2002) Normal fate and
altered function of the cardiac neural crest cell lineage in retinoic acid receptor mutant
embryos. Mech Dev 117: 115-122.

Kaufman MH, Navaratnam V (1981) Early differentiation of the heart in mouse embryos. J
Anat 133: 235-246.

Kaufman MH and Bard JB (1999) The Anatomical Basis of Mouse Development. Academic
Press, London.

Kelly RG, Brown NA, Buckingham ME (2001) The arterial pole of the mouse heart forms
from Fgf10-expressing cells in pharyngeal mesoderm. Developmental cell 1: 435-440.

Kirby ML (2002) Molecular embryogenesis of the heart. Pediat. Dev. Pathal. 5(6), 516-543.

Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M (2001)
Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.
Developmental biology 230: 230-242.

Komiyama M, Ito K, Shimada Y (1987) Origin and development of the epicardium in the
mouse embryo. Anat. Embryol. (Berl) 176,183-89.

Krug EL, Mjaatvedt CH, Markwald RR (1987) Extracellular matrix from embryonic
myocardium elicits an early morphogenetic event in cardiac endothelial differentiation. Dev
Biol 120: 348–355.

35

Lawler S, Candia AF, Ebner R (1994) The murine type II TGF-beta receptor has a coincident
embryonic expression and binding pregerence for TGF-beta 1. Development 120:165-175.

Lee RY, Luo J, Evans RM, Giguere V, Sucov HM (1997) Compartment-selective sensitivity of
cardiovascular morphogenesis to combinations of retinoic acid receptor gene mutations.
Circulation research 80: 757-764.

Li P, Pashmforoush M, Sucov HM (2010) Retinoic acid regulates differentiation of the
secondary heart field and Tgfβeta-mediated outflow tract septation. Dev Cell 18:480–485.

Li P, Pashmforoush M, Sucov HM. Mesodermal retinoic acid signaling regulates endothelial
cell coalescence in caudal pharyngeal arch artery vasculogenesis. Developmental Biology
361(2012) 116-124.

Loretta L Hoover, Elizabeth G. Burton Bonnie A Brooks, Steven W Kubalak (2008) The
Expanding Role for Retinoid Signaling in Heart Development. TheScientificWorldJOURNAL,
vol. 8, pp. 194-211.

Mariano JM, Montuenga LM, Prentice MA (1998) Concurrent and distinct transcription and
translation of transforming growth factor beta type I and type II receptors in rodent
embryogenesis. Int J Dev Biol 42:1125-1136.

Martinsen BJ, Bronner-Fraser M (1998) Neural crest specification regulated by the helix-loop-
helix repressor. Science 281(5379), 988-991.

Massagué J (2012) TGFβ signaling in context. Nat. Rev., Mol. Cell Biol. 13, 616-630.

Mendelsohn C, Lohnes D, Decimo D, Lufkin T, LeMeur M, Chambon P, Mark M (1994)
Function of the retinoic acid receptors (RARs) during development (II). Multiple abnormalities
at various stages of organogenesis in RAR double mutants. Development (Cambridge,
England) 120: 2749-2771.

Molin DG, Bartram U, Van Der Heiden K (2003) Expression patterns of Tgfbeta1-3 associate
with myocardialisation of the outflow tract and the development of the epicardium and the
fibrous heart skeleton. Dev Dyn 227:431-444.

Mummery CL (2001) Transforming growth factor beta and mouse development. Microsc Res
Tech 52:374-386.

Ninomiya-Tsuji J, Kishimoto K, Hiyama A, Inoue J, Cao Z, Matsumoto K (1999) The kinase
TAK1 can activate the NIK-KappaB as well as the MAP kinase cascade in the IL-1 signaling
pathway. Nature 398, 252-256.

Ono K, Ohtomo T, Ninomiya-Tsuji J, Tsuchiya M. (2003). A dominant negative TAK1
inhibits cellular fibrotic responses induced by TGF-beta. Biochem. Biophys. Res. Commun.
307,332 - 337.
36

Danielian PS , Muccino D, Rowitch DH, Michael SK, McMahon AP (1998) Modification of
gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.
Curr. Biol.8 pp. 1323–1326.

Saija M Savolainen, Julie F Foley, Susan A Elmore (2009) Histology Atlas of the
Developing Mouse Heart with Emphasis on E11.5 to E18.5. Toxicol Pathol 37: 395.

Sanford LP, Ormsby I, Gittenberger-de Groot AC, Sariola H, Friedman R, Boivin GP, Cardell
EL, Doetschman T (1997) Tgfβeta2 knockout mice have multiple developmental defects that
are non-overlapping with other Tgfβeta knockout phenotypes. Development (Cambridge,
England) 124: 2659-2670.

Sato S, Sanjo H, Takeda K, Ninomiya-Tsuji J, Yamamoto M, Kawai T, Matsumoto K,
Takeuchi O, Akira S (2005) Essential function for the kinase TAK1 in innate and adaptive
immune responses. Nat Immunol 6(11):1087-95.

Shibuya H, Iwata H, Masuyama N, Gotoh Y, Yamaguchi K, Irie K, Matsumoto K, Nishida
E, Ueno N (1998) Rolet of TAK1 and TAB1 in BMP signaling in early Xenopus
development. EMOB J. 17, 1019-1028.

Song Z, Liu C, Iwata J, Gu S, Suzuki A, Sun C, He W, Shu R, Li L, Chai Y, Chen Y
(2013) Mice with Tak1 deficiency in neural crest lineage exhibit cleft palate associated with
abnormal tongue development. J Bio Chem 288(15):10440-50

Soriano P (1999) Generalized LacZ expression with the ROSA26 Cre reporter strain. Nat.
Genet. 21, 70–71.

Srivastava D (2006) Making or breaking the heart: from lineage determination to
morphogenesis. Cell 126: 1037-1048.

Waldo KL, Hutson MR, Stadt HA, Zdanowicz M, Zdanowicz J, Kirby ML (2005) Cardiac
neural crest is necessary for normal addition of the myocardium to the arterial pole from the
secondary heart field. Dev Biol 281: 66 – 77.

Ward C, Stadt H, Hutson M, Kirby ML (2005) Ablation of the secondary heart field leads
to tetralogy of Fallot and pulmonary atresia. Developmental biology 284: 72-83.

Webb S, Qayyum SR, Anderson RH, Lamers WH, Richardson MK (2003) Septation and
separation within the outflow tract of the developing heart. Journal of anatomy 202: 327-342.

Wilson JG, Warkany J (1949) Aortic-arch and cardiac anomalies in the offspring of vitamin
A deficient rats. Am J Anat 85: 113-155.

Jiang X, Rowitch DH, Soriano P, McMahon AP, Sucov HM (2000) Fate of the mammalian
cardiac neural crest. Development 127 pp. 1607–1616.
37

Yamaguchi K, Shirakabe K, Shibuya H, Irie K, Oishi I, Ueno N, Taniguchi T, Nishida E,
Matsumoto K (1995) Identification of a member of the MAPKKK family as a potential
mediator of TGF-beta signal transduction. Science 270,2008 -2011.

Zhang YE (2009) Non-Smad pathways in TGF-beta signaling. Cell Res 19:128–139.

Zhang D, Gaussin V, Taffet GE, Belaguli NS, Yamada M, Schwartz RJ, Michael LH, Overbeek,
PA, Schneider MD (2000) TAK1 is activated in the myocardium after pressure overload and is
sufficient to provoke heart failure in transgenic mice. Nat. Med. 6,556 -563.

Zhou B, Ma Q, Rajagopal S, Wu SM, Domian I, Rivera-Feliciano J, Jiang D, von Gise A,
Ikeda S, Chien KR, Pu WT (2008) Epicardial progenitors contribute to the cardiomyocyte
lineage in the developing heart. Nature 454: 109-113.

Abstract (if available)

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Proliferation and maturation events in second heart field cells during cardiovascular development activated by the Delta like ligand-4 mediated notch signaling

PDF

A novel role for post-migratory neural crest cells in cardiac outflow tract alignment: insights into the molecular basis of concomitant congenital cardiac and cleft malformations

PDF

Lineage boundaries and cell migration in the patterning of the mammalian skull

PDF

Searching for mitogenic factors from the epicardium: PDGFA, Igf2 and more

PDF

Chromatin remodeling factors Chd7 and Phf6 in craniofacial and heart development

PDF

Opposing function of chd7 and phf6 in zebrafish craniofacial development

PDF

Elucidating the functional role of CHD7 associated nuclear PDH complex and other associated proteins on neural crest development

PDF

TNNI3K expression affects nucleation of cardiomyocytes and skeletal myoblasts

PDF

Derivation, expansion and characterization of human hippocampal primordial cells from normal and diseased iPSCs

PDF

Tissue interactions & molecular pathways in specification of the ectomesenchyme from cranial neural crest

PDF

Characterization of transgenic zebrafish lines for studying role of platelet derived growth factor (PDGF) signaling on coronary vessel formation in regenerating zebrafish heart after cryoinjury

PDF

Neurogenic placodes provide migratory enteric sensory neural progenitors in response to endothelin signaling pathway

PDF

Tools to study the epicardium's response during cardiac regeneration

PDF

Functions of Msx2 and Msx1 during mouse skull and hair follicle development

PDF

TWIST1 functions in both mesoderm and neural crest derived cranial tissues to establish and maintain coronal suture patency

PDF

The roles of Tnni3k in heart regeneration, cardiac conduction system defects and cardiomyopathy

PDF

Epigenetic checks and balances: PHF6 activity restricts neural crest migration

PDF

Effects of electronic cigarettes on viability and proliferation of lung mesenchymal stem cells

PDF

The role of Wnt signaling in organogenesis: limb and lung

PDF

Characterization of Cxcr7 in zebrafish cardiac lymphatic vessel development

Asset Metadata

Creator Ma, Man Cheong (author)

Core Title Retinoic acid and TGFβ signaling regulate cardiovascular development

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 08/06/2013

Defense Date 06/10/2013

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

Tag cardiovascular development,OAI-PMH Harvest,retinoic acid,TGFβ

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Sucov, Henry (committee chair), Lien, Ching Ling (Ellen) (committee member), Pashmforoush, Mohammad (committee member)

Creator Email mamancheong@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-318647

Unique identifier UC11293782

Identifier etd-MaManCheon-1979.pdf (filename),usctheses-c3-318647 (legacy record id)

Legacy Identifier etd-MaManCheon-1979-0.pdf

Dmrecord 318647

Document Type Thesis

Format application/pdf (imt)

Rights Ma, Man Cheong

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