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Neural fate regulated by extrinsic signaling and epigenetics

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Neural fate regulated by extrinsic signaling and epigenetics

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Neural fate regulated by extrinsic signaling and epigenetics
by
Jingyang Zhong

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
(Neuroscience)

August 2013

Copyright 2013 Jingyang Zhong

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ACKNOWLEDGEMENTS

Over the past six years I have received support, inspiration and encouragement
from a great number of individuals. I would like to express my sincere gratitude to
their valuable assistance that helped me overcome the all the difficulties that I
have in pursing my scientific goal and developed me as a person.

My advisor Dr. Wange Lu has been a strong, supportive and trustworthy advisor
to throughout these years. I have always been interested in developmental
biology and he has created this opportunity for me to join his lab and provided
me with great freedom to explore many aspect of developmental neurobiology.
During the beginning years, I doubted my ability because I wasn’t successful with
the first project and Dr. Lu demonstrated his trust in my ability to rise to the
occasion by giving me the time and freedom to practice and explore. During the
time of submitting my fist paper, Dr. Lu has not only spent hours and hours in
revising my manuscript but more importantly supported my work with respect,
confidence and optimistic attitude. Through my second project, Dr. Lu has
supported me to pursue independent research of cutting edge topics, through
independent learning and collaborations. Through the rounded training in his lab,
I have learned how to be an independent and critical thinker, vigorous researcher,
and open-minded collaborator. Moreover, Dr. Lu also supported me with
generous help and understanding when I wanted to make a change in my career
path, I believe that I will benefit from all abilities and skills that I learned from this
lab throughout my future career and life.
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Secondly, I would like to thank my committee members Dr. Chien-Ping Ko, Dr.
Le Ma, Dr. Gage Crump for their generous scientific advices and help. Dr. Ko
provided with me valuable advice ever since my first semester in NGP, when he
was the academic advisor for the first year graduate student at that time. Dr. Ma
and Dr. Gage all have provided important technique support for several times
during my research throughout the years.

I have been especially fortunate to establish collaborations with several great
scientists. My study would not go so far without learning new techniques and
obtaining advice from them. I would like to thank Dr. Hyoung-tai Kim, Dr. Si Ho
Choi, and Dr. Seth Frietze for teaching me various critical techniques that are
required to my research. Dr. Kim has trained me as a neurodevelopmental
biologist, Dr. Choi and Dr. Frietze helped me to conquer my barrier with
epigenetic studies. I would like to thank Dr. Masato Nakafuku for his generous
scientific input for my first paper, Dr. Jennifer Banya for providing me with LacZ
staining protocol and Dr. Charles Nicolet for helping with generating second-
generation sequencing data and his encouragement to me. I deeply appreciate
their generosity, patience and kindness.

I would like to thank many former and current Lu lab members for their help
during my entire PhD period. The friendship that I have gained in the lab has
companied me in many difficult situations. I would like to deliver my special thank
to Dr. Kim, Dr. Choi, Dr. Wei, Wen-Hsuan Chuang, and many others, with whom
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I have shared numerous up and downs during life and research though these
years. I also learned a lot through them.

Finally I most want to deliver my deepest gratitude to my family members. My
parents have always supported me unconditionally. I want to thank my husband
Dr. Kornelius Rácz for his love, sacrifice and inspiration over the six years.

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TABLE OF CONTENTS
ACKNOWLEDGEMENTS 2

LIST OF FIGURES 7

ABSTRACT 9

CHAPTER 1: GENERAL INTRODUCTION 11

1.1 Neural development overview 11
1.2 Neural progenitor cell self-renewal and proliferation 13
1.3 Neural progenitor cell specification and differentiation 27
1.4 Epigenetic regulation of neural cell fate specification 42

CHAPTER 2: WNT-RYK SIGNALING REGULATES THE CELL
FATE SPECIFICATION OF GABAERGIC NEURON
VERSUS OLIGODENDROCYTE
49

2.1 Abstract 49
2.2 Introduction 50
2.3 Methods and Materials 53
2.4 Results 56
2.4.1 Expression of Ryk receptor 56
2.4.2 Phenotypes of Ryk knockout mice 57
2.4.3 In vitro differentiation of Ryk knockout NPCs 68
2.4.4 Cell-autonomous effects of Ryk receptor 71
2.4.5 Wnt proteins regulate the differentiation of NPCs 73
2.4.6 Ryk-ICD is necessary and sufficient for the
regulation of cell fate switches during differentiation
76
2.5 Discussion 79

CHAPTER 3: HISTONE H2A DEUBIQUITINASE MYSM1
REGULATES CORTICAL NEUROGENESIS
84

3.1 Abstract 84
3.2 Introduction 85
3.3 Method and Materials 89
3.4 Results 93
3.4.1 Expression of MYSM1 93

3.4.2 Cortical neurogenesis is impaired in the MYSM1
knockout mouse
96

3.4.3 Neural progenitor self-renewal and proliferation is
not impaired upon Mysm1 deletion
98

3.4.4 Increased apoptosis in basal progenitor cells of
the MYSM1 knockout mice
104
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3.4.5 Enhanced astrogenesis in new born
MYSM1 knockout mice
106

3.4.6 Cell fate tracing of E14.5 neural progenitor cell
in vivo
108

3.4.7 In vitro differentiation of MYSM1 knockout neural
progenitor cells
110

3.4.8 Cell-autonomous effect of MYSM1 in regulating
neuronal differentiation
114

3.4.9 ChIP-Seq analysis of histone modification
indicates a global impact on neuronal genes by
MYSM1 deletion
114
3.5 Discussion 129

CHAPTER 4: CONCLUSIONS AND PERSPECTIVES 135

4.1 Ryk receptor and ventral NPC output 136
4.2 Dissection of the cellular mechanism of Ryk signaling 136
4.3 Molecular mechanism of Ryk mediated Wnt signaling 137
4.4
Histone H2A deubiquitinase MYSM1 and cortical
neurogenesis
138
4.5 MYSM1 regulates neuronal cell fate specification 139
4.6
Global histone modifications defects upon MYSM1
deletion hint impaired differentiation capacity
139
4.7 Future directions 140

BIBLIOGRAPHY 142

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LIST OF FIGURES
Figure 1.1: Classification of progenitor cells in the developing brain 16

Figure 1.2: Cortical neurogenesis 33

Figure 2.1: Ryk knockout mice exhibit increased oligodendrogenesis
during brain development
60

Figure 2.2: Ryk knockout mice exhibit increased oligodendrogenesis
during brain development
61

Figure 2.3: Impaired GABAergic neuron development in Ryk knockout
mice
64

Figure 2.4: Enhanced oligodendrocyte differentiation without impaired
proliferation in Ryk knockout mice
67

Figure 2.5: Ryk knockout NPC cultures give rise to reduced neurons
and increased numbers of oligodendrocytes though a cell-
autonomous mechanism
70

Figure 2.6: Ryk receptor is required for the Wnt3a-induced GABAergic
neuron differentiation and inhibition of oligodendrocyte
differentiation
75

Figure 2.7: Ryk intracellular domain is necessary and sufficient to
mediate the cell-fate change
78

Figure 3.1: Morphological defects of MYSM1 knockout embryonic brain
and adult brain
95

Figure 3.2: Expression of MYSM1 in the developing mouse brain 100

Figure 3.3 MYSM1 knockout mice exhibit severely impaired
corticoneurogenesis at E14.5
101

Figure 3.4 Impaired corticoneurogenesis in MYSM1 knockout mice
does not show layer preference
102

Figure 3.5 The neural progenitor cell pool is maintained in MYSM1
knockout mice
103

Figure 3.6 TUNNEL assay revealed increased apoptosis of neuronal 105
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precursor cells in the MYSM1 knockout mice

Figure 3.7 Enhanced production of astrocytes in the new born MYSM1
knockout cortex
107

Figure 3.8 Cell fate tracing of E14.5 neural progenitor cells in the
MYSM1 knockout mice
109

Figure 3.9 In vitro differentiation of wild-type and MYSM1 knockout
NPCs
112

Figure 3.10 Overexpression of MYSM1 is sufficient to rescue the
differentiation phenotype of MYSM1 knockout neural
progenitor cells in vitro
113

Figure 3.11 Validation of histone modification antibodies for ChIP assay
in mouse ESCs
119

Figure 3.12 Occupancy of important histone markers at the Neurod2
locus in wild-type primary neural progenitor cells
120

Figure 3.13 Summary and examples of ChIP-Seq datasets 121

Figure 3.14 Genome-wide location analysis of active and repressive
histone marks H3K4me2 and H3K27me3 in primary wild-
type and MYSM1 knockout NPCs
126

Figure 3.15 Gene ontology analysis of a core subset of the H3K4me2
differentially bound regions
127

Figure 3.16 Validation of ChIP-Seq results by ChIP qPCR at selective
targets
128

Figure 3.17 Model of the MYSM1 regulated neuronal differentiation 134

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Abstract
The mammalian central nervous system is the most complex organ among all the
mammalian organs and we have just began understanding the highly
orchestrated but nonlinear process of brain development. Brain development
involves many various cellular behaviors including progenitor self-renewal,
proliferation, patterning, differentiation, migration etc. My dissertation research in
Dr. Wange Lu’s lab has mainly focused on one core question in the field of neural
development: to uncover the mechanisms regulating the cell fate specification of
neural progenitor cells during brain development. My research involves the
investigation of two independent but closely related aspects of this core question.

In my thesis, I have first investigated the role of an atypical Wnt receptor Ryk in
regulating the cell fate choice between the neuronal differentiation and
oligodendrogenesis and demonstrated that Wnt3a-Ryk-mediated signaling
promotes GABAergic neuron production, while inhibiting oligodendrocyte
differentiation through a Ryk intracellular domain-dependent mechanism.

Secondly, I have investigated the biological function of a histone H2A
deubiquitinase MYSM1 in cortical neurogenesis. Both in vivo and in vitro
analyses demonstrate that MYSM1 plays a critical role in neuronal cell fate
specification without affecting the maintenance of the cortical progenitor pool. To
further elucidate the underlying molecular mechanisms, a ChIP-Seq analysis of
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genome-wide occupancy of repressive histone mark H3K27me3 and active
histone mark H3K4me2 in both wild-type and MYSM1 knockout primary neural
progenitor cell culture under proliferating conditions was conducted: it shows that
a group of genes regulating neuronal development exhibit a more repressive but
less active chromatin status upon loss of MYSM1. This research supports the
role of MYSM1 in regulating transcriptional activation of neuronal gene to
facilitate the neuronal cell fate specification, likely through removing repressive
histone mark H2Aub1 and cross-talk with other histone modifications.

This research is of great importance not only for our understanding about the
extremely complicated establishment process of the central nervous system, but
also provides a potential application for developing stem cell based therapy.

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CHAPTER 1 GENERAL INTRODUCTION

1.1 Neural development overview
The vertebrate central nervous system (CNS) originates from the neural plate of
the ectoderm, which is located at the opposite side of the primitive streak. During
neurulation, part of the dorsal ectoderm is specified into neural ectoderm or
neural plate, which is quickly folded and formed the neural tube at around
embryonic day 8 (E8) in the mouse. The neural tube is initially composed of a
single layer of neuroepithelial cells, but eventually develops into the entire central
nervous system including the brain and spinal cord. After a phase of fast planar
expansion of neural epithelial cells, neuroepithelial cells transit into radial glial
cells, and neurogenesis starts at the same time, around E9 in the mouse.
Through asymmetric cell division, radial glial cells, as the primary progenitors,
give rise to most neurons, as well as two types of glial cells, including
oligodendrocytes and astrocytes throughout the entire CNS.

At the early stage of neurogenesis, the neural tube is patterned into dorsal and
ventral domains by extrinsic cues, which determine a completely differentiated
cell fate output in each domain. Dorsal neural progenitors give rise to various
types of excitatory cortical projection neurons in a chronological order, which
forms the six cortical layers(Kwan, Sestan and Anton). At the late stage of the
neurogenic phase, around E18 in the mouse, the competence of neural
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progenitors switches from neurogenic to gliogenic by mainly giving rise to
astrocytes. The timing of the neuronal-to-glial switch significantly influences the
number of neurons that can be produced during cortical development. In the
ventral domain, neural progenitors give rise to distinct types of neurons, including
inhibitory GABAergic neurons, cholinergic neurons and oligodendrocytes.
However, the regulation of production of various cell types is itself heavily
regulated by a location-based mechanism. A distinct feature of ventrally derived
cell types is that they undergo a tangential migration from the ventral domain to
the dorsal cortex and eventually populate the cortex and form connections there.
Unlike the neuronal-to-astroglial switch occurring in the dorsal progenitors, there
is no clear cell fate switch in the ventral progenitors. The production of
oligodendrocytes and ventral neurons exhibits a similar time window.

With the continuously increasing interest in understanding neurological and
neurodegenerative diseases, intensive studies over the past several decades
have made significant contributions to the understanding of the extraordinarily
complex network of CNS development that encompasses signaling,
transcriptional and epigenetic mechanisms. However, neuronal and glial subtype
specification, maturation mechanisms and the differences in regulation between
human and mouse CNS development remain poorly understood. Therefore,
basic research in these areas will significantly facilitate the understanding of the
root cause of neurological and neurodegenerative diseases and the design of
rational strategies to develop clinical relevant models and ultimately, therapies.
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1.2 Neural progenitor cells self-renewal and proliferation
1.2.1 Bipolar Progenitors: Neuroepithelial cells, Radial glial cells, oRG cells
Based on the morphology during the M-phase of the cell cycle, neuroepithelial
cells and the radial glial cells are considered bipolar progenitors in the cortical
development (Fig. 1.1). At embryonic day 8 (E8), the neural tube is composed of
a single layer of cells: neuroepithelial (NE) cells, which appear layered because
of the interkinetic nuclear migration (INM). The bipolar nature of NEs is reflected
by the bipolar morphology of NEs with its apical membrane forming the
ventricular surface and its basal process contacting the basal lamina. NEs
demonstrate characteristic features of epithelial cells: They express CD133 at the
apical plasma membrane and have tight junctions and adherens junctions at the
apical end of the lateral plasma membrane (Farkas and Huttner; Rowitch and
Kriegstein). Neuroepithelial cells mainly divide in a symmetrical manner, which
generates two identical neuroepithelial cells in order to expand the stem cell
population. This “expansion phase” of NEs is sustained until around E9/E10
(Miyata, Kawaguchi, Kawaguchi, et al.).

With the switching-on of neurogenesis and thickening of the neural tube, NEs are
transformed into another type of bipolar progenitor cells: the radial glial cells
(RGCs), which extend their radial glial fibers but still maintain the attachment to
the basal lamina and obtain more glial properties while losing some of their
epithelial character. RGCs do not have tight junctions but still have adherens
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junctions and show CD133 expression. Astroglial markers begin to be expressed
by RGCs, including GFAP, GLAST and S100β (Rowitch and Kriegstein).
Therefore, RGCs exhibit properties of both epithelial cells and astroglias. Notably,
two common features of NEs and RGCs are that both of them show an obvious
apical-basal polarity and interkinetic nuclear migration. Upon the division
pattern’s switch to asymmetric division, RGCs enter a “neurogenic phase”
(Miyata, Kawaguchi, Kawaguchi, et al.). This transition occurs from E10 to E12.
At E12 and later, the ventricular zone (VZ) is dominated by the cell body of RGCs.
Studies based on retroviral labeling and time lapse imaging of cultured brain
slides show that RGCs mainly divide asymmetrically, which allows RGCs to self-
renew and give rise to more differentiated daughter cells, either post-mitotic
neurons or more-differentiated progenitor cell types, which utilize the radial glial
fiber as a guide to migrate toward the basal side of VZ (Noctor, Martinez-
Cerdeno and Kriegstein; Noctor, Martinez-Cerdeno, et al.; Noctor, Flint, et al.).

1.2.2 Monopolar progenitors: oRG cells
Recent studies have identified a new class of bipolar progenitor cells: outer-
subventricular-zone radial glial like cells (oRG) or outer subventricular zone
(OSVZ) progenitor cells. This subtype of bipolar progenitor cell was first identified
and characterized in humans and ferrets (Hansen et al.; Fietz et al.), and later
were also found to exist in developing cortex (X. Wang, J. W. Tsai, B. LaMonica,
et al.). OSVZ progenitor cells have been found to be originally derived from the
AP cells and delaminated from the apical surface, and mainly reside in the SVZ
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and OSVZ region. Despite the lack of attachment to the apical surface of the
ventricular zone, studies revealed that oRG cells still maintain a bipolar
morphology and the attachment to the basal lamina (Fig.1.1). The predominant
existence of oRG progenitor cells and their daughter cells have created a
significant expanded OSVZ zone in human and higher primate compared to
mouse, and therefore significantly increased the capacity of neuronal output
during neurogenesis (X. Wang, J. W. Tsai, B. LaMonica, et al.; Hansen et al.;
Fietz et al.).

1.2.3 Nonpolar progenitors: Basal/intermediate progenitors
Through asymmetric division, RGCs give rise to neurons, more-differentiated
progenitors, basal progenitors (BPs) or subventricular zone progenitors (Noctor,
Martinez-Cerdeno, et al.; Miyata, Kawaguchi, Saito, et al.; Haubensak et al.;
Farkas and Huttner). Unlike NEs and RGCs, basal progenitors retract their
extensions to the apical surface (Fig. 1.1). Right after their birth at VZ, they
migrate toward the basal side of VZ, become multipolar and finally undergo
mitosis at the basal side of the VZ. Hence, BPs can be distinguished from NEs
and RGCs, which divide at the apical surface of the VZ. BPs usually divide only
once, symmetrically, and generate two post-mitotic neurons (Noctor, Martinez-
Cerdeno, et al.; Miyata, Kawaguchi, Saito, et al.; Haubensak et al.). Therefore, as
the production of BPs continues, the size of the subventricular zone (SVZ) and
the capacity of neuron production dramatically increase. In the dorsal
telencephalon, where cortical projection neurons are produced, the character of
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RGC and BPs has been well defined. BPs express more differentiated and fate-
committed genes including Tbr2, Cux1/2. NEs and RGCs are Pax6+/Tbr2-/Cux1-
(Farkas and Huttner; Zimmer et al.; Nieto et al.; Englund et al.). However, the
BPs located in the ventral SVZ have not been clearly defined in terms of their
gene expression profiling and their markers.

Figure 1.1 Classification of progenitor cells in the developing brain.
The neural progenitor cells can be classified based on the morphology when they
divide, the absence or presence of polarized morphology at M phase.I,
interphase; M, M-phase. Obtained from the review (Fietz and Huttner).

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The differentiation capacity or competence of these three types of mitotic
progenitor cells has also been characterized. We know from Cre-loxp
recombination-mediated fate mapping studies that a vast majority of neurons in
all brain regions are derived from RGC. Time-lapse imaging of Tis21 transgenic
mice demonstrated that most BPs generally divide symmetrically to generate two
neurons, which later contribute to all layers of cerebral cortex (Noctor, Martinez-
Cerdeno, et al.; Haubensak et al.). However, it has been suggested that a small
fraction of rodent BPs are capable of proliferative symmetric divisions to expand
the BP pool in the SVZ (Noctor, Martinez-Cerdeno and Kriegstein; Noctor,
Martinez-Cerdeno, et al.; Hodge et al.).

Despite all the morphological, functional and marker expression diversity and the
newly discovered progenitor subtypes in higher primates, the three major cell
types that are shared during mammalian cerebral cortex development, i.e. NEs,
RGCs, and BPs, can be divided into two major groups: Apical progenitors (NEs
and RGCs) and Basal progenitors. The main differences between two groups are
the loss of polarity and the attachment in BPs. Since the vast majority of self-
renewal and proliferative division happens in apical progenitors, neural progenitor
self-renewal and proliferation are most intensively studied in Aps [citations?].
Below are several key aspects that have been suggested to govern the self-
renewal and proliferation of neural progenitor cells.

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1.2.4 Apical-basal Polarity
As mentioned above, APs are highly polarized cells, and the signaling controlling
the proliferation/differentiation of APs residing in the apical side or in the
ventricular fluid. Although neural stem cells can be maintained in proliferation
condition without any polarity in vitro, this does not reflect what is happening in
vivo, since proliferation, self-renewal, and differentiation are tightly regulated and
coupled in vivo. Disruption of the attachment of radial glial end-feet to the basal
lamina does not affect the proliferation and peak neurogenesis, which suggests
that critical signals regulating the self-renewal/differentiation choice are not
located in the basal end-feet but rather in the apical end-feet (Haubst et al.).
Indeed, the apical membranes harbor adherence junctions as well as the polarity
machinery Par3/Par6/aPKC etc. (Siller and Doe). Utilizing a conditional knockout
for a critical component of adherences junction N-cadherin results in a complete
loss of apical-basal polarity of the APs. However, this does not appear to affect
the self-renewal and differentiation of APs (Noles and Chenn; Kadowaki et al.).
Furthermore, recent studies have demonstrated that Numb serves as a major
regulator of adherens junction maintenance and AP cell polarity (Rasin et al.; Kim
and Walsh). The conditional knockdown of Numb leads to disruption of adherens
junctions and apical-basal polarity, disrupting laminar organization, but has no
significant effect on sequential neocortical neurogenesis or gliogensis (Rasin et
al.; Kim and Walsh). In summary, it seems that the integrity of AP polarity is
critical for the general organization of cortex formation, but does not seem to be
! 19!
required for self-renewal and differentiation, suggesting a strong cell-intrinsic
program.

1.2.5 Interkinetic nuclear migration
Another hallmark of APs is the migration of the nucleus during the cell cycle, a
perfect coupling of nuclear location with cell cycle (Fietz and Huttner): mitosis
occurs at the apical surface of VZ, apical-to-basal migration during G1 phase,
basal-to-apical migration during G2 phase (Taverna and Huttner). The
relationship between cell cycle and INM has been investigated through the study
of specific inhibition of cell cycle and INM. It has been demonstrated that INM is
dispensable for cell cycle progression, since specific inhibition of INM due to a
treatment of cytochalasin B and myosin II inhibitor, which inhibit F-actin
polymerization, does not affect cell-cycle progression and the length of various
phases of the cell cycle (Messier; Murciano et al.; Calegari et al.). Conversely,
INM has been shown to be dependent on cell cycle progression, as
pharmacological cell-cycle arrest in the S or G2/M phase results in inhibition and
arrest of INM (Baye and Link; Ueno et al.). Collectively, it seems that the primary
function of INM is to achieve pseudostratification of the VZ in order to maximize
the number of AP mitoses per available apical space, which helps to expand the
AP pool (Taverna and Huttner). Consistent with this interpretation, interference
with INM has been found to reduce the AP pool (Ge et al.; Xie et al.). The
mechanism behind INM influence on AP cell fate is still under investigation,
though it has been hypothesized that INM regulates AP cell fate by determining
! 20!
the time the AP nucleus is exposed at the apical surfaces, a critical signal for
self-renewal and differentiation (Del Bene et al.).

1.2.6 Asymmetric cell division
As mentioned before, NEs initially go through a phase of massive expansion,
during which the progenitor pool quickly increases through symmetric division.
Later on, when neurogenesis starts, NE cells switch to RGC cells and undergo a
phase of self-renewing asymmetric division (SRAD), which results in one
daughter cell that remains an RGC and another daughter cell that is either a
neurogenic BP or a mitotic neuron. It has long been thought that the orientation
of the mitotic spindle dictates the choice between symmetric vs. asymmetric
division, thereby regulating the determination of cell fate. Basically, the
differential inheritance of the apical or basal epithelial structure is important for
daughter cells to either self-renew or differentiate. However, this conclusion has
faced serious challenge (Siller and Doe). It has been proposed that cleavage
planes that bisect the crucial apical and basal membrane result in a symmetric
distribution of critical molecules and therefore generate two identical AP cells
(Kosodo et al.). On the other hand, cleavage planes that included tilted vertical
cleavage and horizontal cleavage will result in partition of the apical domain into
one of the daughter cells, leading to asymmetric division and generation of one
AP and one neurogenic cell(Kosodo et al.). This correlation between the partition
of apical membrane domain and cell fate has been demonstrated by Tis21
knock-in mice(Kosodo et al.). Therefore, it has been believed for a long time that
! 21!
spindle orientation determines the orientation of the cleavage plane, and the
positioning and alignment of the spindle is critical for the cell fate because minor
changes in spindle orientation may decide whether the cleavage plane would
bisect or bypass the small apical domain and result in equal or unequal
inheritance of the apical domain which is the putative cell fate determinant
between the daughter cells. However, this conclusion has been put into question
based on several recent discoveries. En-face time lapse imaging of sub-apical
membrane labeled with an EGFP-ZO1 reporter revealed that equal apical
surface inheritance is frequently followed by the retraction of one of the apical
attachments, which suggests that one daughter cell remains an apical
progenitors, while the other daughter cell has been adopted a different cell fate
(Shitamukai, Konno and Matsuzaki). This provides strong evidence that
asymmetric cell division is not correlated with the inheritance of the epithelial
structure of the RGCs. Moreover, this current conclusion is also supported by the
recently discovered fact that the inactivation of LGN function randomizes the
spindle orientation during the early NE proliferative phase in the mouse and chick,
but failed to effectively induce defects in cell fate determination and neurogenesis
(Konno et al.). Taken together, it is likely that indeed spindle orientation controls
the inheritance of the apical epithelial structure but does not directly influence cell
fate of the daughter cells produced by the division.

Several models that involve both apical and basal localized molecules have been
proposed as the candidate cell fate determinants in order to explain the
! 22!
molecular mechanism underlying asymmetric division. One attractive model
using apically localized molecules as candidates is that the centrosome
asymmetry leads to differential inheritance of cell fate determinants thereby
resulting in cell fate asymmetry during division. The recent discovery of the
functional link between SRAD and centrosome asymmetry in Drosophila and
rodents sheds light on an explanation for the incoherent phenotype observed
during disruption of spindle orientation (Lesage et al.; X. Wang, J. W. Tsai, J. H.
Imai, et al.; Yamashita et al.). Shi’s group demonstrated that the older mother
centriole is preferentially inherited by the renewed RGC, while the newly-made
centrosome is inherited by the differentiating sister cell (X. Wang, J. W. Tsai, J. H.
Imai, et al.). They also showed that centrosome asymmetry not only occurs but
plays a crucial role in SRAD of rodent RGCs. Depletion of Ninein, a component
of the centriolar appendages required for centrioles to mature and acquire
mother centrosome features, results in the depletion of AP pools (X. Wang, J. W.
Tsai, J. H. Imai, et al.). Thinking back actually in those cases that disruption of
spindle orientation resulted in cell fate defects, those genes are actually very
closely related to centrosome functions, including Hook3, Pcm1,Cep120, LIS1,
ASPM which were previously believed to function in the self-renewal VS
differentiation through regulating spindle orientation (Xie et al.; Ge et al.; Yingling
et al.; Fish et al.; Lesage et al.). The severe consequences of the loss of
centrosomal proteins on brain development strongly indicate the functional
relationship between centrosomal activity and self-renewal of AP. Five loci for
which loss of function causes human microcephaly, a condition that produces
! 23!
significantly smaller brains, have recently been identified to encode centrosome-
related proteins (Thornton and Woods). Another model that considers basally
localized molecules as candidate cell fate determinants is that the differential
inheritance of molecules located in the basal process actually is critical for
adopting different cell fates during cell division. For instance, recently beta 3-
intergrin and cycline D2 mRNA and proteins have been found to be located in the
basal process of RGCs and to play critical role in asymmetric cell division
(Glickstein, Alexander and Ross; Glickstein et al.; Tsunekawa et al.; Fietz et al.).
Concluding, although the questions of how division behavior regulates cell fate,
and what actually regulates the division behavior of APs are still fuzzy, it can be
anticipated that future studies will focus on elucidating the mechanism of
asymmetric division, especially in a bigger content of extrinsic signaling and cell
fate determinant transcription factors.

1.2.7 Major signaling pathways regulating self-renewal
Notch signaling: The function of Notch signaling has been heavily examined in
mouse neural development since it plays a prominent role in the maintenance of
neural progenitor self-renewal in Drosophila. Over the past few decades, mutant
mouse studies of the Notch pathway (including receptor, ligands, modulators and
effectors) have elucidated the role of Notch signaling (Yoon and Gaiano). In
general, disruption of any component of the Notch signaling pathway results in
precocious differentiation and a decrease in progenitor markers, which in turn
lead to the prevailing view that Notch maintains the self-renewal ability of neural
! 24!
progenitors. Specifically, knockouts mice of Notch1, Notch2, PS1/PS2 (activator
of Notch receptor), Dll1 (ligand for Notch receptors), Cbf1 (effector of Notch1),
Hes1/Hes5 (main downstream target of Notch/CBP pathway) and Mib1
(Mindbomb1, required for endocytosis-dependent activation of Notch ligand)
have shown a common phenotype of more precocious neuronal differentiation
and depletion of the progenitor pool, with an especially significant loss of RGCs
shown by a reduction of radial glia markers (Yoon and Gaiano). Conversely,
forced expression of a constitutively activated form of Notch1 receptor by
retroviral infection at E9.5 or misexpression of Notch target genes result in an
inhibition of differentiation and acquisition of more RGC characteristics (Ohtsuka
et al.; Sakamoto et al.; Rallu et al.). Hence, it seems that Notch signaling
promotes RGC identity. Notch signaling has also been shown to promote a
terminal differentiation of glia cells, and therefore, Notch signaling appears to act
to promote RGC identity as well as terminal glial differentiation.

Wnt signaling: The neural tube is a heterogeneous structure: at very early
stages of neural development, the neural tube has been patterned into distinct
cell fates (i.e. dorsal vs. central domain). There are a variety of master regulators,
other than Notch signaling, which promote the proliferation and differentiation of
different domains of the neural tube (Michaelidis and Lie). One example is β-
catenin-mediated canonical Wnt signaling, Wnt1 and Wnt3a being typical
components of the roof plate. Compelling data support the role of Wnt signaling
in contributing to the proliferation of neural progenitors and dorsal telencephalic
! 25!
patterning of the neural tube in early embryogenesis. Conditional inactivation of
β-catenin before the onset of neurogenesis leads to the loss of dorsal patterning
marker Emx1/2, Ngn2, as well as ectopic up-regulation of ventral cell fate makers
Gsh2, Mash2 and Dlx2 in the dorsal VZ, suggesting a cell fate shift toward
ventral fate (Backman et al.). Wnt-3a knockout, LEF1 knockout, LRP6 knockout
and conditional inactivation of β-catenin all show significantly disrupted
hippocampus development (Zhou, Zhao and Pleasure; S. M. Lee et al.; Galceran
et al.). These knockouts are specifically affected in the proliferation of precursors
to the hippocampus, but had minimal defects in the cortex. Conversely, over-
expression of a constitutive active and stabilized form of β-catenin in Nestin
expressing APs resulted in a significant enlargement of the brain and VZ and a
large increase in the proliferation of neural progenitors (Chenn and Walsh). This
further demonstrates that β-catenin is involved in the precursor decision between
proliferation and differentiation during mammalian neuronal development.
Although Wnt signaling has traditionally been considered to act on dorsal neural
progenitors, it has recently been demonstrated that conditional inactivation of
canonical Wnt signaling in ventral APs also results in significant reduction of
proliferation of the ventral neural progenitors (Gulacsi and Anderson). Therefore,
Wnt signaling not only specifies the dorsal telencephalic neuronal fate but also
promotes the proliferation of neural progenitors.

! 26!

Shh signaling: Shh signaling is centrally involved in the formation of the ventral
CNS. In Shh null mice, the telencephalon is greatly dysmorphic and reduced in
size. Shh signaling regulates both patterning and cell proliferation of the ventral
progenitors; its distinct function is regulated through dynamic changes in the
temporal competence of the neural progenitors. In general, neural progenitors
experience three different competence windows (Fuccillo et al.). In the first
competence window, from E8.5-E9.5, the ventral fate is first induced through Shh
activity via repression of dorsal patterning marker Pax6 and induction of ventral
patterning gene Nkx2.1, thus establishing a sharp boundary between the MGE
and dorsal domain (Fuccillo et al.). In the second competence window, from
E9.5-E10.5, the second eminence LGE emerges in response to Shh signaling
from the gap between the dorsal Pax6 expressing domain and more ventral
medial Nkx2.1 expressing domain, marked by patterning transcription factor
Gsh2 (Fuccillo et al.). The third competence window starts at around E12.5: at
this stage, the patterning activity of Shh is completed and the main role of Shh is
to regulate the proliferation of ventral progenitors including MGE and LGE. This
is supported by the fact that early removal of Shh activity at E9.5 from the ventral
telencephalon results in extensive patterning alterations, but conditional deletion
of Shh activity in Smo knockout mice from E12.5 onwards results in only in a
reduction in the number of neural progenitors and minor patterning abnormalities
(Fuccillo et al.).

! 27!

1.3 Neural progenitor cell specification and differentiation
The generation of a great diversity of cell types and the establishment of an
extremely complex neural connectivity are probably the most fascinating features
of CNS development. During development, neural stem cells give rise to the
three major neural cell types of the mammalian CNS in a tightly regulated
manner. A fundamental feature of neural development in vertebrates is that
different neural cell types are generated in a precise sequence and from a
regionally restricted population of the neural stem cells. The origin and cell fate
specification process has been intensively studied. The following sections will
mainly discuss the signaling and transcriptional regulation of specification of each
neural lineage.

1.3.1 Cortical projection neurons
The mammalian cerebral cortex is a highly ordered brain structure, mainly
composed of the glutamatergic cortical projection neurons and GABAergic cortial
interneurons. Cortical projection neurons exclusively arise from and are located
in the cerebral cortex, which is organized into six cell layers distinguished by
projection neurons with distinct morphology(Kwan, Sestan and Anton). Within
each layer, projection neurons adopt specific cellular and molecular identities and
project their axons to distinct but appropriate targets. The cell fate specification
of cortical projection neurons seems to follow a sequence of first, the induction of
telencephalic identity, and next, sequential generation of projection neurons
! 28!
belonging to each individual layer. There is compelling data supporting the idea
that the induction of telencephalic fate of the neural tube is mainly governed by
the combining of patterning morphogen gradients; but the ensuing sequential
production of each population of projection neurons belonging to each layer is
largely governed by an intrinsic program through temporal regulation. The
cerebral cortex arises from the anterior portion of the neural tube, the
telencephalon. Specifically, the dorsal telencephalon gives rise to the cerebral
cortex. The dorsal telencephalon is patterned or specified by the “organizing
centers of the forebrain” which include a dorsal source of BMPs and WNTs, a
ventral source of SHH, and rostral and anti-hem regions which are the source of
FGF8 and TGF (Rowitch and Kriegstein). All of these signals provide positional
information through morphogen gradients in the dorsal-ventral, anterior-posterior
and medial-lateral axes. These factors regulate the expression of patterning
genes of the pallium neural progenitors including transcriptional factors Lhx2,
Foxg1, Emx2, Pax6 and nuclear receptor Tlx etc, each of which has crucial roles
in specifying the progenitors that give rise to the projection neurons of the
neocortex (Arai et al.; Porter et al.; Hanashima, Shen, et al.; Roy et al.).

These exists substantial evidence that Wnt signaling plays a critical role in the
specification and proliferation of the dorsal telencephalon, especially the
formation of the hippocampus. Wnt1 and Wnt3a are components of the roof plate.
β-catenin mediated canonical Wnt signaling is one of the master regulators of
proliferation and dorsal cell fate. Conditional inactivation of β-catenin starting
! 29!
before the onset of neurogenesis leads to loss of dorsal patterning marker
Emx1/2, Ngn2 and ectopic up-regulation of ventral cell fate makers Gsh2, Mash2
and Dlx2 in the dorsal VZ suggesting a cell fate shift toward ventral
fate(Backman et al.). Wnt-3a, LEF1, LRP6 knockouts and conditional
inactivation of β-catenin all show significantly disrupted hippocampus
development (Zhou, Zhao and Pleasure; Galceran et al.; S. M. Lee et al.) and
specifically affected proliferation of precursors to the hippocampus but less
defect in cortex. Conversely, when over-expressing the constitutive active and
stabilized form of β-catenin in Nestin expressing APs, the brain and VZ are
significantly enlarged and the proliferation of neural progenitors is significantly
increased (Chenn and Walsh). This further demonstrates that β-catenin can
affect the decision of precursors to proliferate or differentiate during mammalian
corticogenesis. Although Wnt signaling has been traditionally considered to only
act on dorsal neural progenitors, recently it has been demonstrated that
conditional inactivation of the canonical Wnt signaling in ventral APs also results
in significant reduced proliferation of the ventral neural progenitors (Gulacsi and
Anderson). Therefore, Wnt signaling not only specifies the dorsal telencephalic
neuronal fate but also promotes the proliferation of neural progenitors.

BMPs expressed at the dorsal midlines of the telencephalon have also been
demonstrated to play essential roles in the specification of the dorsal
telencephalon. Conditional inactivation of BMP receptor 1a in the mouse dorsal
telencephalon in combination with complete inactivation of BMP receptor 1b
! 30!
results in dramatically reduced size of the dentate gyrus of the hippocampus
reflecting a decreased production of hippocampus neurons (Caronia et al.). It is
possible that BMP and Wnt signaling are functionally redundant in specifying
dorsal cell fate, which is supported by the fact that a telencephalic enhancer of
the Emx2 gene contains requisite binding sites for both Smad and Tcf
proteins(Campbell).

At the transcriptional level, four transcriptional factors expressed by the apical
progenitors Lhx2, Foxg1, Emx2 and Pax6 together establish the neocortical
progenitor domain by keeping the boundary between neocortical progenitors and
dorsal midline and ventral telencephalon (Molyneaux, Arlotta, Menezes, et al.).
Lhx2 and Foxg1 express in a medial high, lateral low fashion. In the absence of
Lhx2 and Foxg1, neocortical progenitors are not correctly specified, instead
cortical hem and archicortex expressed markers, which are usually restricted to
the medial edge of the telencephalon,significantly expanded and covered the
entire dorsal telencephalon of the mutant mice (Bulchand et al.; Hanashima, Li,
et al.). However, in these mutants ventral patterning is not changed. Emx2 and
Pax6 are expressed in the cortical APs with a medial low, lateral high pattern and
serve as the main patterning genes that antagonize the ventral cell fate. In the
absence of Emx2 and Pax6, the dorsal ventral boundary significantly shifted
upwards and ventral cell fate dramatically expanded to the dorsal area with
dorsal progenitors abnormally expressing ventral markers (Muzio et al.; Yun,
Potter and Rubenstein; Kroll and O'Leary). Collectively, all these data support a
! 31!
role of these 4 transcription factors in defining the neocortical domain by
repressing the adjacent cell fate.

The basic-helix-loop-helix (bHLH) pro-neural transcriptional factors have been
shown to play crucial roles in neurogenesis. Ngn1, Ngn2 and Mash1 have been
shown to be necessary and sufficient to induce a full program of neurogenesis in
vivo and in vitro in neural progenitors, Ngn2 and Mash1 can induce rapid and full
neuronal differentiation (Mizuguchi et al.; Nakada et al.). Remarkably, Ngn2 and
Mash1 have also been suggested to dictate a cortical neuronal cell fate during
transdifferentiation and reprogramming of non-neural cells. Moreover, Ngn2 has
been shown to directly activate specific genes required for neuronal
differentiation and migration of cortical projection neurons (Mizuguchi et al.;
Nakada et al.; Berninger, Guillemot and Gotz).

The subtype and laminar specification is mainly governed by a cell-intrinsic
temporally regulated program, in other words, temporally sequential expression
of certain sets of cell fate determinants (Molyneaux, Arlotta, Menezes, et al.).
Various subtypes of cortical projection neurons are produced in an “inside-out”
fashion (Merot, Retaux and Heng), except for the most early born neurons that
form the preplate (PP), which is later separated into layer 1 and layer SP. All the
other layers are formed such that later born neurons radially migrate and pass
the early born neurons and terminate on top of the early born neurons.
Importantly, neurons in the upper layers including layers 2, 3 and 4, project their
! 32!
axon within the cortical hemisphere, while neurons in the deep layers, including
layers 5 and 6, project their axon subcortically toward midbrain, hindbrain and
spinal cord (Molyneaux, Arlotta, Menezes, et al.). Abundant laminar-specific and
subtype-specific markers have been identified, followed with generation of
transgenic mutant mice of these genes, whose function has also been revealed.
Genes found playing a role in the cortical projection neurons subtype and laminar
specification can be divided into two groups. The first group consists of
transcriptional factors that specify the subtype cell fate of projection neurons.
They are usually not involved in the production of these neuron populations, but
rather instruct the projection target of these neurons. For example, Fezf2, Ctip2,
and OTX1 are transcription factors expressed by the deep layer neurons,
including layers V, VI and SP, which are subcortical projection neurons. Loss of
Fezf2, Ctip2 and OTX1did not significantly affected the production of these
neurons but severely impaired the establishment of their subcerebral projection,
axon outgrowth, pathfinding and axon pruning, which suggests a late stage
function of these genes in defining the characteristics of a subcerebral projection
neuron (Arlotta et al.; Weimann et al.; Molyneaux, Arlotta, Hirata, et al.; Chen,
Schaevitz and McConnell). Another group of genes are transcriptional factors
that are expressed by RGCs and intermediate progenitors until the postmitotic
stage and play earlier roles in RGCs in specifying the production of certain
subtypes of projection neurons that belong to certain laminar layer. Brn1/2 are
examples of this type of genes. Loss of Brn1/2 has resulted in decreased
numbers of layer II-V cortical neurons, and those layer II-V neurons that are born
! 33!
with Brn1/2 deletion exhibited abnormalities in migration and arresting in the VZ
and SVZ (Sugitani et al.). However, Brn1/2 are not specific to any subtype or
laminar layer, and hence, it is not clear whether there exist any markers to
distinguish among progenitors that generate different laminar projection neurons
or subtypes and it is not clear whether such lineage-committed progenitors exist
or not.

Figure 1.2 Cortical neurogenesis
Schematic model of the generation of cortical projection neurons, migration and
neuron-glia switch in the mouse neocortex. Obtained from the review (Kwan,
Sestan and Anton), detailed description please the text.

! 34!

Recently, the discovery of fate-restricted Cux2+ neural progenitors has shed light
on this topic. It has been demonstrated the existence of Cux2 expressing RGCs
that is specified to generate upper-layer neurons regardless of birthdate, this
results suggested that molecular fate specification ensures proper birth order,
rather than vice versa (Franco et al.). It is likely that the low level expression of
cell fate determinant is sufficient to determine the competence of RGCs, however
further detailed analysis and verification is needed in order to clarify this novel
hypothesis.

1.3.2 Cortical interneurons
Cortical interneurons comprise around 30% of the neuronal population in the
cerebral cortex. In contrast to cortical projection neurons, they use GABA as their
neurotransmitters and are inhibitory. Research over the past years has provided
compelling evidence that the origin of cortical interneurons is in the subpallium of
ventral telencephalon in the mouse, based on various experimental methods
including slice culture, genetic manipulations and fate mapping. Upon
differentiation, postmitotic interneurons undergo tangential migration along
several different routes before reaching their cortical targets. The development of
cortical interneuons from the ventral telencephalon initiated from the patterning of
ventral neural tube by extrinsic cue Shh signaling similar to the patterning of
dorsal telencephalon by Wnt/BMP signalings, but later on, the diversity of cortical
interneurons is mainly achieved by spatial segregation of neural precursors in the
! 35!
subpallium, which is distinct from the temporal regulation during the generation of
diversity of cortical projection neurons (Flames and Marin; Gelman and Marin).

Shh is secreted from the ventral midline of the forebrain and mainly governs the
establishment of ventral identities in the telencephalon. In Shh null mice, the
telencephalon is greatly dysmorphic and reduced in size, which suggests that
Shh signaling regulates both patterning and cell proliferation of the ventral
progenitors (Chiang et al.; Rallu et al.). However, the regulation of Shh signaling
undergoes dynamic change in different “competence windows” of the ventral
neural progenitors (Sousa and Fishell). Ventral neural progenitors experience
three different competence windows for Shh signaling. The first competence
window (C1) occurs around E8-E9 in the mouse, in which the ventral fate is first
induced by the activity of Shh signaling via repression of dorsal patterning gene
Pax6 and induction of ventral patterning gene Nkx2.1, hence a sharp boundary
between the medial ganglionic eminence (MGE) and dorsal domain is
established. The second competence window (C2) occurs around E9 to E10, in
which the lateral ganglionic eminence (LGE) emerges in response to Shh
signaling from the gap between Pax6 expressing dorsal VZ and Nkx2.1
expressing ventral medial domain, marked by expression of patterning
transcription factor Gsh2. The third competence window (C3) starts at around
E12.5, in which the patterning activity of Shh signaling is completed and the main
influence of Shh signaling is regulating the proliferation of ventral progenitors
including MGE and LGE. This competence window of ventral progenitor model is
! 36!
supported by the fact that early removal of Shh activity at E9.5 from the ventral
progenitors resulted in extensive patterning alterations, whereas conditional
deletion of Shh activity in Smo knockout mice starting from E12.5 onwards
resulted in only a reduction in the number of neural progenitors with minor
patterning abnormalities (Fuccillo et al.).

Although it is still unclear how many different types of cortical interneurons
actually there are, it is generally accepted that a diversity of interneuron does
exist: interneurons can be divided into 4 major classes based on their
neurochemical, anatomical and electrophysiological characters: (1) fast-spiking,
PV+ interneurons; (2) intrinsic burst spiking SST+ interneurons; (3) rapidly
adapting, CR+ or VIP+ interneurons; (4) rapidly adapting NPY+/SST- or
Reeling+/SST- interneurons(Gelman and Marin; Vitalis and Rossier). Although
cortical interneurons also preferentially invade certain layers, due to the
significant level of overlapping, the classification of cortical interneurons differs
from that of cortical projection neurons in that it is based on the interneuron’s
physical location or “layer” rather than its neurochemical marker expression.

Collectively, the current view on cortical interneurons’ origin includes mainly three
domains: the MGE, the caudal ganglionic eminence (CGE), and the preoptic area
(POA) (Flames and Marin; Gelman and Marin). The MGE mainly gives rise to PV
and SST containing interneurons, which account for near 60% of all the cortical
interneurons in rodents, based on fate mapping of PV and SST interneurons and
! 37!
analysis of the NKX2.1 and Lhx6 knockout mice (Lhx6 is a direct target of
Nkx2.1). It has also been demonstrated recently that the dorsal and ventral
domains of the MGE region preferentially give rise to SST and PV containing
interneurons, respectively (Gelman and Marin). Although the cell fate separation
of these domains is not sharp, this phenomenon of segregated progenitor
domain segregation associated with distinct cell fate is very similar to what has
been observed in the spinal cord. This segregation may involve the activity of the
Shh gradient, but the exact mechanism is still under investigation. Sox6 is a
recently identified target of Lhx6 (Batista-Brito et al.), and it seems that Sox6 is
more closely related to the generation of PV-containing interneurons, but less to
SST-containing interneurons, based on the analysis of Sox6 null mice (Azim et
al.; Batista-Brito et al.). This suggests that the MGE contains distinct progenitors
or precursor domains which are differentially specified by additional factors
downstream of Nkx2.1 and Lhx6. The CGE region is the main source of bipolar
and double-bouquet cortical interneurons, which make up around 30-40% of all
cortical interneurons. This interneuron population was not labeled by the fate
mapping of Nkx2.1 and Lhx6 expressing neural progenitors (Gelman and Marin).
Prior to this work, the CGE region was largely defined by anatomical means,
which can cause difficulties in analyzing the contribution of this region to cortical
interneuron production. Recent studies suggest that CGE is not just an extension
of the LGE region but rather a distinct domain with its own unique and defined
molecular profile. Specifically, the transcriptional factor Couptf2 has been
identified as a molecular marker of the CGE region (Kanatani et al.). Couptf2
! 38!
seems to primarily contribute to the migration of CGE-derived interneurons, but
the mechanism underlying the cell fate specification of CGE derived interneurons
remains largely unknown. In addition, the preoptic area (POA) region is a novel
origin of cortical interneurons, and has been suggested to produce up to 10% of
cortical interneurons (Gelman and Marin). The characteristic Nkx2.1+ /Lhx6-
expression profile can distinguish POA progenitors from MGE progenitors which
are Nkx2.1+/Lhx6+. Fate mapping of this domain revealed that POA progenitors
give rise to NPY and or reeling containing cortical interneurons, and may also
give rise to a small fraction of Lhx6 independent PV and SST cortical
interneurons (Vitalis and Rossier; Gelman and Marin).

In summary, the emergence of the physically segregated MGE, LGE, CGE and
POA progenitor domains, which express distinct patterning markers, reveals that
distinct transcriptional programs for different cell fate outcomes are already set in
place at the onset of neurogenesis of each domain. Additional factors, which
arise later, involve conferring or diversifying the progeny from these domains
later on. Therefore, the spatial segregation of ventral neural progenitors in the
subpallim is tightly associated with the subtype cell fate specification of cortical
interneurons. The contribution of temporal regulation to the cortical interneuron
cell fate diversity has also been suggested, that is the birth date may influence
regional and laminar differences during cortical interneuron migration and
terminal differentiation. However, the birth date periods largely overlap among
the major interneuron subtypes, and the extent to which the birth dates correlate
! 39!
with their final phenotypes is still unclear. A detailed analysis of the functional
correlation between the birthdates of cortical interneurons and subtype cell fate
may shed a light on this interesting topic.

1.3.3 Astrogenesis
Astrocytes provide structural support, regulate water balance, ion distribution,
and maintain the blood-brain barrier. Astrocytes have been shown to associate
with synapses and govern key steps of the synapse formation and plasticity.
Astrocytes arise from the neural progenitors, which reside in the dorsal
telencephalon after neurogenesis. In other words, astrogenesis is the terminal
cell fate output of RGCs. At the later stage of neurogenesis, neural progenitors
switch their competence from neurogenic to astrogenic. Research on the cell fate
specification of astrocytes has been focused on elucidating the neuron-glia
switch, compelling data have indicated that this fate switch is orchestrated by
both extrinsic environment cues and intrinsic mechanisms that decrease
neurogenic and increase astrogenic competence over the embryonic
developmental time.

Intrinsic cues that induce astrogenesis are mainly changes in the epigenetic
status of RGCs. A recent study by Yukiko Gotoh’s study has demonstrated that
neurogenic competence of RGCs is gradually decreased by the polycomb group
complex (Hirabayashi, Suzki, et al.). PcG proteins epigenetically suppress the
ngn1 locus during the astrogenic phase and thus trigger the neurogenic to
! 40!
astrogenic fate switching. Knockout of key components of this complex prolongs
the neurogenic phase and delays astrogenesis (Hirabayashi, Suzki, et al.).

Extrinsic cues have been shown to play critical roles in the induction of
astrogenesis. In particular, CNTF, LIF and CT-1 have been shown to activate
the JAK-STAT pathway that is the master activator of astrocyte cell fate(Bonni et
al.), thereby promoting astrocyte differentiation in vitro. It has been found that
STAT binds to the promoter of GFAP, the major regulator of astrocyte
differentiation, to activate its transcription (Bonni et al.). CT-1 has been shown to
be the endogenous activating cytokine of JAK-STAT pathway to induce the
neuron-glia switch(Namihira et al.). Although CT-1 is highly enriched in
embryonic cortical neurons but this neuron-derived CT-1 is essential for
astrogenesis because mice lacking CNTF and LIF do not exhibit many defects in
astrocyte development, whereas mice lacking CT-1 receptors including gp130
and LIFR have significantly reduced numbers of astrocytes (Namihira et al.).
Notch signaling has also been suggested to be involved in the regulation of JAK-
STAT signaling activity. Committed neuronal cells express Notch ligands and
activate Notch signaling in the neighboring NPCs, in this case activating Notch
target NF1 expression (Deneen et al.; Namihira et al.). NF1A then confers the
competence of astrocytic differentiation via binding to the GFAP promoter and
promoting its demethylation of the GFAP promoter (Deneen et al.; Namihira et
al.).

! 41!

1.3.4 Oligodendrogenesis
Oligodendrocytes are the myelin-forming cells in the CNS. Myelin provides
insulation for neuronal axons and allows saltatory conduction through the nodes
of Ranvier. Oligodendrocyte development in the mouse telencephalon occurs in
three distinct waves: oligodendrocyte precursor cells (OPCs) of the first wave
appear in the MGE and AEP at around E11.5 (Kessaris et al.; Pringle and
Richardson; Olivier et al.; Spassky et al.; Tekki-Kessaris et al.), arrive in the
cortex at around E16, and mature into myelinating oligodendrocytes postnatally
(Richardson, Kessaris and Pringle). A second wave of OPCs originates in the
LGE and/or CGE at around E14.5 (Kessaris et al.). OPCs have also been
observed to emerge in the dorsal telencephalon during postnatal stages
(Kessaris et al.; Yue et al.; Gorski et al.). Obviously, there are huge temporal and
spatial differences among these three distinct waves of oligodendrocytes.
Moreover, there are also huge differences in terms of the transcriptional profile of
their origins (first wave derived from Nkx2.1+ domain, second wave derived from
Gsh2+ domain and third wave derived from Emx2+ domain). Although it is not
clear whether the heterogeneity of oligodendrocytes exist or not , the majority of
OPCs are all considered to differentiate into oligodendrocytes and myelinate
axons regardless of their origin. Hence, whether and to what extent the temporal
and spatial differences of origins of oligodendrocytes contribute to the diversity of
the oligodendrocyte diversity is still unclear. In the past, a significant amount of
! 42!
research has been conducted in order to understand the transcriptional program
for oligodendrocyte induction. MGE and LGE derived oligodendrocytes require
expression of Olig genes in response to Shh signaling. Olig1/2 are basic helix-
loop-helix transcription factors that are expressed broadly the ventral VZ, SVZ, in
committed OPCs and throughout matured oligodendrocytes (Zhou, Wang and
Anderson). Olig2 are considered to be the cell fate determinant gene of this
lineage (Zhou, Choi and Anderson; Q. R. Lu et al.). Early oligodendrogenesis
from the MGE and LGE requires Shh signaling, whereas the third wave of
oligodendrogenesis derived from dorsal telencephalon is Shh-independent. The
molecular mechanism underlying later stage oligodendrogenesis remains unclear.
Interestingly, the Olig2 expressing domain in the ventral telencephalon
significantly overlaps with the GABAergic neurogenic Nkx2.1, Dlx2 and Mash1
expressing domains (Petryniak et al.). Therefore, it is likely that oligodendrocytes
and GABAergic neurons are specified as a net outcome of combination of
neurogenic and oligodendrogenic factors (Petryniak et al.).

1.4 Epigenetic regulation of neural cell fate specification
Epigenetics is the study of mitotically and/or meiotically heritable changes in
gene function or cellular phenotype caused by mechanisms other than changes
in the underlying DNA sequence (Russo). It refers to functionally relevant
modifications to the genome that do not involve a change in the DNA sequence.
Both DNA and histone proteins can be modified to function as part of the
epigenetic regulations. Most well established examples of epigenetic
! 43!
modifications are DNA methylation and histone modifications, both of which can
regulate gene transcriptional activity and gene expression without changing the
DNA sequences. Most of our understanding of the biological function of
epigenetic regulations is based on observations of loss or gain of function studies
of enzymes that acts as epigenetic modifiers, and genome-wide mapping of
these modifications, which can be correlated with certain biological phenomena.

DNA methylation is the most well characterized epigenetic modification of DNA. It
has been shown to play a critical intrinsic role during the neuron-glial switch. The
promoter of astrocyte cell fate determinant gene GFAP is methylated during the
early and mid stage of neurogenic stage so that the GFAP gene is essentially
blocked from transcriptional activation by present extrinsic molecules that directly
regulate the activation of the GFAP gene. At the later stage of embryonic
neurogenic stage, the GFAP promoter is demethylated, which enables the direct
binding of transactivators and gene expression. DNA hypomethylation result from
conditional deletion of Dnmt1, the maintenance DNA methylase cause
precocious astrocyte differentiation both in vivo and in vitro likely caused by
prematurely demethylation of GFAP promoter and other genes encoding the
glialgenic pathway.

Genome-wide DNA methylation patterns are established and maintained by the
coordinated action of three DNA methytransferaces, DNMT1, DNMT3a and
DNMT3b. However, loss of function studies in central nervous system reveals
! 44!
distinct actions with regards to neuronal development. Conditional deletion of
DNMT1 demonstrated that DNMT1 seems to be involved in the maturation
process of neurons postnatally, but it is not required for the maintenance or the
stability of matured neurons. Similarly, DNMT3a also exhibits a late stage
biological function. Mice with conditional deletion of DNMT3a are grossly normal
at birth and only exhibit abnormal gait and reduced motor coordination, which
suggests a late stage function in the maturation of the CNS. On the contrary,
DNMT3b, which is expressed in the neural progenitors early on, appears to be
involved in early stage neurogenesis supported by the prominent neural tube
defects between E13.4 and E16.5 (MacDonald and Roskams).

Histone modifications display high levels of complexity and diversity compared
with DNA methylation. The role of chromatin status conferred by the dynamic
histone modifications has been intensively studied and is better understood in
vitro in the context of embryonic stem cells (ESCs) differentiation process. In
pluripotent ESCs, it has been well accepted that key developmental regulators
are silenced or reduced to very low expression level, in order to maintain the
undifferentiated and pluripotent identity of ESCs. However, since these genes
are not completely silenced but rather maintained at a poised state or reversible
silencing level, they can respond to developmental cues quickly. This is
considered a critical feature of the ESCs. A specific “bivalent” chromatin
modification pattern has been shown to be a characteristic of the poised genes.
Genome-wide ChIP-Seq analyses have shown that developmental genes are
! 45!
trimethylated at the histone H3 lysine 27 (H3K27me3) by Polycomb repressor
complexes (PRCs) and trimethylated at the histone H3 lysine 4 (H3K4me3) by
the trithorax group (TrxG) proteins MLL (T. I. Lee et al.; Boyer et al.; Azuara et
al.; Bernstein et al.). Both H3K27me3 and H3K4me3 modifications are thought to
associate with gene repression and activation, respectively, and the combination
of H3K27me3 and H3K4me3 at the same time is referred to as a “bivalent”
chromatin mark, which is believed to maintain the specific gene at a
transcriptional “poised” status. Neural specifying genes like Pax6, Sox1, Nkx2.2
and Mash1 are bivalent in ESCs but derepressed upon neural lineage
differentiation (Mikkelsen et al.). On the contrary, genome-wide DNA methylation
analysis demonstrated that DNA methylation mediated long term silencing occurs
preferentially at tissue specific genes, for example astrocytes genes GFAP and
S100β (Hirabayashi and Gotoh).

During the transition from ESCs to NPCs, both the pluripotency related genes
and previously bivalent genes that are not associated with neural lineage
development become silenced. Bivalent genes that are not associated with
neural lineage lose their poised state and transform into repressed states,
whereas the promoters of bivalent neural lineage genes lose the H3K27me3
modification and become more accessible to for transcriptional
activation(Mikkelsen et al.). Interestingly, a subset of genes that are expressed
only in terminally differentiated neurons gain both H3K27me3 and H3K4me2
marks during the transition from ESCs to NPCs (Mikkelsen et al.; Mohn et al.).
! 46!
The newly established bivalent states of neuronal genes allow a quick response
to the neuronal differentiation cues and therefore confer a terminal neuronal
phenotype. Moreover, a subset of neuronal genes that function in terminally
differentiated neurons is repressed in ESCs and non-neuronal cells by the
transcriptional repressor REST, also known as NRSF, through interacting with
the DNA response element RE1 (Chong et al.; Ballas et al.). This interaction
recruits chromatin modifiers including HDAC1/2 and Sin3A and form a repressor
complex. It has been shown that loss of Rest resulted in derepression of
neuronal genes like calbindin1, which are directly regulated by REST, but did not
affect upstream neural lineage cell fate determinant genes like Sox1, Mash1 and
Ngn1 (Jorgensen et al.). It has been suggested that REST can also induce long-
term silencing of its target neuronal genes through inducing H3K9 methylation,
and DNA methylation (Lunyak et al.; Ballas and Mandel; Ballas et al.). Therefore,
it is likely that tissue specific progenitor cells like ESCs also have their own
defined chromatin modification pattern and their own set of poised developmental
genes that together shape the competence of NPCs.

Recently, great efforts have been made to understand the in vivo contribution of
epigenetic mechanisms in neural specification and differentiation. For example,
conditional deletion of the major players of Polycomb complex members Ring1b
and EZH2, respectively, during cortical neurogenesis were demonstrated to
result in loss of repressive histone marks at pro-neural genes including Ngn1 and
significantly extended the neurogenic phase and delayed the onset of astrocytes
! 47!
differentiation (Hirabayashi, Suzki, et al.). However, the roles of histone
modifications in vivo are much more complex than it appears initially. Conditional
deletion of Ezh2 at a slightly earlier developmental stage using a different Cre
mouse line, which allows the ablation of Ezh2 gene prior to the onset of
neurogenesis, resulted in the opposite phenotypes including accelerated
neurogenesis and an early onset of gliogenesis (Pereira et al.). The only
difference between these two studies is that in the previous study, a Nestin
promoter driven Cre line that deleted Ezh2 at the beginning of neurogenesis was
used, while the later study used the Emx promoter driven Cre line. The dramatic
different phenotypes upon Ezh2 deletion might be due to a dynamic role of
Polycomb proteins in regulating major developmental transitions in cortical
progenitor cells. One the other hand, conditional deletion of Ring1b in RGCs did
not severely impair the self-renewal and maintenance of the neural progenitor
pool, which is consistent with the hypothesis that PcG proteins are not essential
for the maintenance of self-renewal of progenitors but are essentially involves in
regulating differentiation potential (Hirabayashi, Suzki, et al.). However, knockout
and knockout down of Bmi1, another critical member of the PRC1, resulted in
significantly impaired neural progenitor pool and increased astrogenesis both in
vivo and in vitro (Zencak et al.). Conversely, overexpression of Bmi1 promoted
the self-renewal and proliferation (S. He et al.; Fasano et al.). Hence, both gain
and loss of function studies have suggested Bmi1’s critical roles for the
maintenance of self-renewal and proliferation capacity of neural stem cells, which
is very different from the function of Ring1b. Therefore, further studies are
! 48!
needed to reveal the mechanism underlying the discrepancy of PRC1
components during neural development.
In summary, current epigenetic studies have accumulated abundant insights
about the contribution of epigenetic regulations during the restriction of
differential potential of neural progenitor cells. However, due to the
heterogeneous and dynamic features of the developing CNS, our knowledge
about the precise mechanisms of epigenetic regulation, the causal relationships
between the epigenetic regulations, signaling pathways and extrinsic cues during
neural progenitors differentiation is still in its infancy.

! 49!

CHAPTER 2
WNT-RYK SIGNALING REGULATES THE CELL FATE SPECIFICATION OF
GABAERGIC NEURON VERSUS OLIGODENDROCYTE

2.1 Abstract
GABAergic neurons and oligodendrocytes originate from progenitors
within the ventral telencephalon. However, the molecular mechanisms controlling
neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-
fate changes, are poorly understood. We discovered that the Wnt receptor Ryk
promotes GABAergic neuron production while repressing oligodendrocyte
formation in the ventral telencephalon. We demonstrated that Ryk controls the
cell-fate switch by negatively regulating expression of the intrinsic
oligodendrogenic factors Olig2 while inducing expression of the interneuron fate
determinant Dlx2. In addition, we demonstrated that Ryk is required for
GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a
stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk
is sufficient to regulate the cell-fate switch by regulating the expression of
intrinsic cell-fate determinants. These results identify Ryk as a multi-functional
receptor able to transduce extrinsic cues into progenitor cells, promote
GABAergic neuron formation, and inhibit oligodendrogenesis during ventral
embryonic brain development.
! 50!

2.2 Introduction
Multipotent neural progenitor cells (NPCs) located in the ventral ventricular zone
(VZ) of the telencephalon follow complex differentiation paths. Two distinct types
of neural cells, the inhibitory GABAergic neurons and the oligodendrocytes, that
eventually help populate the dorsal telencephalon, arise from this spatially,
temporally and molecularly delimited “common” pool of NPCs (Le Bras et al.;
Miller; Wonders and Anderson; Kessaris et al.; Yue et al.). Generation of these
different cell types in proper ratios and at correct times is important for normal
development of the telencephalon. Both extrinsic and intrinsic molecular
regulators combine to assign uncommitted progenitor cells to specific neural
lineages and facilitate their differentiation. Major progress has been made in
understanding the intrinsic transcriptional regulators of cell-fate determination.
However, how these intrinsic factors are integrated with extrinsic signals at the
progenitor cell level to regulate the spatiotemporal pattern of neuro/gliogenesis is
less well understood.

Mouse embryonic neurogenesis begins around E8.5 and peaks around E14.5
(Bayer and Altman). GABAergic neurons originate from the subpallium, including
the medial ganglionic eminence (MGE), the lateral ganglionic eminence (LGE),
and the anterior entopeduncular area (AEP) (Wonders and Anderson).
Postmitotic GABAergic neurons migrate tangentially to the cortex and the basal
! 51!
ganglia and then integrate into the cortical projection neuron network, eventually
maturing postnatally (Wonders and Anderson). Oligodendrocyte development in
the mouse telencephalon occurs in three distinct waves: oligodendrocyte
precursor cells (OPCs) of the first wave appear in the MGE and AEP at around
E12.5 (Kessaris et al.; Pringle and Richardson; Olivier et al.; Spassky et al.;
Tekki-Kessaris et al.), arrive in the cortex at around E16, and mature into
myelinating oligodendrocytes postnatally (Richardson, Kessaris and Pringle). A
second wave of OPCs originates from the LGE and/or caudal ganglionic
eminences at around E14.5 (Kessaris et al.). OPCs have also been observed to
emerge in the dorsal telencephalon during postnatal stages (Kessaris et al.; Yue
et al.; Gorski et al.). Compared to dorsal telencephalic NPCs, which have been
intensely studied, little is known about the regulation of NPC differentiation in the
ventral telencephalon and the neuron-versus-glia fate decisions made in the
ventral forebrain.

Βeta-catenin-mediated canonical Wnt signaling has traditionally been considered
to be critical for the development of the dorsal telencephalon, including dorsal
NPC proliferation, patterning, and neuronal differentiation (Galceran et al.;
Hirabayashi, Itoh, et al.; Israsena et al.; S. M. Lee et al.; Grove et al.). Recent
discoveries show that β-catenin-dependent Wnt signaling is activated in the
ventral telencephalon (Shimizu et al.; Gulacsi and Anderson; Ye et al.) and is
associated with ventral tissue growth (Gulacsi and Anderson). Moreover, β-
catenin-dependent Wnt signaling is recently showed to play a critical inhibitory
! 52!
role in specification and maturation of oligodendrocytes in the telencephalon (Ye
et al.; Fancy et al.). Thus, a more complex role for Wnt signaling in growth and
differentiation of ventral NPCs is beginning to be appreciated. However,
questions including whether Wnt signaling is involved in ventral neuronal fate
specification, whether β-catenin-independent Wnt signaling is involved in the
development of the ventral telencephalon, and whether Wnt signaling acts on
different lineages concurrently at the progenitor level remain unclear.

Discovery of the Wnt receptor Ryk revealed further complexities to the Wnt
signaling network (W. Lu et al.; Inoue et al.). Ryk has been shown to have
various functions in the CNS; for example, Ryk is required for Wnt3a-induced
neurite outgrowth of dorsal root ganglion neurons (W. Lu et al.), for Wnt5a-
induced axon outgrowth (Azim et al.), for repulsion of cortical axon guidance by
Wnt5a (Keeble et al.), for Wnt3-mediated repulsive axon guidance (Schmitt et al.),
and for Wnt3-induced neuronal differentiation of dorsal telencephalic NPCs (Lyu,
Yamamoto and Lu).

Immunohistochemical analysis showed that Ryk is expressed in Nestin-positive
progenitors, in neurons, and in oligodendrocyte progenitors in the rodent central
nervous system (Kamitori et al.; Lyu, Yamamoto and Lu). These data strongly
indicate that Wnt signaling acts through Ryk receptors expressed on NPCs to
regulate NPC differentiation; however, it is not known if regulating neuronal
differentiation is the only function of Ryk-mediated Wnt signaling in the
! 53!
developing brain.

Here we report that during development of the ventral telencephalon, Ryk
receptor regulates cell-fate choice between the oligodendrocytic and GABAergic
neuronal differentiation pathways in ventral progenitor cells. Ryk does this
through a cell-autonomous mechanism by regulating the expression of the key
cell-fate determinants Dlx2 and Olig2. We further demonstrate that Ryk receptor
is responsible for Wnt3a-induced promotion of GABAergic neuronal
differentiation and inhibition of oligodendrocyte differentiation. Ryk intracellular
domain (ICD) is required and sufficient to suppress oligodendrocyte
differentiation and induce GABAergic neuronal differentiation, suggesting that
nuclear signaling of Ryk-ICD governs the neuronal versus oligodendroglial
differentiation of NPC.

2.3 Methods and Materials
2.3.1 Animals
All animals were maintained and handled according to the guidelines and
regulations of the Institutional Animal Care and Use Committee and the National
Institutes of Health. Maintenance and genotyping of Ryk
+/-
and Ryk
–/–
mice were
carried out as described (Halford et al.). Embryos and pups of both wild-type and
knockout mice were collected from timed, mated pregnant females.

2.3.2 Neural progenitor cell (NPC) culture
! 54!
NPCs were derived from E14.5 mouse telencephalons. Tissues were dissected
and dissociated as described (Reynolds and Weiss; Schmitt et al.). Cells were
cultured in serum-free Neurobasal medium (Invitrogen) supplemented with
mouse recombinant epidermal growth factor (20 ng/ml, Sigma-Aldrich), basic
fibroblast growth factor (20 ng/ml, R&D), B27 (Invitrogen), 1% penicillin-
streptomycin (Sigma-Aldrich), 1% L-glutamine, and 1% glutamax in T25 culture
flasks (Nunc) in a humidified 5% CO
2
/95% air incubator at 37°C. For
differentiation, neurospheres were dissociated into single cells and plated onto
poly-L-lysine/laminin-coated chamber slides (Nunc) at approximately 10
4
cells
per well in the presence of mitogen. After one day, medium was changed to NPC
medium without mitogen. After several additional days, cells were analyzed by
immunocytochemistry.

2.3.3 Immunohistochemistry and immunocytochemistry
For immunohistochemistry, embryonic brains were dissected and fixed in 4%
parafomaldehyde (PFA) at 4
o
C and cryoprotected in 30% sucrose. Samples
were embedded and frozen in Tissue Tek OCT compound, and sections were
then cut at a thickness of 12 µm. For immunocytochemistry, cell cultures were
fixed with 4% PFA in PBS for 15 minutes and immunostained after
permeabilization in 0.2% Triton X-100. Immunostaining for membrane proteins
was performed without prior permeabilization. The following antibodies were
used: anti-β Galactosidase (1:1000, Abcam); Anti-Nestin (1:400, BD); anti-tubulin
III (1:1000, Sigma-Aldrich); anti–glial fibrillary acidic protein (GFAP) (1:1000,
Sigma-Aldrich); anti-Olig2 (1:400, Chemicon); anti-NG2 (1:400, Millipore); anti-
! 55!
PDGFRα receptor (1:200, Santa Cruz); anti-O4 (1:200, Chemicon); anti-
Galactosidase-C (GAL-C) (1:400, Sigma-Aldrich); and anti-myelin basic protein
(MBP) (1:400, Abcam); anti-Nkx2.1 (1:300, Millipore); anti-Islet1(1:100,
Hybridoma Bank); anti -Shh (1:1000, Hybridoma Bank); anti-Dlx2 (1:400, Abcam);
anti-GABA (1:3000, Sigma); anti-Gad67 (1:1000, Millipore); anti-Calbindin
(1:1000, Swant); Mash1 antibody (1:10), kindly provided by Dr. David Anderson;
Gsh2 antibody (1:2000), a kind gift from K. Campbell. For BrdU labeling, cells
were treated with 2 N HCl for 30 min and detected by using mouse anti-BrdU
(1:50, Beckton Dickinson) or rat anti-BrdU (1:350, Accuratech) antibody. TUNEL
assays were performed using the In Situ Cell Death Detection Kit (Roche)
following the manufacturer’s instructions. BrdU- and TUNEL-positive cells were
counted and percentages relative to total DAPI-stained cells were calculated in
several independent experiments. Immunolabeled cells were detected by using
fluorescently-labeled secondary antibodies (1:250, Jackson Immunoresearch
Laboratories, West Grove, PA). Viable cells were identified by Hoechst nuclear
staining.

2.3.4 Clonal assay
In the clonal assay, wild-type and knockout NPCs were mechanically dissociated
into single cells and grown as clonal colonies in the presence of 0.9% (w/v)
methylcellulose matrix under proliferation conditions for one week. Individual
neurospheres were then picked and transferred to 24-well plates (1 neurosphere
per well), and cultured for another week. These colonies were collected and
! 56!
plated onto plastic chambers. Subsequently, differentiation was carried out as
described above. Six days later, differentiation of neurons and glia of individual
clones were examined by immunocytochemistry. More than 100 clones were
examined.

2.3.5 BrdU labeling
For in vivo BrdU labeling, time-mated pregnant females were injected
intraperitoneally with 50 µg per g body weight of 5-bromo-2’-deoxyuridine (BrdU)
(Sigma). Mice were culled 30 minutes later and embryos were collected, fixed in
4% parafomaldehyde and embedded in OCT compound for sectioning. Sections
were stained with anti-BrdU antibody. For in vitro BrdU labeling, dissociated
NPCs were plated onto coated slides in the presence of mitogen. Twenty-four
hours later, mitogen was removed by changing the medium, and cells were
treated with BrdU (10 µM) for 3 h and cultured for an additional 2 days to allow
NPC differentiation.

2.4 Results
2.4.1 Expression of Ryk receptor
We investigated the expression of Ryk in NPCs in vivo by performing
immunohistochemistry on coronal sections of brains from wild-type,
heterozygous and homozygous Ryk knockout mice at various embryonic stages.
In the Ryk knockout allele, part of the Ryk gene from exon 2 to exon 6 was
replaced by a expression cassette LacZ gene; thereby, the endogenous Ryk
! 57!
promoter drives expression of LacZ, allowing identification of Ryk-expressing
cells (Halford et al.). LacZ staining, and by inference Ryk expression, in the
mouse telencephalon is detectable at E10.5 (data not shown), peaks at E12.5,
and is sustained until E14.5 (Fig. 2.1 A); it then decreases at around E16.5,and is
barely detectable by E18.5 (data not shown). Staining at E12.5 and E14.5
indicates that Ryk is highly expressed in cells in the VZ, in both the dorsal and
ventral regions (Fig. 2.1 A). Particularly high expression was detected in the
MGE and LGE from E12.5 to E14.5 (Fig. 2.1 A). Ryk was also expressed in the
cortical plate, which consists mainly of newly differentiating neurons at E14.5 (Fig.
2.1 A), indicating that Ryk is expressed in both proliferating mitotic and
postmitotic cells at this stage. LacZ expression in the VZ of the MGE and LGE
showed co-localization with Nestin (Fig. 2.1 B), a marker of NPCs, suggesting
that Ryk is expressed by the primitive ventral progenitor pool. Nestin expression
was equivalent between heterozygous and homozygous knockout mice at E14.5
(data not shown). LacZ is also expressed in Nestin-negative cells located in the
subventricular zone (SVZ) and mantle zone (Fig. 2.1 C), suggesting that Ryk is
expressed in a differentiated cell population derived from ventral VZ progenitor
cells. The finding of Ryk expression in ventral NPCs raised the possibility that
Ryk participates in the development of ventrally derived cell lineages, including
oligodendrocytes and GABAergic neurons.

2.4.2 Phenotypes of Ryk knockout mice
By observing the patterns of Ryk expression, we proceeded to test whether Ryk
! 58!
mutants have any defects in oligodendrogenesis. Using immunohistochemistry
analysis of OPC markers, we found that Ryk knockout mice exhibited increased
numbers of OPCs and pro-oligodendrocytes in the forebrain throughout
embryonic development. Staining for PDGFRα, one of the earliest OPC markers,
in E12.5 brain sections revealed significantly more PDGFRα+ cells in the mantle
zone of the MGE of knockout mice than of wild-type mice (Fig. 2.2 A, B). Analysis
of Olig2 staining showed that Olig2 expression was enhanced in the MGE of the
mutant (Fig. 2.2 A). At E18.5, 2.5-fold more PDGFRα+ cells were seen in both
the cortex and striatum of knockout mice compared to wild-type controls (Fig. 2.2
C, D). At E18.5, staining of NG2, another OPC marker, showed a two-fold
increase in the number of NG2+ cells in the cortex of knockout mice compared to
the control (Fig. 2.2 C, D). Immunostaining of oligodendrocyte markers in P0
mouse brains showed that the numbers of cells expressing NG2, O4, and GalC
(pro-oligodendrocyte markers) were all significantly increased in the cortex and
striatum of knockout mice compared to wild type (Fig. 2.2 E, F). Besides OPC
markers, we also examined expression of the mature oligodendrocyte marker
myelin basic protein (MBP), which begins to be expressed in myelinating
oligodendrocytes one week after the P0 stage. No MBP staining was detected in
either wild-type or knockout P0 mice (data not shown).

We next investigated whether the increase in production of the first wave of
OPCs observed in the MGE region of E12.5 Ryk knockout mice is caused by
altered timing of differentiation. We examined the timing of the first wave
! 59!
oligodendrogenesis by performing PDGFRα staining on sections of E9.5, E10.5
and E11.5 wild-type and knockout samples. No PDGFRα+ cells were detected in
wild-type or knockout E9.5 and E10.5 brains. The first detectable PDGFRα +
cells in both wild-type and knockout brains were found in E11.5 samples.
Interestingly, the numbers of PDGFRα+ cells per section in knockout mice were
double those of wild type. Therefore, our data suggest that the timing of MGE-
derived oligodendrogenesis is not significantly changed, but production of first-
wave OPCs is increased in the mutant.

Based on the fact that oligodendrogenesis occurs as three distinct waves, we
examined whether the second wave of oligodendrogenesis originating in the LGE
is affected in Ryk knockout mice. Immunostaining of Olig2 at E14.5 in the LGE
region showed significantly more Olig2+ cells migrate out of the LGE VZ into the
SVZ and mantle zone in the knockout mice compared to controls. We then
proceeded to perform BrdU injection of 15.5-day pregnant mother mice and
analyzed the OPCs differentiated from these labeled cells 2 days later. NG2 and
BrdU double staining was carried out to identify OPCs that were labeled by BrdU
at E15.5. Most NG2+/BrdU+ OPCs were detected in the septum area (data not
shown). Interestingly, quantification of NG2+/BrdU+ cells showed a two-fold
increase in the knockout mice compared to wild-type controls, suggesting that
more OPCs are being produced at E15.5 in the mutant. These results
demonstrate that deletion of the Ryk gene causes increased numbers of OPCs to
be generated in both the first and second waves of OPC production in the ventral
! 60!
telencephalon.

Figure 2.1 Ryk knockout mice exhibit increased oligodendrogenesis during
brain development

(A) Lac Z staining of coronal sections of E12.5 and E14.5 wild-type,
heterozygous, and knockout mouse brains. Nuclei are stained by DAPI. V,
ventricular zone; LGE, lateral ganglionic eminence; MGE, medial ganglionic
eminence.(B) Co-immunostaining of LacZ and Nestin plus DAPI staining in E14.5
heterozygous brain in the ventricular zone of the LGE region indicates Ryk and
Nestin co-expression (arrowheads). (C) Co-immunostaining of LacZ and Nestin
plus DAPI staining of E14.5 heterozygous brains in the mantle zone of LGE
indicates that Ryk is expressed by Nestin-negative differentiated cells. Scale bars
are 100 µm.

! 61!
Figure 2.2 Ryk knockout mice exhibit increased oligodendrogenesis during
brain development

(A) PDGFRα and Olig2 immunostaining of the MGE region of E12.5 wild-type
and Ryk knockout brains. Dashed line indicates the VZ boundary. (B)
Quantification of PDGFRα+ and SVZ-located Olig2+ cells per area. (C) PDGFRα
immunostaining of striatal sections and NG2 immunostaining of cortical sections
of E18.5 brains. V, ventricular zone; St, striatum; Cx, cortex. (D) Quantification of
PDGFRα+ and NG2+ cells per area. (E) NG2, O4 and GC expression in P0
brains. (F) Quantification of NG2+, O4+ and GC+ cells per area in P0 brains.
Scale bars are 50 µm. ** P< 0.01, *** P< 0.001.

! 62!
Formation of GABAergic neurons is reduced in Ryk knockout mice
It has been shown using clonal analysis that OPCs and GABAergic neurons
likely derive from a common ventral telencephalic progenitor (Yung et al.; W. He
et al.). Given the substantial increase in oligodendrocyte numbers in Ryk
knockout mice, we next examined if production of GABAergic neurons is also
affected in the mutant.

We analyzed neuron production in the ventral forebrain through immunostaining
of the GABAergic neuronal markers GAD67 and GABA, and found a significant
reduction in expression of GAD67 and GABA in the striatum and septum area of
Ryk knockout mice at E14.5 compared to controls (Fig. 2.3 A). Reduced numbers
of migrating GABA+ cells were found in the piriform cortex of Ryk knockout mice
(Fig. 2.3 A). We also observed reduced expression of Dlx2 in the ventral MGE
and LGE region of E14.5 Ryk knockout brains relative to controls (Fig. 2.3 B).
Dlx1 and Dlx2 (Dlx1/2) have been shown to play a critical role in controlling
neuro/oligodendrogenesis in the developing telencephalon (Petryniak et al.;
Parras et al.): Dlx1/2 mutant mice showed a dramatic increase in OPC formation
at the expense of GABAergic neuron differentiation. In Ryk knockout mice,
expression of the GABAergic neuronal maker Lhx6 was also found to be reduced
(Fig. 2.3 B). Moreover, the numbers of migrating Dlx2+, Lhx6+, Calbindin+
(GABAergic neurononal marker) cells in the piriform cortex were also significantly
reduced in Ryk knockout mice (Fig. 2.3 B, C). As a consequence of impaired
neuron formation at this early stage, labeling for GABA and Calbindin revealed a
! 63!
significant reduction in the density of cortical GABAergic neurons in the cortex of
Ryk knockout mice at E18.5 (Fig. 2.3 D, E). Thus, increased OPC formation
occurs concomitantly with reduced GABAergic neuron formation in the ventral
telencephalon of Ryk knockout mice. Our data suggest that Ryk promotes
GABAergic neuron generation while repressing oligodendrogenesis by regulating
Dlx2 expression.

Cell proliferation and cell survival of Ryk knockout mice are not affected
We next examined if progenitor proliferation or cell survival is affected in Ryk
mutant embryos. We first detected S-phase cells by administration of BrdU to
12.5-day pregnant mother mice 30 minutes before sacrifice. As expected, BrdU-
labeled S-phase cells in the forebrain of E12.5 embryos were located basally in
the VZ, whereas robust staining of Ki67, a marker of proliferating cells, was
detected in the apical layer of the VZ (Fig. 2.4 A). The number of BrdU-labeled S-
phase cells and Ki67+ proliferating cells in the MGE was not visibly altered by the
absence of Ryk (Fig. 2.4 A, B).

! 64!
Figure 2.3 Impaired GABAergic neuron development in Ryk knockout mice

(A) Immunostaining of GABAergic neuronal markers GAD67 and GABA in the
ventral telencephalon of E14.5 wild-type and Ryk knockout mice. Areas indicated
by arrowheads are enlarged to the right. Se, septum; St, striatum; Pcx, piriform
cortex. (B) Immunostaining of Dlx2 and Lhx6 in the ventral telencephalon (left
panels) and cortex (right panels) of E14.5 wild-type and Ryk knockout mice. (C)
Histogram depicts the percentage of Dlx2+, Lhx6+ and Calbindin+ cells in the
knockout E14.5 cortex relative to wild-type cortex. (D) Immunostaining of GABA
and Calbindin in the cortex of E18.5 wild-type and Ryk knockout mice; white
boxes indicate the insets. Cx, cortex; CP, cortical plate; MZ, marginal zone; IZ,
intermediate zone. (E) Histogram depicts the percentage of GABA+ and
Calbindin+ cells in the knockout E18.5 cortex relative to wild-type cortex. Scale
bars are 100 µm. * P< 0.05, ** P< 0.01, *** P< 0.001.

! 65!
To further examine proliferation versus differentiation, we conducted
immunohistochemical staining of BrdU and PDGFRα. Most PDGFRα+ cells were
BrdU-negative in both wild-type and knockout brains (Fig. 2.4 C). While the
numbers of BrdU-labeled proliferating cells were similar between wild-type and
knockout mice, the number of PDGFRα+ oligodendrocyte precursors in the MGE
was significantly increased in E12.5 Ryk knockout mouse embryos (Fig. 2.4 C).
Since OPCs are mitotic cells, increased generation of OPCs in the MGE region
can be due to enhanced proliferation of OPCs, we have performed double
immunostaining of PH3 (phosphorylated histone 3, an M phase marker) and
Olig2 at E12.5, however the percentage of PH3+/Olig2+ cells in mantle located
Olig2+ cells is not changed (data not shown) which suggested that OPC
proliferation is not likely the cause of the increased OPCs induction. To further
examine Ryk’s role in regulating NPC differentiation rather than proliferation, we
performed in vitro culture of primary NPCs. We isolated NPCs from both wild-
type and mutant E14.5 telencephalon, plated them onto coated slides, treated
the cells with BrdU for 3 hours after removing the mitogen, and cultured the cells
under differentiation conditions for an additional 2 days. During the 3-hour BrdU
treatment, newborn cells were labeled. The cell fates of these BrdU-labeled cells
during differentiation were evaluated by staining for the lineage markers GFAP,
TuJ1, and O4 (Fig. 2.4 D). Whereas the percentage of GFAP+/BrdU+ cells
among total BrdU+ cells was not changed in knockout cultures (Fig. 2.4 E), the
percentage of TuJ1+/BrdU+ cells was decreased and the proportion of
O4+/BrdU+ cells was significantly increased (Fig. 2.4 E), suggesting that, in the
! 66!
absence of Ryk, greater numbers of NPCs were fated to become
oligodendrocytes rather than neuronal cells. Overall, our in vivo and in vitro
results suggest that Ryk gene deletion affects differentiation instead of
proliferation of NPCs in the ventral VZ.

We next employed TUNEL staining to assess apoptosis in the MGE at E12.5 in
vivo. Few apoptotic cells were detected in both wild-type and knockout brains at
this stage, and there was no significant difference between the two groups (data
not shown). Similar results were found in the LGE region at E14.5 as well (data
not shown). To rule out the possibility of different rates of apoptosis in neuronal
versus oligodendroglial lineages, we performed TUNEL analysis in combination
with staining of the lineage markers Nestin, GFAP, TuJ1 and O4 on differentiated
cell cultures. The percentages of apoptotic cells showed no significant difference
between the neural lineages (data not shown).

! 67!
Figure 2.4 Enhanced oligodendrocyte differentiation without impaired
proliferation in Ryk knockout mice

(A) Co-immunostaining of Ki67 and BrdU plus DAPI staining in the MGE region
of E12.5 brains. (B) Quantification of percentage of Ki67+ and BrdU+ cells in Ryk
knockout mice relative to wild-type. (C) Co-immunostaining of PDGFRα and
BrdU in the MGE of E12.5 brains. Dashed line is the VZ boundary. (D) Co-
immunostaining of GFAP, TuJ1, O4, and BrdU in dissected telencephalic cells
treated with BrdU for 3 h and cultured under differentiation conditions for 2 days.
(E) Quantification of the percentage of GFAP+/BrdU+, TuJ1+/BrdU+, O4+/BrdU+
cells and total BrdU+ cells (n=3). Scale bars are 50 µm.

! 68!
2.4.3 In vitro differentiation of Ryk knockout NPCs
We further examined the role of Ryk in cell-fate specification by using
neurosphere culture. NPCs were isolated from the telencephalon of E14.5 wild-
type and Ryk knockout mice and maintained in proliferation conditions in the
presence of epidermal growth factor (EGF) and basic fibroblast growth factor
(FGF). Neurospheres from floating culture were then plated on slides and
cultured under differentiation conditions. On differentiation day 2, most cells were
Nestin+ in both wild-type and Ryk knockout cell cultures, with no noticeable
morphological differences between the two cultures (data not shown). On
differentiation day 6, the number of TuJ1+ cells was decreased in the Ryk
knockout cell culture. Tuj1 is an early neuron marker. In addition, TuJ1+ cells
exhibited an immature shape with shortened dendrites in the Ryk knockout cell
culture (Fig. 2.5 A). Consistent with increases of NG2+ and Olig2+ cells, the
number of O4+ cells was also significantly increased in Ryk knockout cultures
(Fig. 2.5 A). However, the number and morphology of GFAP+ astrocytes were
similar in Ryk knockout and wild-type cultures (Fig. 2.5 A). Quantitative analysis
of cells of all three lineages, (i.e., neurons, oligodendrocytes, and astrocytes) on
differentiation day 6 confirmed the visual observations (Fig. 2.5 B). We also
examined the expression of the mature-oligodendrocyte marker MBP in vitro:
immunostaining of NPC cultures on differentiation day 9 showed that the number
of MBP+ cells was increased by 2.5-fold in knockout cells compared with wild-
type controls (Fig. 2.5 C, D). These results demonstrate that Ryk signaling
inhibits oligodendrocyte lineage differentiation during NPC differentiation in vitro.
! 69!
This observation is further supported by expression profiling of neurogenesis and
oligodendrogenesis genes. Quantitative PCR of NPC cultures on differentiation
day 6 showed a significant increase in expression of Nkx2. 2 and Olig2, both of
which control oligodendrocyte differentiation, and a decrease in expression of
Ngn1 and Ngn2, which regulate neuronal differentiation (Fig. 2.5 E). Together,
results from these in vitro experiments demonstrate that Ryk inhibits
differentiation of NPCs into oligodendrocytes.

! 70!
Figure 2.5 Ryk knockout NPC cultures give rise to reduced neurons and
increased numbers of oligodendrocytes though a cell-autonomous
mechanism

(A, B) Immunostaining and quantification of TuJ1, O4, GFAP and GABA in wild-
type and Ryk knockout cell cultures on differentiation day 6 (D6) (C) MBP
expression in wild-type and knockout cell cultures at D9. (D) Histograms show
normalized fold changes in MBP+ cells. (E) Expression of cell fate determinant
genes at D6 in wild-type and knockout cell cultures as determined by quantitative
RT-PCR and normalized. (F) Neurospheres subjected to the clonal assay:
different kinds of clones were identified by immunostaining for GFAP/TuJ1 and
GFAP/O4. Images to the left show four different types of clones: tri-potent clones
(N/O/A), bi-potent clones (O/A, N/A), and uni-potent clones (O-alone). The table
to the right compares wild-type and Ryk knockout NPCs in the clonal assay. The
parameters used to evaluate the differentiation properties of each clone are
shown on the top row. The percentage of clones for each parameter is listed
below for both wild-type and Ryk knockout cells. More than 100 clones were
tested. (G) Mixtures of knockout NPCs transduced with a GFP-expressing
construct and an excess of wild-type NPCs were cultured under differentiation
conditions at D6 and then subjected to immunostaining for TuJ1 and O4.
Percentages of GFP+/ TuJ1+ and GFP+/O4+ cells were plotted. Scale bars are
100 µm. * P< 0.05, ** P< 0.01, *** P< 0.001.

! 71!
2.4.4 Cell-autonomous effects of Ryk receptor
The fact that the Ryk knockout mice exhibited opposite phenotypes with respect
to neuronal differentiation and oligodendroglial differentiation raises the
possibility that the altered oligodendrocyte differentiation pattern is due to a cell-
non-autonomous mechanism which is secondary either to defects of tissues
other than NPCs or to the defect in neuronal differentiation. To address the first
aspect of this issue, we carried out clonal assays to examine whether the altered
differentiation pattern occurs at the progenitor level (Sugimori et al.; Gritti, Galli
and Vescovi; Reynolds and Weiss). We let both wild-type and knockout single
neural progenitor cells grow as clonal colonies in methylcellulose matrix, and
subsequently, individual colonies were isolated and subjected to differentiation
assay. Differentiated cells were detected and analyzed by immunostaining with
lineage markers TuJ1, O4 and GFAP (Fig. 2.5 F). Under our conditions, three
kinds of clones can be found: tri-potent clones (N/O/A: N stands for neuron, O
stands for oligodendrocyte, A stands for astrocyte); bi-potent clones (N/O, N/A
and O/A clones); and very few uni-potent clones (N-alone, O-alone, and A-alone
clones) (Fig. 2.5 F). Because each clone or each clonal neurosphere is formed
without any contact with other cells, the differentiation properties reflect the
intrinsic characteristics of the initial progenitor. The percentage of tri-potent
clones in total was similar between wild-type and knockout cultures; however,
N/A bi-potent clones decreased 53% while O/A bi-potent clones increased 63%
in the knockout cultures (Fig. 2.5 F). Moreover, uni-potent O-alone clones, which
can differentiate only into oligodendrocytes, were significantly increased, to 5%,
! 72!
in the knockout culture compared with the wild-type culture, which contained 0%
(Fig. 2.5 F). In general, the number of N-containing clones was reduced in the
knockout culture by 15% and the number of O-containing clones was increased
by 21% (Fig. 2.5 F). The number of A-containing clones was not changed (Fig.
2.5 F). Therefore, the results demonstrate that the observed opposite changes in
the generation of neurons and oligodendrocytes in the Ryk knockout NPC
populational assay are indeed attributable to abnormal cell-fate commitment of
NPCs at the progenitor level. These data also indicate that defects in Ryk
mutants occur in NPCs, not secondary to abnormality of other cell types or
tissues.

The second aspect of the cell-non-autonomous possibility is the potential
existence of a feedback signal released from differentiated neurons that can
inhibit oligodendrocyte formation and lead to the opposite phenotypes of neuron
and oligodendrocyte differentiation in knockout mice. To address this, we labeled
knockout NPCs with a GFP-expression construct using lentivirus and mixed them
with an excess amount of unlabeled wild-type NPCs, then cultured them under
differentiation conditions for 6 days. Lentiviral infection efficiency for knockout
NPCs reached 100% in this case. Immunostaining of the lineage markers TuJ1
and O4 in the culture showed that GFP-labeled knockout NPCs differentiated into
11.5% (±0.9%) TuJ1+ neurons and 18.7% (±2.2%) O4+ oligodendrocytes (Fig.
2.5 G; cf. B), suggesting these cells exhibited the same differentiation pattern as
the pure knockout cell culture (TuJ1+11.1%, O4+18.8%). Thus, wild-type cells
! 73!
cultured in the same dish did not rescue the abnormal cell-fate commitment of
knockout cells to any extent. Therefore, it is unlikely that differentiated neuronal
cells release any type of inhibitory signal to suppress oligodendrocyte formation
and change the differentiation pattern in the knockout NPCs. In summary, our
data demonstrate that the altered cell-fate commitment in Ryk knockout mice
occurs in progenitors, and is not secondary to abnormality of other cell types or
to defects in any cell-cell interactions, rather it is a result of a change of cell-fate
decision.

2.4.5 Wnt proteins regulated differentiation of NPCs
To test the hypothesis that Ryk mediates Wnt signaling in modulating neuronal
versus glial cell-fate specification, we treated wild-type and knockout NPC
cultures with Wnt3a recombinant protein during the differentiation assay. Wnt3a
has been shown to bind to Ryk receptor to promote neurite outgrowth (W. Lu et
al.), and Wnt3a can induce neuronal differentiation of NPCs in both the forebrain
and spinal cord (Muroyama et al.; Lie et al.; Yu et al.; Kalani et al.). Wnt3a was
recently shown to up-regulate gene expression of the oligodendrocyte inhibitors
Id2 and Id4 and to down-regulate expression of myelin genes such as MBP in
hippocampus-derived adult neural progenitor cells and oligodendroglial cell
cultures (Ye et al.). Interestingly, Wnt3a treatment increased the percentage of
GABAergic neurons which were GABA+/TuJ1+ by 50% in the wild-type cell
culture, but had no such stimulatory effect on knockout cell cultures (Fig. 2.6 A),
suggesting that Ryk receptor is required for GABAergic neuron differentiation
! 74!
induced by Wnt3a. We then asked whether the interaction between Wnt3a and
Ryk receptor is critical for this phenomenon. We found that applying Ryk
antibody not only brought down the percentage of GABA+/TuJ1+ neurons in the
wild-type cell culture to the level observed in the knockout culture, but also
almost completely eliminated Wnt3a-induced generation of GABA+/TuJ1+
neurons in the wild-type culture (Fig. 2.6 A). Therefore, we conclude that Ryk
receptor plays a crucial role in Wnt3a-induced differentiation of GABAergic
neurons in vitro.

Wnt3a treatment induced the opposite effect on O4+ oligodendrocyte
differentiation. A 25% decrease in O4+ cells was found following Wnt3a
treatment of the wild-type culture. Such a suppressing effect of Wnt3a, however,
was not significant in the knockout cell culture (Fig. 2.6 B). As expected, applying
Ryk antibody doubled the production of O4+ cells, yielding numbers very close to
the level observed in the knockout cell culture (Fig. 2.6 B). Application of Ryk
antibody also eliminated the inhibitory effect of Wnt3a. Taken together, our data
indicate that Ryk receptor plays a critical role in Wnt3a-induced inhibition of
oligodendrocyte differentiation.

! 75!

Figure 2.6 Ryk receptor is required for Wnt3a-induced GABAergic neuron
differentiation and inhibition of oligodendrocyte differentiation

(A) Ryk receptor is required for GABAergic neuron differentiation stimulated by
Wnt3a. GABAergic neurons were identified by co-immunostaining of TuJ1 and
GABA. (B) Ryk receptor is critical for Wnt3a-induced suppression of
oligodendrocyte differentiation demonstrated by O4 immunostaining. * P< 0.05, **
P< 0.01, *** P< 0.001.

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2.4.6 Ryk-ICD is necessary and sufficient to regulate cell fate switch during
differentiation
Previously, we discovered that cleavage of Ryk and subsequent nuclear
localization of the cleavage product, Ryk ICD, constitutes a novel mechanism of
Wnt signaling (Lyu, Yamamoto and Lu). To investigate the role of Ryk nuclear
signaling in regulating GABAergic versus oligodendrocyte differentiation,
constructs expressing (1) wild-type Ryk, (2) Ryk NLS-ICD, in which ICD is fused
with a nuclear localization signal peptide, (3) Ryk RC, which is a chimeric
construct that cannot be cleaved, or (4) control GFP alone, were transduced into
Ryk knockout NPCs by lentivirus. Transduced NPCs were cultured under
differentiation conditions for 6 days (Fig. 2.7 A). As expected, overexpression of
wild-type Ryk was sufficient to rescue the excess oligodendrocyte differentiation
from knockout cultures, as shown by O4 staining compared with the control (Fig.
2.7 A). Interestingly, transduction of Ryk NLS-ICD, like wild-type Ryk, led to a
>80% reduction in O4+ oligodendrocyte differentiation compared with the control
(Fig. 2.7 A). However, Ryk RC-transduced cells gave rise to similar numbers of
O4+ cells as the control (Fig. 2.7 A). Quantification of O4 and GFP double
positives cells further supports that cleavage of Ryk is important for Ryk’s role in
oligodendrogenesis, and nuclear localization of Ryk ICD is sufficient to inhibit
oligodendrogenesis (Fig. 2.7 B, left panel). A similar result was obtained from
MBP immunostaining (Fig. 2.7 B, right panel). Moreover, while repressing
differentiation of O4+ cells, overexpression of wild-type Ryk and Ryk NLS-ICD
! 77!
can rescue the defect in differentiation of GABAergic neurons detected by
Calbindin immunostaining, but not Ryk RC (Fig. 2.7 C). These data demonstrate
that Ryk ICD-mediated nuclear signaling is required and sufficient for
suppression of oligodendrocyte lineage differentiation and promotion of the
GABAergic neuron lineage from NPCs.

To investigate whether ICD-mediated Ryk nuclear signaling regulates the
expression of key genes involved in cell-fate specification of GABAergic neurons
and oligodendrocytes, Ryk knockout NPCs were cultured under proliferation
conditions and transduced with a construct expressing doxycycline-inducible Ryk
ICD tagged with GFP or a construct expressing doxycycline-inducible GFP only
as a control, and time-dependent changes in the mRNA levels of Dlx1 and Olig2
were examined (Fig. 2.7 D). Two hours after application of doxycycline, Dlx2
gene expression was stimulated by 4-fold compared with the control; however,
Olig2 gene expression was inhibited by 2-fold in the same time window (Fig. 2.7
D). This suggests that the end effect of Ryk nuclear signaling in the cell-fate
determination of NPCs is to regulate expression of key differentiation genes.

! 78!

Figure 2.7 Ryk intracellular domain is necessary and sufficient to mediate
the cell-fate change

A) Ectopic expression of wild-type Ryk or Ryk NLS-ICD rescues the excess
oligodendrocyte formation in the knockout cultures. Knockout NPCs transduced
with lentivirus expressing either control GFP, wild-type Ryk, ICD with nuclear
localization signal (Ryk NLS-ICD) or uncleavable Ryk mutant (Ryk RC) were
cultured under differentiation conditions for 6 days. Differentiated NPC cultures
were then analyzed by immunostaining with GFP, O4. (B) Percentages of
O4+/GFP+ and MBP+/GFP+ cells in total GFP+ cells are shown in the
histograms. (C) Differentiated NPC cultures from panel A were analyzed by
immunostaining with Calbindin. Percentages of Calbindin+/GFP+ cells in total
GFP+ cells are shown in the histograms. (D) Quantitative PCR showed that
inducible ectopic expression of Ryk ICD regulates the expression of Dlx2 and
Olig2 in NPC culture under proliferation conditions with the expression of
inducible GFP as a control. Cells were collected after doxycycline (Dox)
treatment at 0 h, 2 h, 4 h, 8 h, 12 h and 24 h. Scale bars are 100 µm. * P< 0.05,
** P< 0.01, *** P< 0.001.

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2.5 Discussion
Although regulation of fate determination of NPCs has been intensely studied,
very little is known about how the production of neuronal versus oligodendroglial
cells is balanced in the common progenitors and how cell-fate decisions are
made and regulated by extrinsic signaling in the ventral telencephalon. In this
paper, we demonstrate that a novel function of Ryk-mediated Wnt signaling
regulates the cell-fate decision of ventral NPCs to differentiate as either
GABAergic neurons or oligodendrocytes during telencephalon development.

Ryk receptor regulates neuronal versus oligodendroglial cell fate
We previously reported that Ryk plays a critical role in promoting neuronal
differentiation in the dorsal telencephalon during corticogenesis (Lyu, Yamamoto
and Lu). Here we provide three lines of evidence that Ryk receptor also has a
central role in repressing oligodendrocyte differentiation while promoting
GABAergic neuronal differentiation in the ventral telencephalon. First, the first
wave of MGE-derived OPC production is increased significantly in Ryk knockout
cortices from E11.5 to E12.5 without changing the differentiation timing and
location. The LGE-derived second wave of oligodendrogenesis is also likely to be
enhanced as well. Second, development of MGE- and LGE-derived GABAergic
neurons is impaired in Ryk knockout mice from E14.5 to E18.5, as evidenced by
in vivo analysis of GABAergic neuron markers and expression of genes required
for determination of GABAergic neuronal cell fate. Third, excessive production of
oligodendrocytes is observed in primary cultures and clonal neurosphere assays
! 80!
of Ryk knockout NPCs. We also found a significant reduction in GABA+/TuJ1+
neurons but a significant increase in O4+ oligodendrocytes in the knockout cell
culture. These results have revealed an unexpected function of Ryk receptor in
controlling GABAergic neurogenesis and oligodendrogenesis during embryonic
brain development. This suggests that normal development of the ventral
forebrain requires Ryk receptor to promote neurogenesis while restricting
concurrent oligodendrogenesis.

In principle, the aforementioned phenotypes of Ryk mutant NPCs could be a
consequence of secondary effects of altered DV patterning, abnormal
proliferation or maturation of ventral cells, cell death, or non-cell-autonomous
effects. Our in vivo and in vitro data do not support any of these possibilities.
Rather, our in vivo and in vitro data collectively support a model in which Ryk
regulates the fate choice between GABAergic neurons and oligodendrocytes of
ventral telencephalic NPCs.

Our data also suggest that Ryk may regulate cell fate by modulating the
expression of Dlx2 and Olig2. Members of the Dlx gene family are known to be
key factors in GABAergic interneuron specification, migration and differentiation
(Cobos, Borello and Rubenstein; Panganiban and Rubenstein). Dlx1/2 were first
reported as homeobox transcriptional factors involved in coordinating GABAergic
neuron versus oligodendrocyte specification in bi-potent progenitor cells
(Petryniak et al.). Although whether Ryk receptor can directly regulate expression
! 81!
of Dlx2 is currently unknown, our in vivo and in vitro data suggest that extrinsic
signal molecules and receptors may work through transcriptional factors to
regulate progenitor cells in order to coordinate differentiation of different lineages.
To our knowledge, Ryk is the first receptor shown to coordinate OPC and neuron
specification in the mammalian telencephalon.

Activity of Ryk-mediated Wnt signaling in the ventral telencephalon
Because of the cell-fate-change phenotype that we observed in Ryk knockout
mice and NPC cultures, and the fact that Ryk receptor is known to bind multiple
Wnt proteins (e.g.,Wnt1, Wnt3, Wnt3a, Wnt4, Wnt5a), we sought to identify the
Wnt ligand(s) responsible for this effect by employing a series of Wnt treatment
experiments. Wnt3a is known to promote neuronal differentiation of NPCs of the
forebrain and spinal cord (Muroyama et al.; Lie et al.; Yu et al.; Kalani et al.). Our
in vitro study showed that Wnt3a promotes GABAergic neuron differentiation and
that Ryk receptor is required for this effect.

Previous studies showed that Wnt signaling inhibits differentiation and maturation
of oligodendrocytes in both the spinal cord and telencephalon, and that the β-
catenin-mediated canonical pathway has been implicated in this action (Ye et al.;
Fancy et al.; Shimizu et al.). Our study shows that Ryk receptor also plays a
critical role in Wnt3a-induced inhibition of oligodendrocyte differentiation.
Therefore, downstream of Wnt, both the β-catenin-mediated canonical and Ryk
receptor-mediated atypical Wnt signaling pathways appear to be involved in
! 82!
repressing oligodendroglial cell-fate choice. Our data illustrate that Wnt proteins
act through Ryk to promote neurogenesis in both the dorsal and ventral
telencephalon and to antagonize simultaneously oligodendroglial fate in the early
stages of brain development.

Ryk-ICD regulates both neuronal and oligodendroglial cell differentiation
How does Wnt3a influence the differentiation of NPCs? Our previous data
indicate that cleavage of Ryk and ICD nuclear localization are required for Wnt3-
induced neuronal differentiation (Lyu, Yamamoto and Lu). In the present study
we found that, during Wnt3a-induced GABAergic neurogenesis, both Ryk wild-
type and Ryk NLS-ICD, but not the non-cleavable form Ryk RC, can rescue the
supernumerary formation of oligodendrocytes in Ryk mutant cell culture.
Therefore, we conclude that ICD-dependent Ryk signaling serves as a common
mechanism for both promoting neuronal differentiation and inhibiting
oligodendrocyte differentiation. However, Ryk signaling does not seem to affect
the level of β-catenin or the activity of TCF significantly in NPCs in vitro (Lyu,
Yamamoto and Lu), suggesting that Ryk ICD-mediated β-catenin-independent
signaling induces GABAergic neuron generation and represses oligodendrocyte
specification.

Our study reveals for the first time that Ryk-mediated atypical Wnt signaling is
involved in the neuronal differentiation from the dorsal pallium and also in
differentiation of ventral cell types, including GABAergic neurons and
! 83!
oligodendrocytes, during forebrain development. Our results provide strong
evidence that Ryk signaling is critical for controlling the neuron-versus-
oligodendrocyte fate decision. This activity in ventral NPCs may promote both
dorsal and ventral neurogenesis while antagonizing the induction of other cell
types from the ventral NPCs during early brain development.

! 84!
CHAPTER 3
HISTONE H2A DEUBIQUITINASE MYSM1 REGULATES CORTICAL
NEUROGENESIS

3.1 Abstract
Covalent histone modifications have been shown to play critical roles in
regulating gene transcriptional activity. Compelling data suggest that the histone
H2A mono-ubiquitination is likely to serve as an important mechanism to regulate
the repression of chromatin status and gene expression in embryonic stem cells
and tissue specific progenitors. However, the functional relevance of histone H2A
mono-ubiquitination during mammalian embryonic neural development is poorly
understood. In this study, we found that embryonic neural progenitor cells
express a histone deubiquitinase MYSM1 and the loss of MYSM1 resulted in
severely impaired specification of basal progenitors during corticogenesis and
enhanced astrogenesis. To uncover the molecular mechanism of MYSM1
mediated neuronal differentiation, we have utilized primary cultured NPCs and
performed a series of mechanistic studies. We have proven that MYSM1 has a
intrinsic cell-autonomous role in regulating NPC differentiation. Our ChIP-Seq
analysis of the genome-wide occupancy of repressive histone mark H3K27me3
and active histone mark H3K4me2 in both wild-type and MYSM1 knockout NPCs
under proliferating conditions demonstrated that a group of critical neuronal
development regulating genes are bound by more repressive but less active
histone modifications upon loss of the MYSM1. Our results support a role of
! 85!
MYSM1 in regulating transcriptional activation of neuronal gene to facilitate the
neuronal cell fate specification likely through removing repressive histone mark
H2Aub1 and through cross talk with other histone modifications.

3.2 Introduction
The development of mammalian embryonic cerebral cortex is a complex but
highly organized choreography of progenitor self-renewal and differentiation.
Multipotent cortical neural progenitor cells (NPCs) first undergo an initial
expansion phase in which they only divide symmetrically, then enter a
neurogenic phase in which they divide asymmetrically to generate various types
of cortical neurons in a fixed order (Kriegstein and Alvarez-Buylla). At the late
stage of embryogenesis, the competence of NPCs switches from neurogenic to
gliogenic by purely differentiating into astrocytes (Kriegstein and Alvarez-Buylla).
This tightly regulated restriction of NPCs’ “competence” depends on coordinated
activation of cell fate determinant gene and is likely to be the result of the
combination of extrinsic cues and intrinsic networks (Martynoga, Drechsel and
Guillemot). Epigenetic regulations including histone modifications and DNA
methylation have been demonstrated by compelling data to be one crucial level
of mechanism that interacts with transcriptional networks and extrinsic cues to
eventually determine the transcriptional activity of genes during neural stem cell
differentiation (Hirabayashi and Gotoh). While various histone marks have been
identified and recognized to play critical roles in gene transcriptional activity
(Strahl and Allis), however, our understanding about the role of histone
! 86!
modifications during cortical development has just begun.

Histone modifications come in various forms including methylation, acetylation,
ubiquitination, SUMOylation and phosphorylation(Shilatifard). Most of our
knowledge of the functional roles of these histone modifications came from the
phenotype analysis of knockout or mutant mice of specific corresponding
epigenetic modifiers. Histone H2A monoubiquitination is abundant and highly
conserved at Lysine 119 in mammalian cells(Higashi, Inoue and Ito). Unlike
polyubiquitination of peptides, H2A monoubiquitination does not lead to protein
degradation but has been suggested to be stable and play a critical role in
regulating transcriptional silencing, which is controlled by the activities of
ubiquitin-ligating and deubiqutinaing enzymes (Higashi, Inoue and Ito). There are
two main H2A ubiquitin E3 ligases: Ring1b of the polycomb repressor complex
1(PRC1) and Brca1 (H. Wang et al.; Cao, Tsukada and Zhang; Q. Zhu et al.).
Ring 1b is essential for the stem cell identity maintenance, differentiation and
tissue development (Surface, Thornton and Boyer), Ring 1b deletion in mouse
ES cells leads to derepression of PRC1 target genes and differentiation of mouse
ES cells (Stock et al.; Endoh, Endo, Endoh, Fujimura, et al.; Endoh, Endo, Endoh,
Isono, et al.). Current available data in mouse ES cells, it suggests that H2Aub1
co-localize well with Ring1b, H3K27me3 and PRC2 components at a core group
of PRC target genes (Stock et al.; Brookes et al.; Endoh, Endo, Endoh, Isono, et
al.). Moreover, Ring1b conditional deleted mice exhibited derepression of
neuronal genes including Ngn1 and an extended neurogenic phase and a
! 87!
delayed astrogenic phase (Hirabayashi, Suzki, et al.). Another H2A ubiquitin
ligase Brca1 has been identified recently as ubiquitinase of Histone H2A at the
satellite repeats in neural tissues. Loss of Brac1 caused impaired integrity of
constitutive heterochromatin and disruption of gene silencing at the tandemly
repeated DNA regions (Q. Zhu et al.). However, the biological role of histone
H2A monoubiquitination during brain development remains unclear. One of the
reasons is the difficulty in reconciling the different phenotypes upon deleting
critical components of PRC1, which include Ring1B and Bmi1 (Zencak et al.;
Hirabayashi, Suzki, et al.). Another reason is the lack of systmetic analysis of
occupancy of H2Aub1 during differentiation. In summary, while increased an
increasing amount of data suggests a critical role of histone H2A
monobuquitination in repressing gene expression in various biological contents,
the biological function of this histone modification remains poorly understood.

So far eight proteins have been identified as histone H2A deubiquitinases,
including six members of the ubiquitin specific protease family (Usp3, 12, 16, 22
and 46), BAP1 and the Mysm1 (Joo et al.; Vissers et al.; Scheuermann et al.; P.
Zhu et al.). Biochemically they have been shown to deubiquitinate H2A
specifically and to be involved in various biological processes like cell cycle, DNA
repair and transcription (Clague, Coulson and Urbe). However, the key biological
and molecular functions of the large class of histone H2A deubiquitinases have
just begun to be explored. Mysm1 was the first member that has been reported
to have critical roles in tissue development including B cell development and
! 88!
hematopoiesis and lymphocyte differentiation (Jiang et al.; Nijnik et al.). However
many questions related to how H2A ubiquitination and deubiquitination work in
regulating stem cell activity still remain unclear. These questions furthermore
include the genome-wide localization of H2Aub1 in these tissue specific
progenitor cells, whether H2Aub1 functions through interacting or affecting other
histone modifications, and what are identity of MYSM1’s target genes in these
progenitor cells. Si-Yi Chen’s group have shown that Mysm1 binds to cell fate
determinant of B cell development Ebf1 gene and regulates its transcriptional
activation likely through a H2A ubiquitination mediated mechanism that involves
modulating other histone modifications like H3K4me3 and H3K27me3 (Jiang et
al.; Higashi, Inoue and Ito). However, genome-wide unbiased analysis is yet to
be performed to substantially uncover the molecular function of MYSM1.

MYSM1 null mice suffer from a low survival rate after birth, severe ataxia and trail
truncation among surviving individuals. Therefore, we have hypothesized that
MYSM1 may play a critical role during the neural development. In particular the
MYSM1 mediated histone H2A deubiquitination may serve as a critical
counteracting force to the histone H2A ubiquitination mediated bivalent gene
repression, allowing proper transcriptional activation of these genes at the right
time or full activation of these genes. The MYSM1 knockout mice serve as a
good model to test our hypothesis. Here,, we report that deletion of the histone
H2A deubiquitinase MYSM1 resulted in severe defects in the specification of
neuronal cell fate and enhanced production of astrocytes likely due to inactivation
! 89!
of neuronal genes. We have provided further evidence showing that MYSM1
mediates a cell-autonomous mechanism in regulating neuronal differentiation
and that loss of MYSM1 leads to an impaired histone modification-binding pattern
at a group of genes that are involved in neuron development based on our
genome-wide analysis of critical histone modifications.

3.3 Methods and Materials
Animals
MYSM1 knockout mice were kindly provided by Dr. Si-Yi Chen’s laboratory. All
animals were maintained and handled according to the regulations of the
University of Southern California Institutional Animal Care and Use committee. In
all experiments wild-type littermates were used for controls. Embryos and pups of
both wild-type and knockout mice were collected from timed, mated pregnant
females.

Immunohistochemistry and immunocytochemistry
For immunohistochemistry, embryonic brains were dissected and fixed in 4%
parafomaldehyde (PFA) in PBS at 4
o
C and cryoprotected in 30% sucrose.
Samples were embedded and frozen in Tissue Tek OCT compound, and
sections were then cut at a thickness of 12 µm. Antigen retrieval was performed
for immunohistochemistry of nuclear expressed proteins. For
immunocytochemistry, cell cultures were fixed with 4% PFA in PBS for 15
minutes and immunostained after permeabilization in 0.2% Triton X-100.
! 90!
Immunostaining for membrane proteins was performed without prior
permeabilization. The following antibodies were used: anti-β Galactosidase
(1:1000, Abcam); anti-tubulin III (1:1000, Sigma-Aldrich); anti–glial fibrillary acidic
protein (GFAP) (1:1000, Sigma-Aldrich); anti-PDGFRα receptor (1:200, Santa
Cruz); anti-Map2 (1:400, Sigma) anti-Tbr1 (1:1000 Millipore), anti-Tbr2 (1:1000
Millipore), anti-Cux1 (1:1000 Santa Cruz), anti-Ctip2 (1:1000 Millipore and
Abcam), anti-Pax6 (1:500 Abcam), anti-Ki67 (1:400 Covance), anti-PH3 (1:1000),
anti-CD133 (anti-Mouse CD133 PE, clone 13A4). TUNNEL assays were
performed using the In Situ Cell Death Detection Kit (Roche) following the
manufacturer’s instructions. Immunolabeled cells were detected by using
corresponding fluorescent-labeled secondary antibodies (1:400, Jackson
Immunoresearch Laboratories). Viable cells were identified by Hoechst nuclear
staining.

BrdU labeling
For in vivo BrdU labeling of S phase cells, time-mated pregnant females were
injected intraperitoneally with 50 µg per g body weight of 5-bromo-2’-
deoxyuridine (BrdU) (Sigma). BrdU-labeled mothers or pubs were culled and
tissue were collected 30 minutes after injection, fixed in 4% parafomaldehyde
and embedded in OCT compound for sectioning. Sections were treated with 2 N
HCl for 30 min followed with neutralizing with Borate buffer for 10 minutes. BrdU
is detected by using rat anti-BrdU (1:350, Accuratech) antibody.

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Neural Progenitor Cells (NPCs) isolation and culture
Primary NPCs were derived from E11.5 wild-type and knockout mouse forebrains.
Tissues were dissected and dissociated as described (Reynolds and Weiss;
Schmitt et al.). Cells were cultured in serum-free DMEM/F12 (Invitrogen),
supplemented with mouse recombinant basic fibroblast growth factor (Fgf) (20
ng/ml, R&D), 2% B27 (Invitrogen), 1% penicillin-streptomycin (Sigma-Aldrich) on
POL and fibronectin-coated culture dishes in a humidified 5% CO
2
/95% air
incubator at 37°C. For differentiation, Fgf is withdrawn by changing the medium.
After additional days under differentiation conditions, cells were analyzed by
immunocytochemistry. During that period, the medium was changed every 2
days.

ChIP-qPCR assays
For the ChIP assay, primary NSCs cultured in proliferation conditions were cross-
linked with 1% formaldehyde for 10 minutes at room temperature. Cross-linking
was stopped by the addition of glycine to the 125 mM final concentration for 5
minutes and cells were washed twice with cold PBS. The cell pellet was then
resuspended with the lysis buffer. Samples were sonicated for 1 hour, with 30
second pulses, 45 seconds resting, using the Bioruptor sonicator to produce
chromatin fragments of 250bp to 500bp on average. After clarification by
centrifugation, 2 µl of antibody were added to 100 µg chromatin sample (50 ul of
ubH2A antibody to 450 ug chromatin) and samples were rotated vertically at 4 C
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for 12-16 hours. The primary antibodies used for ChIP-qCR and CHIP-Seq were:
rabbit anti-H3K4me2 antibody (Millipore #07-030), rabbit anti-H3K27me3
(Millipore #07-449) and mouse anti-H2Aub1 (Millipore #E6C5). For ubH2A ChIP,
30 µl rabbit anti-mouse IgM secondary antibody was added to the primary
antibody bound chromatin sample in 4
o
C with vertical rotation for 2-4 hours.
Fresh blocked 50 µl Millipore magnetic beads slurry was added into each ChIP
sample and rotated vertically at 4
o
C for 4 hours. Beads were washed with pre-
chilled RIPA buffer for 5 times and 1 time with TE buffer with 50 mM NaCl on ice.
Precipitates were resuspended in 210 µl Elution Buffer at 65
o
C for 15 minutes,
followed with reverse crosslinking at 65
o
C for 12 hours. Eluted DNA was
recovered from the eluate using the PCR purification kit (Zymo). qPCR was
performed to validate the success of ChIP assay.

ChIP-Seq library construction and data processing
After qPCR-confirmed enrichment of target sequences in ChIP versus input
samples, a bar-coded ChIP library was constructed following Peggy Farnham’s
lab protocols with minor modifications. After adaptor ligation, step libraries were
run on a 2% E gel, and the 200-500bp fraction of the library was extracted and
purified followed by 15-cycle PCR amplification. Libraries’ enrichment was
confirmed by QPCR of the target sequence and then enrichment-confirmed
libraries were analyzed by Nanodrop and bioanalyzer and quantified by the
KAPA-Quant kit. Successfully constructed libraries were sequenced by the
Illumina GAIIx of the Epigenome Core Facility at the University of Southern
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California. Sequence reads were aligned to the UCSC mouse genome assembly
MM9. The Sole-search tool was used to identify peaks for modified histones.
Replicate reads were removed before peak calling.

Quantitative PCR
Total RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA synthesis was
carried out with 500 ng of total RNA using the SuperScript III qRT-PCR kit
(Invitrogen). Real-time quantitative RT-PCR (qPCR) was performed with an ABI
PRISM 7900 Sequence Detection System using SYBR Green Master Mix (ABI).
Samples were run in triplicate and relative levels of each mRNA were examined
by comparing Cycle Threshold (Ct) values for each reaction among samples
using the ACTB gene as a reference. Relative mRNA expression levels were
measured from at least 3 independent experiments.

3.4 Results
3.4.1 Expression of MYSM1
We have observed that MYSM1 knockout mice have significantly smaller
forebrains and olfactory bulbs, in addition to the obvious truncated trail
morphology, as early as E16.5, when the body size between wild-type and
knockout embryos are very comparable (Fig 3.1 left panels). This prominent
phenotype sustained until adult stage postnatal day 35 (P35) among surviving
knockout mice (Fig. 3.1, right panel). MYSM1 P35 knockout mice have
significantly smaller forebrains and olfactory, whereas midbrains and hindbrains
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size appeared to be less altered (Fig. 3.1 right panel). This morphological
observation together with the prominent ataxia exhibited in newborn and adult
knockout mice lead to our hypothesis that MYSM1 is likely to play a critical role in
the neural development and as early as the embryonic stages. Therefore, we
carried out a series of tests to investigate the potential biological function of
MYSM1 during cortical development. We first examined the expression of
MYSM1 in the developing brain by performing LacZ immunohistochemistry. In
the knockout allele, a LacZ gene cassette was inserted between the exon 2 and
exon 3 of MYSM1 gene, which allows the endogenous MYSM1 promoter to drive
the expression of LacZ gene, which in turn can be used for identifying MYSM1-
expressing cells. LacZ immunohistochemistry was performed using the above-
described protocol. As expected, the LacZ staining in the wild-type control
sections demonstrated a minimal level of background signal and exhibited the
strongest signal in the knockout sections (Fig. 3.2 A, B). At the early stage of
neurogenesis E13.5, LacZ immunostaining demonstrated that MYSM1 is strongly
expressed in the neural progenitor layer or the ventricular zone (VZ) (Fig. 3.2 A).
The expression of MYSM1 sustained until mid-stage neurogenesis E14.5 (Fig.
3.2. B). Interestingly, LacZ immunostaining also indicated that MYSM1 is
expressed by not only the NPCs in the dorsal VZ but also ventral VZ in the MGE
and LGE as well (Fig. 3.2 A, B).

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Figure 3.1 Morphological defects of MYSM1 knockout embryonic brain and
adult brain

The left panels are representative images of E16.5 wild type and knockout
embryos; blue arrows indicate the tail morphology. The boxed areas are images
of the brains of these embryos. The right panel is a representative image of the
brain of a P35 wild-type (left) and a P35 knockout mouse (right).

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3.4.2 Cortical neurogenesis is impaired in the MYSM1 knockout mice
The morphological defects of MYSM1 knockout forebrain and the expression
pattern of MYSM1 in embryonic NPCs suggest that MYSM1 may be involved in
the cortical development and NPC activities. To explore this possibility, we
examined the numbers of progenitors and neurons in the wild-type and MYSM1
knockout E14.5 embryos by immunostaining of specific lineage markers.

The morphology of coronal sections from both wild-type and knockout brains
showed that although MYSM1 knockout embryos had a comparable forebrain
size at E14.5 comparing to controls, the thickness of the knockout cortex was
significantly reduced by 20%, compared to the wild-type controls (Fig. 3.3 E).
Immunostaining of early neuronal marker TuJ1 showed that significantly reduced
numbers of TuJ1+ neurons were located in the cortical plate of the MYSM1
knockout mice, compared to the wild-type control (Fig. 3.3 A, F). Immunostaining
of matured neuronal maker Map2 demonstrated that matured neuronal
population was reduced in the MYSM1 knockout cortex as well (Fig. 3.3 B).
Images of Tuj1 and Map2 positive neuronal populations in the wild-type mice
clearly exhibited a “layered” structure, indicating that at this stage the first group
of early born neurons (preplate) has been split into two separate layers (Layer I
and Layer 6) and the cortical plate is being formed in the middle of the split
“preplate” (Fig. 3.3 A, B). However, this phenomenon was not shown or not
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obvious at this stage in the knockout mice, which suggests that the separation of
layer 1 and layer 6 is likely to be delayed due to reduced production of
postmitotic neurons in the cortical plate (Fig. 3.3 A, B). Consistent with this,
immunostaining of postmitotic neuron marker Tbr1 that also labels deep layer
neurons demonstrated that the Tbr1+ neurons in the MYSM1 knockout mice
were reduced by one third, compared to the wild-type controls (Fig. 3.3 C, G).
More importantly, basal progenitor marker Tbr2 immunostaining has
demonstrated that knockout mice have an over 20% reduced critical intermediate
neuronal precursor population, compared to that of the wild-type control (Fig. 3.3
D, H). It is well established that basal progenitors arising from the primitive radial
glial cells are the “direct” source of most postmitotic neurons. Therefore, the
reduction of the Tbr2+ population is a strong indication of impaired neurogenesis
and likely to directly contribute to the reduced neurogenesis capacity in the
MYSM1 knockout mice. In summary, our data suggests a functional role of
MYSM1 during mammalian cortical neurogenesis.

We then asked two questions: first, whether the reduced neuronal production is
coupled with defected neuronal migration; second, whether Mysm1 preferentially
affects the neuronal production and/or migration of specific cortical layers. To
address these questions, we labeled the wild-type and MYSM1 knockout E18.5
cortex sections with three cortical layer markers, including upper layer marker
Cux1, middle layer marker Ctip2 and deep layer marker Tbr1. The results
showed that the numbers of neurons of all three layers were all severely reduced
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(Fig. 3.4 A-C). There is no obvious difference in terms of the level of impairment
among the upper, middle, and deep layers, which suggests that the function of
MYSM1 is not restricted to any particular neurogenic phase. In addition, through
comparing the localization of the Cux1, Ctip2 and Tbr1 positive neurons in the
wild-type and MYSM1 knockout cortical plates, we have concluded that MYSM1
deletion did not obviously affect the migration of cortical neurons (Fig 3.4 A-C). In
summary, MYSM1 is likely to affect the production of cortical neurons throughout
the whole neurogenic phase without affecting the neuronal migration.

3.4.3 Neural progenitor self-renewal and proliferation is not impaired upon
Mysm1 deletion
The reduction in differentiated neurons and neuronal precursors in the MYSM1
knockout mice can result from a preciously exhausted progenitor pool, due to
defects in neural progenitor cells’ self-renewal and/or proliferation. To address
these possibilities, we first examined the maintenance of the NPC pool in the
knockout mice. Performing immunostaining of NPC marker Pax6 in wild-type and
knockout brains, our results showed no visible difference in Pax6+ NPCs
between the knockout and the control (Fig. 3.5 A). Quantifying the number of the
Pax6+ neural progenitor cells, no significant difference was found between
MYSM1 knockout mice and the wild-type controls, indicating that neural
progenitor cells are largely well-maintained at this state upon MYSM1 deletion
(Fig. 3.5 A, E). We then performed Ki67 immunostaining to label all the cells that
are proliferating, our results showed no significant difference between the wild-
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type and knockout mice (Fig. 3.5 C, G). We further examined the cells that are in
the M phase of the cell cycle at both apical and basal positions, by performing
pH3 (phosphorylated histone3) staining (Fig. 3.5 B). The pH3 staining suggested
that the total number of dividing cells in the MYSM1 knockout mice is equal to
that of wild-type controls (Fig. 3.4 B, F). Both apical and basal progenitors are
comparable between wild-type and knockout mice, with slightly increased
number of basal progenitors in the MYSM1 knockout mice. Furthermore, to
address the self-renewal and proliferation level of the MYSM1 knockout mice, we
performed pulse BrdU labeling. We labeled the time-mated 12.5 days pregnant
mice with BrdU for half an hour in order to label the cells that are in the S phase
of the cell cycle. BrdU immunostaining showed that the S-phase cell numbers in
wild type and knockout embryos did not exhibit any significant difference (Fig 3.4
D, H). In summary, our results suggested that the self-renewal and proliferation
capacity of NPCs are not significantly affected upon MYSM1 deletion.

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Figure 3.2 MYSM1 expressions in the developing mouse brain

(A-B) LacZ staining of coronal sections of E13.5 and E14.5 wild-type,
heterozygous, and MYSM1 knockout mouse brains. Nuclei are stained by DAPI.
V is ventricular zone; Scale bars are 100µm.

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Figure 3.3 MYSM1 knockout mice exhibited severely impaired
corticoneurogenesis at E14.5

(A-D) Representative images of immunohistochemistry of coronal sections of
E14.5 wild-type and knockout brains. TuJ1 is a newborn neuron marker, Map2 is
a mature neuron marker, Tbr1 is a postmitotic neuron marker, Tbr2 is a basal
progenitor marker. Dashed lines indicate the boundary of the tissue. White line
and adjacent red lines indicates the thickness of positive population. (E)
Histogram showing the thickness of the knockout cortex normalized with the WT
controls. (F-H) Histogram show the average numbers of TuJ1, Tbr1 and Tbr2
positive cells in 100um cortex column in both WT and KO respectively. All data
are based on three individual little of animals. Scale indicates 100 µm.

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Figure 3.4 Impaired corticoneurogenesis in the MYSM1 knockout mice did
not show layer preference

(A-C) Representative images of immunohistochemistry of different cortical layer
markers in E18.5 coronal sections of both wild-type and MYSM1 knockout brains.
Cux1 labels upper layer (layer 2 and layer 3) cortical neurons; Ctip2 labels
middle layer (layer 4) cortical neurons; Tbr1 labels deep layer (layer 5) cortical
neurons. Dashed lines indicate the upper edge of the cortex. (E-G) Histograms of
the quantification of positive cell numbers per 100 µm in both wild-type and
knockout brains. All data are collected from three individual litters of mice.

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Figure 3.5 The neural progenitor cell pool is maintained in the MYSM1
knockout mice

(A) Image of immunostaining of Pax6 in E14.5 wild-type and knockout cortex.
Pax6 is a neural progenitor marker. Dashed lines indicate the boundary of the
cortex. (B) Immunostaining of PH3 in E14.5 wild-type and knockout cortex. PH3
labels M phase dividing cells. (C) Immunostaining of Ki67 in E14.5 wild-type and
knockout cortex. Ki67 labels proliferating cells. (D) Immunostaining of BrdU in 30
minutes pulse-labeled E14.5 wild-type and knockout cortex. BrdU labels
proliferating cells that are in S phase of their cell cycle. (E-H) Histogram of the
quantification of positive cells. Scale bars are 100 µm.

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3.4.4 Increased apoptosis in basal progenitor cells of the MYSM1 knockout
mice
Since our results indicated that the progenitor pool is largely maintained in the
MYSM1 knockout mice, it is likely that the defected neuronal production occurs
along with mis-specification of neural progenitor cells toward other lineage fate
and/or degeneration of progenitor cells. To test this hypothesis, we first
investigated whether apoptosis is increased in the MYSM1 knockout cortices, by
performing TUNNEL staining, which specifically labels apoptotic cells. As
expected, the results showed that wild-type cortex has very few TUNNEL labeled
cells. Interestingly, TUNNEL staining at E14.5 revealed that the numbers of
apoptotic cells in the MYSM1 knockout cortices were increased, compared to the
wild-type control (Fig. 3. 6 A, A’, B, B’, E). To further analyze the identity of the
apoptotic cells in the knockout cortices, TUNNEL and Tbr2 double staining were
carried out. The results showed that significantly more Tbr2+ basal progenitor
cells or neuronal precursors are indeed undergoing apoptosis in the knockout
cortices (Fig. 3. 6 C, F). In summary, our analysis of cell death in the embryonic
brain tissue suggested that despite the largely normal progenitor self-renewal
and proliferation, MYSM1 deletion resulted in increased apoptosis in the
developing cortex, particularly in the Tbr2+ neuronal precursor cells.

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Figure 3.6 TUNNEL assay revealed increased apoptosis of neuronal
precursor cells in MYSM1 knockout mice

(A, A’) Representative images of TUNNEL staining of E14.5 wild-type cortex,
dashed lines indicate the boundary of the cortex. (B,B’) Representative images of
TUNNEL staining of E14.5 MYSM1 knockout cortex; dashed lines indicate the
boundary of the cortex. (C) Co-immunostaining of TUNNEL, Tbr2 and DAPI of
E14.5 MYSM1 knockout cortex. Boxed area, enlarged to the right, indicating cells
co-stained by TUNNEL and Tbr2. (D) Histogram shows the normalized fold
change of TUNNEL+ cells in knockout cortex compared with wild-type control. (E)
Histogram shows the normalized fold change of TUNNEL+/Tbr2+ cells in the
knockout cortex compared to wild-type control. Scale bars indicate 100 µm.

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3.4.5 Enhanced astrogenesis in the MYSM1 knockout mice
We then investigated the possibility of mis-specification of neural progenitor cells
during differentiation at the expense of neuronal differentiation. We examined the
neuron-glia switch timing and the production of astrocytes in the wild-type and
MYSM1 knockout cortex by performing immunostaining of astrocytes marker
GFAP at E14.5, E18.5 and P7 (Fig. 3.7). GFAP positive cells were detected
neither in wild-type controls nor in knockout brains at E14.5 (data not shown). At
E18.5 in both wild-type and knockout brains, a small number of GFAP+
astrocytes were observed, but we did not detect any significant over-production
of GFAP+ astrocytes in the knockout cortex, compared to the wild-type cortex
(Fig. 3.7 A, A’). Similarly, no significantly enhanced astrocyte production was
observed at the SVZ region in the MYSM1 knockout mice at E18.5 (Fig. 3.7 B,
B’). However, we found dramatically increased numbers of GFAP+ astrocytes
located in the MYSM1 knockout cortex over wild-type control at P7 (Fig. 3.7 C,
C’). At this stage, astrocytes only invaded the top layer of neocortex in wild-type
neocortex (Fig. 3.7 C, E), whereas astrocytes have occupied all cortical layers in
the knockout neocortex (Fig. 3.7 C’, E’). Moreover, there are significantly
increased numbers of astrocytes located in the hippocampus of the MYSM1
knockout brain, compared to the wild-type controls as well at P7 (Fig. 3.7 D, D’).
We quantified the GFAP+ cells in the neocortex, which showed at least triplicate
astrocytes in the MYSM1 knockout cortex compared to controls suggesting a cell
fate mis-specification of neural progenitor cells at early stage (Fig. 3.7 F).
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Figure 3.7 Enhanced production of astrocytes in the new born MYSM1
knockout cortex

(A, A’) Immunostaining of GFAP in the cortex of E18.5 wild-type and knockout
brains. Cx is cortex. (B, B’) Immunostaining of GFAP/DAPI in the subventricular
zone (SVZ) of E18.5 wild-type and knockout brains. (C, C’) Immunostaining of
GFAP in the cortex of P7 wild-type and knockout brains. Cx is cortex. (D, D’)
Immunostaining of GFAP/DAPI of the hippocampus of the P7 wild-type and
knockout brains. Hip is hippocampus. (E, E’) Enlarged image of GFAP staining in
the cortex of P7 wild-type and knockout brains. (F) Quantification of the number
of GFAP+ cells in the wild-type and knockout brains at E18.5 and P7. Scale bar
is 100 µm.

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3.4.6 Cell fate tracing of E14.5 neural progenitor cells in vivo
Our observations suggest that upon MYSM1 deletion, the neuronal output of
NPCs has been severely compromised due to impaired differentiation, coupled
with increased apoptosis and glial cells production. To further test this
hypothesis, we traced the cell fate of the E14.5 cortical progenitor cells in vivo in
both wild-type and MYSM1 knockout mice by BrdU birthdating. We have labeled
14.5 day pregnant mice with BrdU and culled the surviving pups at P7, assuming
that most of the E14.5 progenitors gave rise to differentiated daughter cells at P7.
We then carried out co-immunostaining of BrdU and several lineage markers.
Interestingly, we have observed that while 78% of the BrdU labeled E14.5
cortical progenitor cells have been destined to Cux1 positive layer 2-3 neurons,
19% of cortical progenitor cells have differentiated into deep layer neurons that
are Tbr1+, leaving only 3% of them fated into astrocytes in the wild-type controls
(Fig 3.8 A, H). However, significantly reduced numbers of BrdU-labeled neural
progenitor cells have been specified into either Cux1+ neurons or Tbr1+ neurons
in the MYSM1 knockout P7 pups, compared to the wild-type controls, but the
laminar positioning of E14.5 born neurons were largely maintained, suggesting a
relatively normal migration pattern of cortical neurons (Fig. 3.8 C-F). Strikingly,
GFAP+/BrdU+ astrocytes were dramatically increased with at least 3-fold
augmentation, suggesting a significant enhanced specification of E14.5
progenitors have been fated towards glial cell fate (Fig. 3.8 H). GFAP+/BrdU+
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astrocytes only invaded the very top layer of the cortex in the wild-type cortex,
whereas in the MYSM1 null cortex, GFAP+/BrdU+ astrocytes invaded the entire
cortex (not shown). In summary, our BrdU birthdating study has provided strong
evidence for a cell fate switch from neurons towards glial cells.

Figure 3.8 Cell fate tracing of E14.5 neural progenitor cells in the MYSM1
knockout mice

(A, B) Immunostaining of BrdU and DAPI in the BrdU labeled P7 wild-type and
knockout cortex (BrdU injected at E14). (C-C’’ and E-E’’) Co-Immunostaining of
BrdU and Cux1 in the P7 wild-type and knockout cortex. (D-D’’ and F-F’’) Co-
immunostaining of BrdU and Tbr1 in the P7 wild-type and knockout cortex. (G)
Quantification of the total BrdU+ cells in the P7 wild-type and knockout cortex.
(H) Quantification of GFAP+/BrdU+, Cux+/BrdU+ and Tbr1+/BrdU+ cells. Scale
bar is 100 µm.

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3.4.7 In vitro differentiation of MYSM1 knockout neural progenitor cells
To elucidate the molecular mechanisms underlying the MYSM1 mediated
neuronal differentiation, in particular the H2A deubiquitinase activity of MYSM1 in
regulating neural progenitor cells differentiation, we utilized the in vitro monolayer
primary NPC culture system. We isolated primary neural progenitor cells from the
telencephalon of E11.5 wild-type and MYSM1 knockout embryos and cultured
them in monolayer form. As expected, MYSM1 knockout NPCs were able to self-
renew normally and proliferate at similar speed as the wild-type control from
passage to passage (data not shown). This is consistent with what we have
previously observed in the MYSM1 knockout cortices in vivo, which suggests that
MYSM1 contributes to the NPC differentiation process rather than facilitate the
self-renewal status or proliferation. However, when the proliferating neural
progenitor cells were subject to differentiation conditions, their impaired
differentiation capacity was immediately detected. At differentiation day 3, we
fixed the differentiated cell cultures and analyzed them by performing
immunocytochemistry of various linage markers (Fig 3.9 A). Consistent with the
in vivo observations, the percentage of the neurons labeled by neuronal marker
TuJ1 decreased from 22% to 13% in the knockout cells compared with the wild-
type cells (Fig 3.9 A, B). We also measured and normalized coverage of the
TuJ1+ and Map2+ area, using the image analysis software ImageJ. Our results
showed that the TuJ1 and Map2 positive areas in the immunostaining were
significantly reduced for the MYSM1 knockout cell cultures, compared to the
controls (Fig. 3.9 A, C, D). To the contrary, the percentage of astrocytes labeled
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by GFAP mildly increased from 34% to 40% in the knockout cells (Fig 3.9 A, B).
Interestingly, the percentage of oligodendrocytes precursor cells labeled by
PDGFRα was significantly reduced in the MYSM1 knockout cells, compared to
the wild-type controls (Fig. 3.9 A, B). We further examined the gene expression
of important cell fate determinants of the differentiated cells by qPCR. The results
showed that the expression of Neurod2 and Ngn2, which promote neuronal fate
specification, were significantly decreased in the knockout cells. Nkx2.2, which
regulates oligodendrocytes differentiation, was significantly decreased in the
knockout cells as well, while the expression of GFAP and Hes1 that is involved in
regulating astrocyte differentiation, were dramatically increased in the MYSM1
knockout cells (Fig. 3.9 E). Therefore, the above-mentioned NPC population
assays strongly support the functional role of MYSM1 in regulating neuronal cell
specification during differentiation. In summary, our study of the primary wild-type
and knockout NPCs in vitro recapitulated the impaired neuronal differentiation
capacity of the MYSM1 knockout NPCs in vivo and can serve as a good model
for the molecular mechanism investigation.

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Figure 3.9 In vitro differentiation of wild-type and MYSM1 knockout NPCs

(A) Immunostaining of lineage markers in wild-type and knockout differentiated
cell culture at D3. TuJ1 is a neuronal maker, GFAP is an astrocytes marker and
PDGFRα is an oligodendrocyte precursor cell maker. (B) Quantification of the
percentage of each cell types among the entire cell culture. (C, D) Quantification
of the TuJ1+ and Map2+ area by imageJ (E) Normalized fold change in
expression of cell fate determinant genes.

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Figure 3.10 Overexpression of MYSM1 is sufficient to rescue the
differentiation phenotype of MYSM1 knockout neural progenitor cells in
vitro

Co-immunostaining of neuronal marker TuJ1, GFP and DAPI and co-
immunostaining of oligodendrocyte precursor cell marker PDGFRα, GFP and
DAPI in differentiated knockout cells that were transduced with a construct
MYSM1-Flag and knockout cells that were transduced with a control construct
FUIGW. Cells were cultured under differentiation conditions for 3 days. The
histogram on the right side is the quantification of the percentage of Tu1+ and
PDGFRα+ cells among the GFP+ transduced cells.

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3.4.8 Cell-autonomous effect of MYSM1 in regulating neuronal
differentiation
We then asked whether the defects we observed in the MYSM1 knockout cells
are caused by the cell-autonomous effects of MYSM1 in specifying neuronal cell
fate. To address this question, we constructed a lentiviral construct, which
expresses exogenous MYSM1 tagged with Flag, and transduced this construct
into MYSM1 knockout NPCs. If these defects observed in the knockout cells are
due to a cell-autonomous mechanism, the expression of exogenous MYSM1
should be able to rescue these phenotypes. Hence, we differentiated the
knockout cells that were transduced with the MYSM1 construct and control cells
in parallel, and analyzed their differentiation patterns. Our results showed that the
ectopic expression of MYSM1 successfully rescued the differentiation defects of
the knockout cells in terms of the production of neurons and oligodendrocytes
(Fig. 3.10). These results provided convincing data to support a cell-autonomous
role of MYSM1 in regulating neuronal differentiation.

3.4.9 ChIP-Seq analysis of histone modification indicates a global impact
on neuronal genes by MYSM1 deletion
Based on the phenotypes we observed and the gene expression change in the
MYSM1 knockout NPCs upon differentiation, we hypothesized that MYSM1 is
likely to involve in activating neuronal genes that regulate neuronal differentiation
through removing repressive histone marks H2Aub1 and through cross-talk with
other histone modifications. To probe this hypothesis, we carried out a chromatin
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IP (ChIP) assay for H2Aub1 and another two well-established histone
modification marks, including the H3K4me2 as an active mark and the
H3K27me3 as a repressive mark. Prior to ChIP sequencing, we first validated the
ChIP assay protocol and antibodies that were going to be used for ChIP
sequencing. We first established the ChIP protocol and verified the antibodies by
performing ChIP qPCR in mouse ES cells on selective target loci, which have
been previously reported. The results showed that both the H3K4me2 and the
H3K27me3 antibody exhibited very high specificity and efficiency for the ChIP
assay in mouse ES cells (Fig. 3.11 A, B). The H3K27me3 ChIP showed a
thousand-fold enrichment at a representative bivalent gene Dlx1 promoter over
the control IgG, whereas the negative loci Sox2 promoter, which is an actively
expressed gene in mouse ESCs, is completely depleted from the repressive
mark H3K27me3 binding as expected (Fig. 3.11 A). To the contrary, the
H3K4me2 as an active histone mark showed a very high level of enrichment at
the Sox2 promoter region but was depleted from the negative region (Fig. 3.11 B).
While both H3K4me2 and H3K27me3 demonstrated very high binding efficiency
for ChIP assay, the H2Aub1 antibody demonstrated relatively low binding
efficiency and high background. We picked the Neurod2 locus as our tentative
target area and we achieved reproducible results showing a 10-fold enrichment
at the most occupied region, the coding region of Neurod2, compared to the
negative region in mouse ES cells (Fig. 3.11 C). The occupancy of H2Aub1 at
the Neurod2 locus is consistent with previous reports that H2Aub1 mostly
occupies the PCR1 targets, which consist of the core PRC target genes. In
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summary, our optimized protocol and antibodies are suitable for ChIP and ChIP-
Seq assays in mouse ESCs.

After validating the protocol and antibodies in mouse ESCs, we tested this
pipeline in mouse NPCs at the Neurod2 locus. ChIP qPCR results indicated that
that the Neurod2 gene still remained as a bivalent gene in the NPCs that were
cultured under proliferating conditions (Fig. 3.12 A). Neurod2 is not or very lowly
expressed under proliferating conditions and picked up its expression very
quickly upon differentiation in neural progenitor cells. ChIP qPCR of the
H3K4me2 and H3K27me3 in NPCs under proliferation conditions demonstrated
that the Sox2 promoter was highly enriched with the active marker H2K4me2 but
completely depleted from the repressive mark H2K27me3 as expected (Fig. 3.12
A). Interestingly, the Neurod2 locus was bound by both marks, suggesting a
bivalent status. The highest binding area for both histone marks coexisted at the
coding region (Fig. 3.12 A). More importantly, ChIP of the H2Aub1 in mouse
NPCs under proliferation conditions using previous established protocol for
mouse ES cells produced around 10 folds enrichment at the Neurod2 locus,
which is comparable to mouse ES cells (Fig. 3.12 B). The binding pattern of
H2Aub1 in NPCs was very similar to that of ES cells, which suggests an inherited
histone modification profiling for some bivalent genes from ES cell stage to NPCs
(data not shown). In summary, we have proved the success of our protocol and
antibodies for the ChIP assays of histone modifications including H3K4me2,
H3K27me3, and H2Aub1.
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We then performed ChIP assays for H3K4me2, H3K27me3 and H2Aub1, using
primary wild-type and MYSM1 knockout NPCs under the proliferating conditions.
We prepared bar-coded ChIP-Seq libraries of the ChIP samples that have been
confirmed at selective positive and negative sequences by qPCR. The bar-coded
ChIP-Seq library construction protocol was modified based on Peggy Farham’s
laboratory protocol. Prior to sending the self-prepared libraries for sequencing, all
the ChIP-Seq libraries were confirmed by qPCR at selective positive and
negative sequences in order to verify the enrichment of target sequences.
Sequencing of the bar-coded ChIP-seq sample was carried out by the USC
epigenome core and raw reads were downloaded. Since we have loaded equal
amount of libraries to the sequencer, theoretically equal amounts of reads for
each bar-coded sample should be generated by the sequencer. However, we
have found that sample reads varied among samples: we got 6 out of 8 samples
that reached a good amount of reads (above 10 million reads) for ChIP-Seq data
analysis, which include the H3K4me2, H3K27me3 and input for both wild-type
and knockout (Fig. 3.13 A). Although it was not clear what caused the low yield of
both wild-type H2Aub1 and knockout H2Aub1 samples, we are currently
generating new libraries for these two samples. Hence, all the following data is
based on the analysis of the comparison of the genome-wide occupancy of
H3K4me2 and H3K27me3 between wild-type and knockout NPCs. We have
called peaks using the sole-search tool, and generated high numbers of peaks
for each datasets for analysis (Fig. 3.13 A). Simply by visualizing some of these
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datasets on IGB (Integrated genome browser), it became obvious to us that our
ChIP-Seq data is of high quality and demonstrated expected binding profiling (Fig.
3.13 B). The neural stem cell self-renewal gene Sox2 locus was covered with
active histone mark H3K4me2, whereas the mouse ES cell exclusively
expressed gene Oct4 or Pou5F1 locus was completely depleted from the
H3K4me2 binding as expected (Fig. 3.13 B, C). The Neruod2 locus
demonstrated a similar binding pattern as our ChIP qPCR: it was covered by
active histone mark H3K4me2 and highest binding resides at the coding region
(Fig. 3.13 D). We also observed that many very high and sharp H3K4me2 peaks
were localized at the promoter region of genes, which is a typical binding pattern
of the H3K4me2 (Fig. 3.13 E). Taken together, these observations all
demonstrated our ChIP-sequencing and peak calling generated convincing
dataset for further analysis.

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Fig 3.11 Validation of histone modification antibodies for ChIP assay in
mouse ESCs

(A) ChIP of H3K27me3 and IgG at the Dlx1 promoter and Sox2 promoter region
in mouse ESCs. (B) ChIP H3K4me2 at the Sox2 promoter, GFAP promoter and a
negative region in mouse ESCs. (C) ChIP H2Aub1 at the NeuroD2 locus
spanning from the upstream around 5kb to 3’URT, P1-P11 are primers for the
promoter region; NC1 and NC2 are two primers designed for a region around
10KB downstream of the Neurod2 gene.

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Figure 3.12 Occupancy of important histone markers at the Neurod2 locus
in wild-type primary neural progenitor cells

(A) ChIP analysis of the occupancy pattern of H3K27me3 and H3K4me2 at the
NeuroD2 locus in primary wild-type NPCs in proliferation conditions. (B) ChIP
analysis of the H2Aub1 occupancy at the NeuroD2 locus in primary wild-type
NPCs in proliferation conditions. NC1 and NC2 are two pairs of negative primers;
the Sox2 promoter serves as the positive control for H3K4me2 binding, and the
negative control for H3K27me3 binding.

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Figure 3.13 Summary and examples of ChIP-Seq datasets

(A) A table summarized the reads related information about 8 samples. (B) A
track view of the H3K4me2 ChIP-Seq data at Sox2 locus. (C) A track view of the
H3K4me2 ChIP-Seq data at the Pou5f1 (Oct4) locus. (D) A track view of the
H3K4me2 ChIP-Seq data at the Neurod2 locus. (E) A track view of the H3K4me2
suggests the characteristic pattern at the promoter region of genes.

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The sole search based peak-calling generated thousands of peaks for both the
wild-type and the knockout H3K4me2 andnH3K27me3 datasets. Since we
realized that our knockout ChIP-Seq samples were generated from a NPC line
derived from a female embryo that has one inactivated X chromosome, whereas
the wild-type ChIP-Seq samples were generated from the NPC culture derived
from a male embryo that has one activated Y chromosome instead, in order to
avoid the potential biased comparison caused by peaks on the sex chromosome
X and Y, we first subtracted all the H3K4me2 peaks on the Y chromosome from
the knockout dataset and all the H3K27me3 peaks on the X chromosome from
both the wild-type and knockout datasets (Fig. 3.14) . We used the sole-search
tool to identify the peaks that are unique to each sample. Interestingly, we found
that the H3K4me2 ChIP in knockout cells has more peaks to start with, but
generated much fewer unique peaks, whereas the H3K4me2 ChIP in wild-type
cells has fewer total peaks, but 3 times more unique peaks than the knockout
dataset (Fig. 3.14). We then subtracted peaks that are smaller than 10 in peak
density, based on the assumption that bigger peaks are more likely to be
biologically relevant, and obtained similar results: that is, the wild-type H3K4me2
ChIP generated 6 times more unique peaks then that of the knockout H3K4me2
ChIP (Fig. 3.14). Interestingly, we observed the opposite pattern for the
repressive mark H3K27me3 ChIP: the H3K27me3 ChIP in wild-type cells started
with more total peaks but had much fewer unique peaks, whereas the
H3K27me3 ChIP in the knockout cells started with much less total peaks but had
6 times more unique peaks (Fig. 3.14). A similar pattern was observed when we
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cut off the peak density at 10 reads (Fig. 3.14). Taken together, these interesting
results based on unique peak identification lead to the hypothesis that knockout
NPCs’ chromatin may be occupied by significantly more repressive histone mark
H3K27me3, but much less active histone mark H3K4me2.

To test this hypothesis, we further analyzed the overlapping binding regions
between the wild-type and knockout datasets. Since there are large amount of
overlapping binding regions between the wild-type and knockout datasets, we
first identified the differentially bound genomic regions among the overlapped
binding regions, which have twice the peak density in one H3K4me2 dataset,
compared to the other. We named these peaks “enriched peaks”. Interestingly,
we found that the wild-type H3K4me2 ChIP has 3556 peaks that have twice the
peak density, compared to the knockout dataset, while knockout H3K4me2 ChIP
has only 21 peaks that have twice the peak density of the wild-type dataset (Fig.
3.14). The opposite pattern was observed for the H3K27me3 datasets: we found
that H3K27me3 ChIP in knockout NPCs has 919 peaks and have 1.5 times the
peak density of the wild-type dataset, but H3K27me3 ChIP in wild-type cells has
only 104 peaks of 1.5 times higher peak density than the knockout dataset (Fig.
3.14). Our differentially bound peak analysis demonstrated that around 10% of all
the genomic regions that are bound by active H3K4me2 in the wild-type NPCs
have twice the peak density of the same region in knockout NPCs on the
contrary, around 10% of repressive mark H3K27me3 bound genomic regions in
the knockout NPCs have 1.5 times the peak density of the same region in the
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wild-type cells, which suggests a globally altered chromatin modification status
with more repressive and less active histone modifications upon MYSM1 deletion.

We then further analyzed these more than 3000 H3K4me2 differentially bound
genomic regions or “enriched” peaks. Our location analysis showed that around
40% of these differentially bound regions, which have twice the H3K4me2
binding, are located either within the gene body or within 0-2 kb upstream of
genes (Fig 3.15 A). We were particularly interested in this subset of peaks or
genomic sequences, since these H3K4me2 peaks located within the gene body
and proximally upstream are more likely to have significant biological relevance
in the process of regulating gene transcriptional activities. Based on this rationale,
we sorted out this subset of genomic sequences and then identified the
associated genes. Above 500 genes were identified (data not shown), and we
then examined which part of the genes these differentially bound peaks are
located on. Our results showed that the most enriched area is intron 1 (data not
shown), which is usually considered a critical regulatory genomic region for
transcriptional regulation. We then performed a gene ontology study with this
cohort of genes using the David tool. Interestingly, the top 10 categories based
on the P-value suggested that this group of genes were strongly correlated with
neuronal differentiation and neuron development (Fig. 3.15 B), which is
consistent with the in vivo and in vitro phenotypes suggested a potential causal
relationship between the altered histone modification status and defected
neuronal differentiation. These genes include critical developmental regulators
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involved in neuronal differentiation, for example, Wnt family genes, Tubb3,
MATP2, Tbr1, Gsx2 and Nkx2.2 etc. (list not shown). More interestingly, this
cohort of genes are not only functionally enriched in the function categories
related to neuron development, but also expressed in tissues, which belong to
the CNS, which suggests that these genes are indeed correlated with the
development and function of the CNS, indicating a functional significance of the
cohort of genes (Fig. 3.15 C). Moreover, we have performed a ChIP assay of the
H3K4me2 and H3K27me3, respectively, using different lines of primary wild-type
and knockout NPCs in order to verify our bioinformatics analysis. We selected a
few sequences from the bioinformatically identified “enriched” peak regions that
are located on/or near the gene body of several most representative genes
related to neuronal differentiation, including Tbr1, TuJ1 and Neurod2 as the
tentative target genes, and then designed primer sets for the ChIP validation.
The ChIP qPCR results demonstrated the exact expected results in that the
active histone mark H3K4me2 binding at these genes were reduced in the
knockout cells, compared to the wild-type control (Fig. 4.16 A). However, the
repressive mark H3K27me3 binding at these loci were increased in the knockout
cells, compared to the wild-type controls (Fig. 4.16 B). Therefore, our
bioinformatics analysis and ChIP qPCR verification provided convincing evidence
to support the hypothesis that there is a critical cohort of cell-fate specifying
genes involved in neuronal differentiation, which are occupied by a lower amount
of active histone mark H3K4me2, but higher amount of repressive mark
H3K27me3, in the MYSM1 NPCs in vitro.
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Figure 3.14 Genome-wide location analysis of active and repressive histone
marks H3K4me2 and H3K27me3 in primary wild-type and MYSM1 knockout
NPCs

Summary of the analysis and the comparison of H3K4me2 and H3K27me3 peak
numbers after peak-calling by Sole-search in wild-type and knockout NPCs.

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Figure 3.15 Gene ontology analysis of a core subset of the H3K4me2
differentially bound regions

(A) Pie diagram of the distribution of H3K4me2 enriched peaks in the genome. (B)
Top 10 ranked GO functional categories that are enriched by the genes that are
associated with the differentially bound peaks (gene body and 0-2kb upstream),
suggested a strong correlation between the core subset of peaks and neuron
development. (C) Top 10 ranked GO tissue type, by which the core subset of
differentially bound peaks is expressed based on GO study.

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Figure 3.16 Validation of ChIP-Seq results by ChIP qPCR at selective
targets

(A, B) ChIP QPCR of H3K4me2 and H3K27me3 at genes related to neuronal
differentiation, including Tbr1, Tubb3, Gsx2 and Neurod2 locus in wild-type and
MYSM1 knockout NPCs. All primer sets were selected from the differentially
bound region identified through ChIP-Seq analysis.

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3.5 Discussion
Epigenetic regulations play critical roles during stem cell differentiation.
Understanding the biological function of histone H2A deubiquitination is of
essential importance in revealing the epigenetic regulation of gene transcription
during neural stem cell differentiation. However, currently we know very little
about the physiological roles of histone H2A deubiquitination and nothing about
the biological roles of any identified histone H2A deubiquitinases during neural
development. MYSM1 is the first one, which has been reported to play an
essential role in tissue development (Jiang et al.; P. Zhu et al.). In this study, we
have revealed that the histone H2A deubiquitinase MYSM1 is expressed by the
cortical neural progenitor cells and plays an essential role in the neuronal cell
fate specification by activating or derepressing a cohort of target genes involved
in the neuronal cell fate determination. Our genome-wide sequencing results
demonstrated that MYSM1 is likely to activate or derepress its target genes
through orchestrating with other critical histone modifications at the target loci.
Our study has revealed a novel role of the histone H2A deubiquitinase MYSM1 in
cortical development, and further illustrated the molecular mechanism of histone
H2A deubiquitination in general.

We have performed a detailed analysis of the phenotypes of the MYSM1
knockout mice in terms of neural development. Detailed in vivo analysis allowed
us to discriminate between the effects of MYSM1 on self-renewal, proliferation,
cell survival, cell fate specification and migration. Firstly, our cell cycle analysis
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and the quantification of the cells in mitosis in vivo revealed that there is a normal
maintenance of the NPC pool without any overall cell cycle aberrations upon
MYSM1 deletion. Secondly, careful quantification of neurons in different cortical
layers demonstrated that the function of MYSM1 during corticogenesis is not
exclusively restricted to the production of any cortical layers. The results also
suggested that MYSM1’s function is not restricted to the dorsal cortex alone, but
that it is also involved in the production of ventrally derived neuron subtypes,
which is consistent with its broad expression in the ventricular zone. Thirdly, our
cell death analysis and detection of over-production of astrocytes provided
explanations for the consequences of MYSM1 knockout NPCs’ compromised
differentiation capacity. Our analysis showed that the impaired differentiation of
NPCs is likely to result in mild apoptosis of basal progenitor cells and shift of cell
fate output towards the astrogenesis in the later stage. Lastly, our birth dating
[separate words?] study of the E14.5 NPCs in vivo provided convincing results
demonstrating that loss of MYSM1 results in compromised neuronal specification
but increased astrocyte production. In conclusion, loss of MYSM1 caused the
compromised neuronal differentiation capacity of all cortical layers without
affecting the maintenance of the neural progenitor pool.

Our phenotype analysis supports the hypothetical role of H2A mono-
ubiquitination in repressing neuronal developing genes during cortical
neurogenesis. Therefore, the loss of specific H2A deubiquitinase may comprise
the activation of the transcription of these critical developing genes, which will
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result in defected basal progenitor cell specification, rather than compromised
self-renewal and proliferation of NPCs. Therefore, it is reasonable to believe that
both apoptosis and increased production of astrocytes are secondary
consequences to the primary defect in differentiation. Recently, it has been
reported that astrocytes can proliferate postnatally to further populate the cortex.
However, since we have not only counted the total GFAP+ cells but also
quantified the BrdU+/GFAP+ cells, which retained high level of BrdU signal, we
believe that the increased number of astrocytes in the P7 knockout cortex did not
result from postnatal local astrocytes proliferation. It is more likely to result from
the impaired activation of the neuronal transcriptional program, which leads to
the death of Tbr2+ neuronal precursors or switch into glial cell fate at later stages.

Histone ubiquitination of H2A has been indicated to function as one of the
chromatin-repressive epigenetic marks and plays critical roles in gene
transcriptional silencing during neural development. This hypothesis was based
on the fact that deletion of two main histone H2A ubiquitinase Ring1b and Brac1
in mice leads to significant derepression of their own target genes. Ring1B is the
central component of polycomb repressive complex 1 (PRC1), which targets
largely developmental regulators in mouse ES cells and differentiation genes in
progenitor cells. Conditional deletion of Ring1B in RGCs leads to derepression of
Ngn1 at an early stage of neurogenesis, which then causes the extended
neurogenic phase and delayed neuron-glia switch, leaving NPC proliferation
unaffected. However, whether this is due to a histone H2A ubiquitination-
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mediated epigenetic mechanism is not clear and how H2A ubiquitination could
regulate the transcription of the neuronal cell fate determinant genes is unknown.
Our in vitro differentiation analysis and ChIP-Seq results have provided novel
insights into understanding these issues. In particular, our in vitro differentiation
of MYSM1 knockout NPCs suggested only differentiation defects rather than
proliferation and cell survival, which is consistent with our hypothesis based on
the in vivo data. Surprisingly, the defected differentiation potential can be
reflected by the histone marks binding even in proliferating conditions without
given any differentiation signals. Our genome-wide ChIP-Seq analysis of the
active histone mark H3K4me2 and repressive histone H3K27me3 demonstrated
that prior to the initiation of differentiation, more regions of the knockout
chromatin were bound by repressive histone mark H3K27me3, whereas fewer
regions of the knockout NPC chromatin were bound by active histone mark
H3K4me2. More importantly, 10% of H3K4me2 commonly bound regions have
twice the peak density in wild-type cells, compared to knockout cells. Conversely,
10% of H3K27me3 commonly bound regions have 1.5 times the peak density in
knockout cells, compared to wild-type control. Further analysis demonstrated that
the functions of the H3K4me2 differentially bound genes are highly associated
with neuron development and neuronal differentiation. Moreover, the expression
of these genes is also highly enriched in the central nervous system. Although
we are still in the process of generating H2Aub1 ChIP-Seq data, it is highly likely
that there are accumulated H2Aub1 at those loci, which have shown altered
H3K4me2 and H3K27me3 binding, and we hypothesize that MYSM1 may be
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specifically recruited to those neuron development regulating genes and that it
removes H2A monoubiquitination from these genomic regions to facilitate the
establishment of a more active chromatin status through cross talking with other
epigenetic modifier and histone marks (Fig. 3.17). Comparison of H2Aub1
genome-wide occupancy in wild-type and knockout NPCs and analysis of
MYSM1 genom-wide binding profiling will be critical to further elucidate the
mechanism of the histone H2A deubiquitinase activity of MYSM1 mediated
transcription regulation during neuronal differentiation.

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Figure 3.17 Model of the MYSM1 regulated neuronal differentiation

This scheme illustrates our current working model regarding the mechanism of
MYSM1-mediated neuronal differentiation. Loss of MYSM1 in NPCs resulted in a
impaired histone modification-binding pattern on a cohort of critical neuronal
differentiating genes. These genes are more occupied by H3K27me3 and less
occupied by H3K4me2, which are likely caused by accumulated H2Aub1. These
changes together contribute to a more repressed transcriptional activity of
neuronal differentiating genes, which then leads to the impaired neuronal
differentiation.

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CHAPTER 4
CONCLUSIONS AND PERSPECTIVES

Neurological diseases and neurodegenerative diseases are two major
therapeutic areas that have very few effective therapies under the current
treatment. Pluripotent stem cells especially with the discovery of the induced
pluripotent stem cell (iPS) technology have shown great potential in providing
unlimited sources for the neural tissue repair and replacement and in modeling
diseases. However, obtaining large amount of neural cells of very high quality
and purity has always been the bottleneck for the application of stem cell
technology in both regenerative therapy and drug discovery. Therefore, it is of
vital importance to understand the molecular mechanisms underlying the
complex cell fate specification process during neural progenitor cell differentiation.

Embryonic mammalian brain development serves as a great model for us to
study the cell fate specification process of NPCs. During my six years of research
here, I have focused on understanding the cell fate determination of neural
progenitor cells in the developing mammalian forebrain. I have been focused on
investigation of two very important aspects of this complex process, one is the
role of extrinsic signaling in modulating the output from the ventral forebrain, and
the other one is the role of epigenetic regulations in cortical neurogenesis. My
study have mainly provided novel insights in the role of Wnt signaling in
promoting neuronal output but inhibiting glial output at the early stage of ventral
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neurogenesis and revealed the biological functions of a histone deubiquitinase
during corticogenesis and underlying molecular mechanisms for the first time.

4.1 Ryk receptor and ventral NPC output
Our detailed analysis of all developing stages has demonstrated that Wnt
atypical receptor Ryk is highly expressed by the ventral neural progenitors in the
embryonic cerebral cortex at the peak time of ventral neurogenesis. Our results
suggest that Ryk mediated Wnt signaling imposes the opposite effects on the
production of ventral derived neural lineages: the GABAergic neurons and
oligodendrocytes. The deletion of Ryk receptor results in a substantially
decreased production of GABAergic neurons from the ventral NPCs whereas
dramatically increased production of oligodendrocyte precursor cells in vivo.

4.2 Dissection of the cellular mechanism of Ryk signaling
We found that the overproduction of oligodendrocyte precursor cells was not
caused by precocious differentiation of OPCs. Cell cycle analysis by labeling
cells that are the M phase, S phase and cell death analysis have demonstrated
that the phenotypes we have observed were not resulted from abnormal ventral
progenitor proliferation and cell survival defects. Analysis of dorsal-ventral
patterning by labeling patterning transcriptional factors suggested that the cell
fate switch away from neuronal lineage towards glial lineage is not a patterning
defect rather a post-patterning cell fate switch.

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E14.5 telencephalon derived primary cultured Ryk knockout neural progenitor
cells can recapitulate the in vivo Ryk knockout phenotypes. Ryk knockout neural
progenitor cells give rise to significantly reduced numbers of GABAergic neurons,
but doubled amount of oligodendrocytes in vitro. In addition, we have
successfully proved that this opposite phenotypes exhibited by two different
neural lineages were not secondary effects of a non cell-autonomous mechanism
rather caused by the cell-autonomous, in other word, intrinsic impaired
differentiation capacity.

4.3 Molecular mechanism of Ryk mediated Wnt signaling
We have addressed two aspects of the Ryk receptor mediated Wnt signaling in
regulating the cell fate switch during ventral neural progenitor cell differentiation.
First, we have identified the upstream extrinsic cue that is responsible to
biological function of Ryk receptor in the context of binary fate choice between
the GABAergic neuron differentiation versus oligodendrogenesis. We have
treated the primary NPC culture system with various wnt proteins and identified
that Wnt3a can induce GABAergic neuron differentiation while inhibit
oligodendrocyte differentiation at the same time in a Ryk receptor dependent
manner. Therefore, we have demonstrated that Wnt3a is the upstream of Ryk
receptor in modulating the cell fate choice between GABAergic neuron versus
oligodendrocytes. Secondly, we have revealed how Ryk receptor can transduse
the extrinsic signals into the nucleus to regulate cell fate. We found that the Ryk
intracellular domain (ICD) was necessary and sufficient to rescue the defected
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differentiation of neural progenitor cells with regard to both reduced neuronal
differentiation and increased oligodendrocyte differentiation. In addition, we have
also demonstrated that overexpression of Ryk ICD in the Ryk knockout
background induced the expression of GABAergic neuron cell fate determinant
gene Dlx2 expression but represses the expression of oligodendrocyte cell fate
determinant genes. Therefore, it is possible that Ryk ICD could simultaneously
regulate the transcriptional activity of two different sets of target genes via
working together with either coactivators or corepressors.

4.4 Histone H2A deubiquitinase MYSM1 and cortical neurogenesis
We have demonstrated that the histone H2A ubiquitinase MYSM1 is highly
expressed by cortical neural progenitors during embryonic neurogenesis. Loss of
MYSM1 results in reduced forebrain size, which can be observed at E16.5 and
onwards. In postnatal stage, survived MYSM1 knockout mice suffered from
significantly smaller forebrain and severe ataxia and uncoordinated movement.
Detailed analysis of neuronal markers at E14.5 and E18.5 uncovered that
MYSM1 mice exhibited impaired neuronal production of all cortical layers.
However, migration of cortical neurons was not obviously affected since the
positioning of cortical layers was still maintained in the MYSM1 knockout cortex.
Therefore, MYSm1 plays critical roles in promoting the production of cortical
projection neurons.

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4.5 MYSM1 regulates neuronal cell fate specification
We have demonstrated that self-renewal and proliferation capacity of NPCs was
not largely affected by the loss of MYSM1 based on detailed analysis of cell cycle
and proliferation. As a result of compromised differentiation capacity likely due to
the inactivation of neuronal genes, increased level of apoptosis was detected by
the TUNNEL assay, especially in the basal progenitor level. Moreover, although
no obvious precocious differentiation of glial lineage astrocytes was detected at
embryonic stages, dramatically increased amount of astrocytes were detected at
postnatal day 7. The increased apoptosis and production of astrocytes are likely
to be secondary to the impaired neuronal specification. Another line of evidence
is the in vitro NPC population assay, primary MYSM1 knockout neural progenitor
cells proliferate normally but give rise to reduced numbers of neurons and
increased numbers of astrocytes. In summary, MYSM1 is like to specifically be
involved in the neuronal specification process rather than NPC self-renewal and
proliferation.

4.6 Global histone modifications defects upon MYSM1 deletion hint
impaired differentiation capacity
The wild-type and knockout primary neural progenitor cells served as a platform
for the investigation of the underlying molecular mechanism of MSYM1 regulated
neuronal differentiation. We have performed genome-wide active histone mark
H3K4m2 and repressive histone mark H27me3 ChIP-Seq analysis, and
demonstrated that even before neural progenitor cell differentiation starts, the
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loss of MYSM1 has already caused a global altered histone modification state.
Our analysis has demonstrated that more genomic regions are bound by the
repressive mark H3K27me2 while less genomic regions are bound by the active
histone mark H3K4m2 upon MYSM1 deletion. Moreover, a critical group of
neuron development regulators are bound with increased amount of repressive
mark H3K27me3 but less active mark H3K4me2, suggesting a correlation
between impaired “primed” chromatin statuses is likely to result in defected
differentiation potential. We have also confirmed our ChIP-Seq analysis by ChIP-
qPCR of selective genes including Tbr1, TuJ1, Gsx2 and Neurod2. Our ChIP-
qPCR and ChIP-Seq together support a role of MYSM1 in regulating the
transcriptional activation of neuronal gene. Loss of the deubiquitinase MYSM1
may accumulate H2Aub1 at those loci that have shown altered H3K4me2 and
H3K27me3 binding, and MYSM1 may be specifically recruited to those neuron
development regulating genes and removes H2A monoubiquitination from those
genomic regions to facilitate the establishment of a more active chromatin status
through cross talk with other epigenetic modifier and histone marks.

4.7 Future directions
We would like pursuing three future directions in order to further test our
hypothesis regarding MYSM1 mediated activation of specific neuronal genes
during mammalian cortical neurogenesis. First, we are currently in the process of
finishing ChIP-Seq analysis of H2Aub1 in both wild-type and knockout. We
expect that we will obtain high quality datasets of H2Aub1 in the near future, so
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that we could reveal whether indeed loss of MYSM1 caused accumulated
H2Aub1 at a genome wide level and whether the genomic regions with over
accumulation of H2Aub1 are correlated with the groups of genes that were
identified from the H3K4me2 and H3K27me3 ChIP-Seq analysis. Secondly, since
we have found that the altered histone modification status in the knockout NPCs
could even be detected under the proliferation conditions, we believe it is likely
that we could detect MYSM1 direct binding sites using ChIP-Seq using
proliferating NPC culture as well. Hence, we are currently in the process of
conducting Mysm1 ChIP assay in order to identify the target genes of MYSM1.
Due to unavailability of commercial ChIP grade MYSM1 antibodies, we have
been trying to perform ChIP for the Flag of the ectopically expressed MYSM-Flag
on the MYSM1 knockout background. We have generated low level expressed
MYSM1-Flag knockout cells line, which could be used for this purpose. Currently,
we are also testing various recent developed commercial MYSM1 from different
venders in order to identify a ChIP grade Mysm1 antibody. Thirdly, we sought to
discover Mysm1 interacting proteins in the NPCS in order to uncover the
molecular mechanism in which MYSM1 is recruited to a specific set of neuronal
genes.

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Abstract (if available)

Abstract The mammalian central nervous system is the most complex organ among all the mammalian organs and we have just began understanding the highly orchestrated but nonlinear process of brain development. Brain development involves many various cellular behaviors including progenitor self-renewal, proliferation, patterning, differentiation, migration etc. My dissertation research in Dr. Wange Lu’s lab has mainly focused on one core question in the field of neural development: to uncover the mechanisms regulating the cell fate specification of neural progenitor cells during brain development. My research involves the investigation of two independent but closely related aspects of this core question. ❧ In my thesis, I have first investigated the role of an atypical Wnt receptor Ryk in regulating the cell fate choice between the neuronal differentiation and oligodendrogenesis and demonstrated that Wnt3a-Ryk-mediated signaling promotes GABAergic neuron production, while inhibiting oligodendrocyte differentiation through a Ryk intracellular domain-dependent mechanism. ❧ Secondly, I have investigated the biological function of a histone H2A deubiquitinase MYSM1 in cortical neurogenesis. Both in vivo and in vitro analyses demonstrate that MYSM1 plays a critical role in neuronal cell fate specification without affecting the maintenance of the cortical progenitor pool. To further elucidate the underlying molecular mechanisms, a ChIP-Seq analysis of genome-wide occupancy of repressive histone mark H3K27me3 and active histone mark H3K4me2 in both wild-type and MYSM1 knockout primary neural progenitor cell culture under proliferating conditions was conducted: it shows that a group of genes regulating neuronal development exhibit a more repressive but less active chromatin status upon loss of MYSM1. This research supports the role of MYSM1 in regulating transcriptional activation of neuronal gene to facilitate the neuronal cell fate specification, likely through removing repressive histone mark H2Aub1 and cross-talk with other histone modifications. ❧ This research is of great importance not only for our understanding about the extremely complicated establishment process of the central nervous system, but also provides a potential application for developing stem cell based therapy.

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Asset Metadata

Creator Zhong, Jingyang (author)

Core Title Neural fate regulated by extrinsic signaling and epigenetics

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 08/05/2013

Defense Date 02/01/2013

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

Tag histone modification,neural development,neural stem cells,neuron,OAI-PMH Harvest,oligodendrocyte

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Lu, Wange (committee chair), Crump, Gage D. (committee member), Ma, Le (committee member)

Creator Email zhongjingyang5@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-314961

Unique identifier UC11292767

Identifier etd-ZhongJingy-1950.pdf (filename),usctheses-c3-314961 (legacy record id)

Legacy Identifier etd-ZhongJingy-1950.pdf

Dmrecord 314961

Document Type Dissertation

Format application/pdf (imt)

Rights Zhong, Jingyang

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