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Intrinsic mechanisms governing retinal progenitor cell biology:Retinal homeobox transcriptional regulation and the function of forkhead transcription factors during eye development

ProQuest Dissertations and Theses, 2009
Dissertation
Author: Holly E Moose
Abstract:
Understanding the development and maintenance of Retinal Progenitor Cells (RPCs) is critical to understanding normal and disease processes within the neural retina. To understand RPC biology, it is important to understand the transcriptional regulation of known intrinsic regulators, and continue to identify new RPC expressed genes to demonstrate their function during eye development. The studies presented in this work address the transcriptional regulation of the Xenopus laevis Rx gene product, Rx2A, and function of two newly recognized RPC genes, FoxO3 and FoxM1. To further the understanding of transcriptional regulation in RPCs, we characterized the Rx2A promoter in transgenic embryos. Both the distal portion and the proximal portion of the Rx2A promoter are sufficient for expression of a GFP transgene in the developing eyes. We identify a highly conserved element in the distal region of the Rx2A promoter (UCE). Within UCE, an OTX, SOX and POU site act as cis-elements to coordinately specify proper gene expression in the developing eye. We show that the activity of the proximal promoter is dependant on a forkhead-binding element (FBE). In addition, we have shown that the distal region containing the UCE can cooperate with the FBE to maintain robust Rx expression throughout all stages of eye development. The work associated with the transcriptional regulation of Rx furthers our understanding of how this primary retinal transcription factor is regulated. This is applicable to the understanding of RPC development since Rx is one of the first eye field transcription factors expressed in the anterior neural plate as RPCs are specified. The identification of the FBE within the Rx2A promoter led to further investigation of the involvement of the forkhead family of transcription factors in vertebrate eye development. We present a discussion of current data regarding the expression and function of this family of transcription factors during eye development, and we present the expression pattern of 5 forkhead transcription factors (FoxG1, FoxN2, FoxN4, FoxM1 and FoxP1) in the maturing X. laevis retina. These forkhead gene products have not been previously described in the maturing retina of X. laevis . We chose to pursue studies of FoxO3 and FoxM1 in the developing neural retina to further the understanding of RPC regulation by transcription factors of the forkhead family. These two factors were chosen for co-current studies for the following reasons: (1) neither gene product had been ascribed a role in developing RPCs, (2) their known functions suggested they act in opposing ways with regards to the cell cycle, and (3) due to their expression patterns both FoxO3 and FoxM1 serve as candidate factors to regulate the Rx2A promoter through the FBE. To define the role of FoxO3 during vertebrate eye development, we overexpressed FoxO3 RNA in the anterior neural plate of X. leavis embryos. FoxO3 overexpression results in embryos with small eyes. The small eye phenotype is a result of decreased proliferation, induction of apoptosis, and changes in RPCs gene expression. The phenotype can be exacerbated by introducing a threonine to alanine mutation at a conserved PI3K phosphorylation site, which produces a constitutively nuclear form of FoxO3. The changes in gene expression suggest that FoxO3 can function to delay the differentiation of RPCs, although they are properly specified. The data supports our original hypothesis regarding FoxO3 as cell cycle antagonist with the ability to alter the differentiation capacity of RPCs. To investigate the function of FoxM1 in developing RPCs, we performed loss-of-function studies using morpholino oligonucleotides (MO). FoxM1 knockdown in the anterior neural region of X. laevis results in embryos with slightly smaller eyes and clear defects in retinal lamination. Our data reveals that FoxM1 is not necessary for the specification of RPCs, and suggests that differentiation into the Muller glia, and rod photoreceptor lineages is possible with reduced levels of FoxM1. The studies presented concern the transcriptional regulation of the Rx gene product, Rx2A, and a role of FoxO3 and FoxM1 during RPC development. Collectively, they represent an advancement in the knowledge regarding two important intrinsic mechanisms governing RPC development: transcriptional regulation and transcription factor function.

TABLE OF CONTENTS

Abstract...............................................................................................................................ii Acknowledgments................................................................................................................v Dedication..........................................................................................................................vi Vita....................................................................................................................................vii List of Tables....................................................................................................................xii List of Figures..................................................................................................................xiii List of Abbreviations.........................................................................................................xv

Chapter 1: Development and Regulation of Retinal Progenitor Cells by Known Intrinsic Mechanisms.........................................................................................................1 1.1 Overview........................................................................................................................1 1.2 Eye development in vertebrates and specification of Retinal Progenitor Cells.............2 1.3 From RPC to retinal cell types: Molecular regulation of retinal cell types by intrinsic factors.........................................................................................5 1.4 RPC decision making: The balance between proliferation and differentiation.............8 1.5 Xenopus Laevis as a model system to study RPC development..................................11 1.6 The impact of this work relative to the field of retinal development...........................12

Chapter 2: Regulation of Retinal Homeobox Transcription by Cooperative Activity Among Cis-elements........................................................................................................19 2.1 Introduction..................................................................................................................19 2.2 Materials and Methods.................................................................................................22 2.3 Results..........................................................................................................................26 2.3.1 Identification and conservation of the Rx2A regulatory sequence...............26 2.3.2
The
Rx2A
regulatory
sequence
directs
gene
expression
in








 























regions
of
endogenous
Rx
expression...................................................................28
 2.3.3
Spatial
and
temporal
specificity
of
Rx
expression
are
determined

















 









by
regions
within
the
Rx2A
regulatory
sequence............................................29


ix
























Analysis
of
5’
end
promoter
deletions ...........................................29 Analysis
of
internal
deletions......................................................................30
 



































Analysis
of
Hsp
constructs............................................................................32
 2.3.4
The
SOX
and
OTX
sites
within
UCE
act
cooperatively
to
provide









 










spatial
specificity
to
Rx
expression........................................................................33
 2.3.5
Rx2A
promoter
activity
in
the
mature
retina....................................................34
 2.3.6
Cognate
factors
that
bind
the
Rx2A
cis‐elements............................................35
 2.4 Discussion....................................................................................................................37 2.4.1 Rx genes in X. leavis are similarly regulated................................................37 2.4.2 Expansion of the knowledge of Rx cis-regulatory elements........................37 2.4.3 The central region of the Rx2A promoter contains a repressor element......38 2.4.4 The UCE and FBE cooperate to activate Rx2A promoter activity...............39 2.4.5 Putative trans-acting factors of the Rx2A promoter.....................................41 POU factors..........................................................................................41 Otx family member..............................................................................42 Sox family members............................................................................45 Forkhead family members...................................................................47 2.4.6 Coordinated regulatory activity of gene families implicated in Rx2A promoter activity...........................................................................................47 2.4.7 Future directions...........................................................................................50

Chapter 3: Seeing Eye to Eye: Forkhead Transcription Factors During Eye Development.....................................................................................................................68 3.1 Introduction..................................................................................................................68 3.2 Methods........................................................................................................................69 3.3 Results and Discussion................................................................................................69 3.3.1 Forkhead transcription factors in anterior eye structures.............................69 3.3.2 Forkhead transcription factors in the developing neural retina...................71 3.3.3 Forkhead transcription factors in differentiated retinal cell types...............73 3.3.4 Forkhead transcription factors in retinal progenitor cells............................74 3.3.5 Retinal forkhead function in eye development............................................77

x

3.3.6 Retinal forkhead transcription factors hold enormous promise to advance our knowledge of retinal progenitor cell biology........................................82

Chapter 4: FoxO3 Perturbs Vertebrate Eye Development by Affecting Differentiation and Proliferation in Retinal Progenitor Cells.....................................86 4.1 Introduction..................................................................................................................86 4.2 Materials and Methods.................................................................................................88 4.3 Results..........................................................................................................................92 4.3.1
X.
laevis
FoxO3
protein
is
highly
conserved

 and
expressed
in
developing
eye
tissue...........................................................92
 4.3.2
Overexpression
of
xlFoxO3
results
in
a
small
eye
phenotype....................95
 4.3.3
Differentiated
cell
types
are
produced
in
FoxO3‐injected
embryos........97
 4.3.4
Retinal
Progenitor
Cells
are
specified
but
exhibit
altered
differentiation


 










in
FoxO3
injected
embryos........................................................................................97
 4.3.5
FoxO3
overexpression
results
in
altered
cell
cycle
in
RPCs........................98
 4.3.6
FoxO3
overexpression
results
in
increased
apoptosis..................................99

 4.4 Discussion..................................................................................................................100 4.4.1 A model for FoxO3 function in RPCs........................................................100 4.4.2 Production
of
differentiated
cell
types
in
the
presence
of
exogenous




 























levels
of
FoxO3.............................................................................................................100
 4.4.3 Cell Cycle alterations in FoxO3-injected embryos...................................101 4.4.4 FoxO proteins as regulators of diverse progenitor cell populations.........103 4.4.5 Is RPCs regulation a conserved function for FoxO3 in vertebrates?........104

Chapter 5: FoxM1 is Necessary for Normal Development and Proper Lamination of the Neural Retina by Retinal Progenitor Cells............................................................118 5.1 Introduction................................................................................................................118 5.2 Materials and Methods...............................................................................................123 5.3 Results........................................................................................................................125 5.3.1 Identification of a FoxM1 Isoform in X. laevis...........................................125

xi

5.3.2 A xlFoxM1 homologue is expressed in RPCs during eye development....126 5.3.3
FoxM1
knockdown
in
X.
laevis
results
in
embryos
with
small
eyes......127
 5.3.4
xlFoxM1
is
necessary
of
proper
retinal
lamination.....................................129
 5.3.5
Differentiated
cell
types
are
associated
with
areas
of
normal
retina


 










lamination
and
regions
of
aberrant
stratification........................................130
 5.4 Discussion and Future Directions..............................................................................131

Chapter 6: The significance of exploring Rx regulation and Forkhead transcription factor function in RPCs.................................................................................................146 6.1 Summary of significant findings................................................................................146 6.2. Exploring the relationship between Rx2A, FoxO3, FoxM1 in RPCs.......................147 6.3 Regulation of RPC development by Homeobox and Forkhead transcription factors .................................................................................148 6.4 Closing remarks.........................................................................................................149 Appendix A......................................................................................................................150 Bibliography....................................................................................................................154

xii

LIST OF TABLES Figure Page Number 2.1 Expression domains of Rx2A promoter deletion construct......................................53 2.2 PCR primers used to isolate the Rx2A regulatory region.........................................64 2.3 Generation of Rx2A deletion constructs...................................................................65 2.4 Primers used in the generation of Rx2A deletion constructs....................................66 2.5 Primers used to test cognate factor binding to cis-elements in the Rx2A promoter.67 3.1 Production of anti-sense probes for gene expression analysis in X. laevis...............69 3.2 Forkhead gene products expressed in eye structures................................................83 3.3 Retinal forkhead expression......................................................................................84 4.1 Production of anti-sense probes for gene expression analysis in FoxO3-injected embryos.......................................................................................90 5.1 Production of anti-sense probes for gene expression analysis in FoxM1 MO-injected embryos............................................................................125

xiii

LIST OF FIGURES Figure Page Number 1.1 Eye development in vertebrates ..................................................................................14 1.2 Retinal lamination and cell types in Xenopus Laevis...................................................15 1.3 Birthdating in the retina and the competence model of retinal development..............16 1.4 Molecular regulation of RPC development.................................................................17 1.5 The eukaryotic cell cycle and RPCs............................................................................18 2.1 Conservation and in silico analysis of the Rx2A regulatory region............................52 2.2 Rx2A 2.8 directs gene expression in the eye fields, hypothalamus and pineal gland....53 2.3 Deletion constructs produced to investigate transcriptional activity of the Rx2A regulatory region...............................................................................54-55 2.4 The Rx2A promoter contains cis-elements required for proper spatial and temporal gene expression of the GFP transgene.........................................................................57 2.5 Internal deletions of the Rx2A promoter reveal additional regulatory region that ensure specificity to the developing eyes.....................................................................58 2.6 Cooperative activity of UCE and the proximal FBE directs gene expression 56 throughout developmental stages.................................................................................59 2.7 POU, Sox and Otx sites within UCE cooperatively regulate Rx expression in the developing eyes of Xenopus leavis embryos......................................................60 2.8 Rx2A promoter activity in the mature Xenopus laevis retina......................................61 2.9 Oct-6, FoxO3 and FoxN4 bind the Rx2A promoter cis-elements in vitro...................62 2.9 Model of Rx2A transcriptional regulation derived from deletion analysis.................63 3.1 Expression of retinal forkhead genes in the CMZ of the maturing neural retina in Xenopus laevis..............................................................................................................85 4.1 Xenopus laevis FoxO3 protein is highly conserved and is expressed throughout eye development.....................................................................................................................105 4.2 xlFoxO3 RNA is targeted to the developing eye fields of X. laevis embryos...........106

xiv

4.3 Overexpression of xlFoxO3 results in small eye phenotype.....................................107 4.4 T30A mutation in xlFoxO3 increased the frequency of eye phenotypes...................108 4.5 T30A mutation in xlFoxO3 increased the severity of the small eye phenotype........109 4.6 Retinal lamination is intact in the presence of exogenous xlFoxO3..........................110 4.7 Normal differentiated cell types are present in xlFoxO3-injected embryos..............111 4.8 RPCs are specified in the presence of xlFoxO3 overexpression...............................112 4.9 Differentiation is altered by xlFoxO3 overexpression...............................................113 4.10 Overexpression of xlFoxO3 results in changes in cell cycle gene expression........114 4.11 Overexpression of xlFoxO3 results in decreased rate of cell cycle in RPCs...........115 4.12 xlFoxO3 overexpression results in increased levels of apoptosis............................116 4.13 Proposed model of FoxO3 function in RPCs...........................................................117 5.1 Xl184i11 encodes a FoxM1 protein...........................................................................136 5.2 xlFoxM1 is homologous to hFoxM1 isoform C........................................................137 5.3 FoxM1 is expressed in the developing eye fields and in proliferative zones of the maturing Xenopus laevis retina..................................................................................138 5.4 FoxM1 morpholino is properly targeted to developing eye fields in Xenopus embryos......................................................................................................................139 5.5 FoxM1 morpholino specifically knocks down FoxM1 target............................140-141 5.6 FoxM1 knockdown causes small eye phenotype in X. laevis....................................142 5.7 FoxM1 morphants with eye phenotype......................................................................143 5.8 FoxM1 knockdown results in small eye with lamination defects..............................144 5.9 Differentiated cell types and abnormal retinal patterning are present in FoxM1 morphant retinas.........................................................................................................145 A.1
Cyclin
D1
is
robustly
expressed
in
the
developing
X.
laevis
retina.........................151
 A.2
N‐myc
is
dynamically
expressed
in
the
developing

 






X.
laevis
eyes
and
retina..............................................................................................................152
 A.3
p27
is
dynamically
expressed
in
the
maturing
X.
laevis
retina.................................153

xv

LIST OF ABBREVIATIONS

RPC retinal progenitor cells MO morpholino oligonucleotide pg picogram mM micromolar µg microgram RPE retinal pigmented epithelium CMZ ciliary marginal zone GCL ganglion cell layer IPL inner plexiform layer INL inner nuclear layer OPL outer plexiform layer ONL outer nuclear layer EFTFs eye field transctipsion factors bHLH basic helix loop helix HD homeodomain CDK cyclin dependant kinase CDKI cyclin dependant kinase inhibitors FKD forkhead RGC retinal ganglion cells EST expressed sequence tag

1

CHAPTER 1 : Development and Regulation of Retinal Progenitor Cells by Known Intrinsic Mechanisms

1.1 Introduction

Charles Darwin, when writing his book, “The Origin of Species”, included his description of the eye in the chapter entitled: “Organs of extreme perfection and complication.” To this day, scientists are continuing to pursue an understanding of this fascinatingly complex and intricate organ. The study of the eye, and more specifically retinal development, has been enhanced by the age of molecular biology and advanced by techniques of genetic manipulation. Our understanding is still remarkably limited, however, as new molecules involved in eye development are still being uncovered, and the mechanisms that underlie their function continue to be elucidated. Progenitor cells are by definition multipotent, and the neurons and glia of the retina are derived from a cell population termed retinal progenitor cells (RPCs). RPC development and function is known to be molecularly regulated, and this regulation occurs on many levels. Initially, expression of RPC specific genes must be regulated so that they are correctly expressed as cell fate determinants. As well, translational and post-

2 translational mechanisms exist in order to dictate that proper levels of functional protein exist within developing RPCs. Mis-regulation of gene expression could result in inappropriate loss or gain of expression of these genes, while altering protein structure or the post-translational modifications could alter the levels of functional protein, resulting in aberrant development or disease. It is therefore of high interest to both (1) study the molecular mechanism of gene transcription of known intrinsic factors and (2) identify and investigate the function of proteins novel to the development and maintenance of RPCs. 1.2 Eye development in vertebrates and the specification of Retinal Progenitor Cells (RPCs) The presumptive eyes are specified during neurula stages, and are comprised of tissue that evaginates from the anterior neural tube (Figure 1.1). This tissue, called the optic vesicle, grows into the surrounding mesoderm towards the surface ectoderm. When the optic vesicle makes contact with the surface ectoderm, reciprocal inductive signals between the optic vesicle and the surface ectoderm, as well as the optic vesicle and mesoderm, occur. As a result, the surface ectoderm thickens and develops into the lens placode, while the neuroepithelium of the optic vesicle becomes patterned into three distinct territories: the dorsal region is the presumptive retinal pigmented epithelium (RPE), the ventral region is the presumptive optic stalk, and the lateral region is determined as the presumptive neural retina. The optic vesicle and the lens placode undergo simultaneous invagination and give rise to the optic cup and the lens vesicle, respectively. Over time, the lens vesicle completely segments from the surface ectoderm, forming the lens proper. The RPE completely surrounds and becomes abutted to the

3 thickening neural retina. The surface ectoderm adjacent to the lens gives rise to the sclera laterally and the corneal epithelium medially. The cells comprising the presumptive neural retina are specified as retinal progenitor cells (RPCs). This population of cells rapidly proliferates and subsequently differentiates to form the neural and non-neuronal cell types of the retina. RPCs are therefore multipotent and constitute a population of cells that can further our understanding of the nature of multipotency, and the relationship between cell division and differentiation. In the mature vertebrate retina, the cell types produced by the RPCs are highly organized in a laminar tissue (Figure 1.2). The cell bodies of the retinal neurons and glia are positioned in three distinct nuclear layers: the ganglion cell layer (GCL) adjacent to the lens, the inner nuclear layer (INL) and the outer nuclear layer (ONL) nearest the apposing RPE (Figure 1.2, A). The neuronal processes extended by the cells within these layers make connections within two plexiform layers: the inner plexiform layer (IPL) and outer plexiform layer (OPL). The inner plexiform layer is positioned between the GCL and INL, and the outer plexiform layer between the INL and ONL. In addition, a specialized region of progenitor cells persists in adults of some vertebrate species, including X. laevis (Figure 1.2, A, brackets). Termed the ciliary marginal zone (CMZ), the RPCs of this zone continue to give rise to differentiated retinal cells during the life of the adult animal. The CMZ is located peripherally, adjacent to the lens on both its dorsal and ventral side. Six
neuronal
and
1
glial
cell
type
are
observed
in
the
mature
vertebrate
 retina
(Figure
1.2,
B).

The
major
phototransducing
cells
of
the
retina,
the
rod
and


4 cone
photoreceptors,
possess
their
cell
bodies
in
the
ONL.

The
specialized
 compartments
for
phototransduction,
the
photoreceptor
inner
and
outer
segments,
 are
juxtaposed
to
the
overlying
RPE.

The
photoreceptor
cells
of
the
ONL
extend
 processes
laterally
into
the
OPL
synapsing
with
the
three
interneuron
cells
types:
 the
amacrine,
bipolar
and
horizontal
cells.

Each
of
these
cell
types
possesses
cell
 bodies
that
reside
in
the
INL,
and
extend
outgoing
processes
into
the
IPL.

The
IPL
is
 the
layer
that
houses
connections
between
the
interneuron
and
the
ganglion
cell
 dendrites.

The
long
axons
of
the
ganglion
cells
extend
into
the
optic
nerve
and
 eventually
make
connections
in
the
visual
processing
center
of
the
brain,
the
optic
 tectum.

The
cell
bodies
of
the
major
glial
cell
type
in
vertebrates,
Muller
glia,
are
 positioned
in
the
INL.

They
extend
processes
to
the
inner
segments
of
 photoreceptor
cells,
creating
the
outer
limiting
membrane,
and
likewise,
end
feet
of
 Muller
glia
create
the
inner
limiting
membrane
just
past
the
ganglion
cell
layer.
 Classic birthdating studies have demonstrated that retinohistogensis by RPCs occurs in a generally conserved order across species, always first with the generation of ganglion cells, with rod photoreceptors, bipolar and Muller glia the last cell types being produced (Carter-Dawson and LaVail, 1979; Cepko et al., 1996; Stiemke and Hollyfield, 1995; Wong and Rapaport, 2009; Young, 1985) (Figure 1.3, A). It was initially speculated that the generation of these cell types in such a stereotypical order was dictated by environmental cues (Turner and Cepko, 1987) (Holt et al., 1988). However, the demonstration that stage specific RPCs predictably produce certain cell types regardless of environmental influences lead to the competence model of retinal

5 development (Figure 1.3, B) (Cepko et al., 1996) The competence model proposes that after specification, RPCs progress through distinct stages. In each stage, RPCs are capable of producing specific subsets of retinal cell types (Cepko et al., 1996; Livesey and Cepko, 2001). This model assumes that (1) competence states of RPCs are intrinsically defined and (2) the capacity to produce cell types are intrinsically regulated by the production of competence states. RPCs of a given competence state are then governed by both intrinsic and extrinsic factors to generate specific retinal cell types (Cepko et al., 1996). 1.3 From RPCs to retinal cell types: molecular regulation of retinal cell types by intrinsic factors But how are the production of these cell types regulated on the molecular level? What governs the specification, differentiation and eventual functionality of developing retinal progenitor cells? According to the competence model, both extrinsic and intrinsic signals govern the development of retinal progenitor cells into differentiated cell types through certain competence states. Certainly, extrinsic factors such as extracellular signaling molecules, mitogens, and neurotransmitters have been shown to be involved in both the specification and differentiation of the neural retina (reviewed in Martins et al., 2007). However, for the purpose of introduction for these studies, it is important to emphasize the intrinsic factors that have been shown to regulate eye development, both in specification of RPCs and during the differentiation of RPCs into terminally differentiated neurons. The group of proteins involved in proper specification of RPCs in vertebrates are termed eye field transcription factors (EFTFs) (Zuber et al., 2003). The EFTF gene

6 products include: retinal homeobox (Rx), paired type homeobox 6 (Pax6), lim-domain containing homeobox 2 (Lhx2), sine oculis homologs 3 and 6 (Six3/6), orthodenticle related homeobox 2 (Otx2), the t-box transcription factor, ET, and the orphan nuclear receptor, (Tlx).The majority of these proteins belong to the homeodomain family of transcription factors (the exceptions being Tlx and ET). Their importance during eye development is underscored by the high level of conservation of these factors from flies to humans (Zuber et al., 2003); in each vertebrate system investigated, these transcription factors are highly expressed in RPCs during retinal specification and neurogenesis (Figure 1.4, A). Gene expression and genetic manipulation experiments investigating these factors have determined that each of the EFTFs is necessary for eye development (Bailey et al., 2004; Bovolenta et al., 1997; Chow and Lang, 2001; Esteve and Bovolenta, 2006; Nishida et al., 2003; Porter et al., 1997; Tetreault et al., 2009; Viczian et al., 2006; Wilson and Houart, 2004; Zuber et al., 2003). Many EFTFs are required, and often capable, for the initiation of gene expression of other genes within the group. For example, Rx null mice, do not express Pax6, suggesting that Pax6 is downstream of Rx (Mathers et al., 1997). The overexpression of Rx in contrast induces Pax6 and Six3, reduces Otx2 (Andreazzoli et al., 1999). As well, Pax6 overexpression results in ectopic eyes with induction of Rx, Otx2, an Six3 (Chow et al., 1999). And Six3 itself is also capable of induction of Rx (Loosli et al., 1999) and Pax6 (Loosli et al., 1999) (Bailey et al., 2004; Bovolenta et al., 1997; Chow and Lang, 2001; Esteve and Bovolenta, 2006; Nishida et al., 2003; Porter et al., 1997; Tetreault et al., 2009; Viczian et al., 2006; Wilson and Houart, 2004; Zuber et al., 2003). Tlx overexpression induces the expression of Pax6, Six3 and Lhx2, and conversely, Pax6

7 and Six3 overexpression upregulates Tlx (Bailey et al., 2004; Bovolenta et al., 1997; Chow and Lang, 2001; Esteve and Bovolenta, 2006; Nishida et al., 2003; Porter et al., 1997; Tetreault et al., 2009; Viczian et al., 2006; Wilson and Houart, 2004; Zuber et al., 2003). It is apparent from these studies that the EFTFs form a self-regulating network of factors necessary for the specification of the RPCs in the anterior neural plate. After specification, a combinatorial code of transcription factors dictates the cell types that differentiate from RPCs of a given competence state (Wang and Harris, 2005; Harada et al., 2007)). Two major families of transcription factors are involved: basic helix-loop-helix (bHLH) (Vetter and Brown, 2001), and homeodomain (HD) proteins (Lupo et al., 2000). In general, at least one bHLH and HD are co-expressed in progenitors as the produce a certain cell type (Figure 1.4)(Harada et al., 2007). For example, Ath5 and Pax6 specify the production of ganglion cells (Brown et al., 2001; Marquardt et al., 2001; Wang et al., 2001), or Ath3 and Pax6/Six6 specify the production of horizontal cells (Marquardt et al., 2001)(Tomita et al., 2000). 

 It is well established that perturbing these combinations can alter the distribution of cell types within the retina. Collectively, the data concerning the coordination of these protein families suggests that homeodomain factors regulate layer specificity while bHLH proteins dictate cell fate within the homeodomain factor-specified layers (Harada et al., 2007). A significant amount of work has been done to understand the molecular regulation of eye development. The work presented in this dissertation expands the knowledge of known RPC regulators by (1) furthering the understanding of the molecular regulation of Rx genes, (2) definitively recognizing a third family of transcription factors, the forkhead family, as regulators of RPC development and function, and (3)

8 characterizing a role for two forkhead proteins, FoxO3 and FoxM1, neither of which have been previously attributed a role during vertebrate eye development. 1.4 RPC Decision-making: The balance between proliferation and differentiation The RPCs of the neuroepithelium in the optic cup undergo several rounds of rapid proliferation to produce the cellular diversity of the laminated neural retina. It is during these rounds of cell division that the retinal cell types are specified and differentiate. Therefore, it is not surprising that the differentiation of RPCs is tightly linked to proliferation. Several lines of evidence support this notion: (1) loss of function experiments concerning the EFTFs demonstrate reduced proliferation rates, (2) EFTFs can act as direct or indirect modulators of cell cycle machinery and (3) aberrations in cell cycle genes result in changes in cell type specification in the developing eye. In mice, targeted mutagenesis of Otx1, Otx2, Lhx2, and Tlx results in reduced proliferation rates in the developing eye region (Martinez-Morales et al., 2001; Miyawaki et al., 2004; Porter et al., 1997). The use of morpholino knockdown in Xenopus and zebrafish has demonstrated reduced proliferation in the absence of Lhx2 and Six3, and Rx, respectively (Ando et al., 2005) (Nelson et al., 2009). These studies clearly demonstrate that the EFTFs are responsible for the necessary proliferation of RPCs after specification in the neuroepithelium. Progenitor cells, including RPCs, make the decision to terminally differentiate or continue to proliferate during the G1 phase of the cell cycle (Figure 1.5, A). If a neuron decides to differentiate and enter G0, it is thought to be restricted from re-entering the cell cycle. An exception is represented by the Muller glial cells of the retina, which proceed to G0, but retain the capacity for entering the cell cycle. Several groups of

9 molecules are active in progenitor cells during early G0 acting as intrinsic regulators of differentiation decisions. First, specific cyclin/cyclin dependent kinase (CDK) enzyme complexes are active during early G1 to dictate the decision between G0 or continued proliferation: Cyclin D with CDK4 or CDK6, and Cyclin E with CDK 1 or 2 (Welcker and Clurman, 2005). Secondly, Cyclin-dependent kinase inhibitors exert an additional layer of regulation; these molecules directly alter the activity of cyclin/CDK enzymes complexes. There are two major families of cyclin-dependant kinase inhibitors: the INK family (p15, p16, p18, p19) and the Cip/Kip family (p21, p27, p57). In Xenopus, only Cip/Kip family member has been identified, which shares sequence and functional characteristics of all three Cip/Kip proteins (Su et al., 1995). Cyclins, CDKs and CDK inhibitors are expressed in dynamic tissue-specific manners. The balance of activity among them determines the phosphorylation state of members of the Rb family, that ultimately dictate an RPCs choice between cell division and cell cycle exit. The expanding amount of literature supporting the link between EFTFs and the cell cycle machinery is presented. Studies of EFTF function have shown changes in cell cycle gene expression as well as confirming cell cycle genes as direct targets of EFTFs. In Xenopus, Rx controls cell proliferation by inhibition of the cell cycle inhibitor p27Xic1 (Andreazzoli et al., 2003). Moreover, embryos injected with Xrx1 mRNA demonstrate significantly increased levels of Cyclin D1, the major D-type cyclin expressed in RPCs (Casarosa et al., 2003). Pax6 null mutant mice have decreased expression of a number of cell cycle inhibitors, p27(kip1), p57(kip2), and p21(cip1) (Duparc et al., 2007). Six6, binds to the promoter of the p27Kip1 in retinal cells, suggesting that Six6 may be a direct repressor of

10 p27Kip1 transcription in RPCs. p27Kip1 mRNA and protein is also upregulated in the Six6-null retina (Li et al., 2002). The related EFTF protein, Six3, has been shown to be upstream of both cyclin D1 and p27 (Gestri et al., 2005). Collectively, these data suggest that several of the EFTFs have the ability to alter the cell cycle during RPC development. It has been shown that altering cell cycle components within the retina directly results in changes in neuronal diversity. Cyclin D1 null mice have hypocellular retinas, attributed to decreased proliferation (Sicinski et al., 1995), and it has recently been shown that early born cell types (ganglion and photoreceptor cells) are increased (Das et al., 2009). Interestingly, replacing cyclin D1 with Cyclin E1 is sufficient to restore normal retina structure and cell type specification, suggesting that there is some functional redundancy between active cyclins in the retina (Das et al., 2009). On data regarding CKIs, overexpression of the cell cycle inhibitor p27(Xic1) results in the increased numbers of ganglion cells (Ohnuma et al., 1999), while overexpression of p27 is sufficient to maintain RPCs in the cell cycle and produce later born cell types (Ohnuma et al., 1999). Additionally, targeted mutagenesis of p57 in mice alters cell fate specification by increasing the proportion of amacrine cell sub-populations (Dyer and Cepko, 2000; Dyer and Cepko, 2001). Together, these studies demonstrate the relationship between proliferation and differentiation in the RPC population. As neuroepithelial cells of the developing retina proceed through the cell cycle, the nuclei migrate between the apical and basal surfaces in phase with the cell cycle, a process termed interkinetic nuclear migration (Figure 1.5, B) (Baye, 2007). M-phase nuclei are located at the apical surface, adjacent to the RPE. As the cells proceed to G1- phase, the nuclei migrate toward the basal surface, and progress into S-phase at their most

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Abstract: Understanding the development and maintenance of Retinal Progenitor Cells (RPCs) is critical to understanding normal and disease processes within the neural retina. To understand RPC biology, it is important to understand the transcriptional regulation of known intrinsic regulators, and continue to identify new RPC expressed genes to demonstrate their function during eye development. The studies presented in this work address the transcriptional regulation of the Xenopus laevis Rx gene product, Rx2A, and function of two newly recognized RPC genes, FoxO3 and FoxM1. To further the understanding of transcriptional regulation in RPCs, we characterized the Rx2A promoter in transgenic embryos. Both the distal portion and the proximal portion of the Rx2A promoter are sufficient for expression of a GFP transgene in the developing eyes. We identify a highly conserved element in the distal region of the Rx2A promoter (UCE). Within UCE, an OTX, SOX and POU site act as cis-elements to coordinately specify proper gene expression in the developing eye. We show that the activity of the proximal promoter is dependant on a forkhead-binding element (FBE). In addition, we have shown that the distal region containing the UCE can cooperate with the FBE to maintain robust Rx expression throughout all stages of eye development. The work associated with the transcriptional regulation of Rx furthers our understanding of how this primary retinal transcription factor is regulated. This is applicable to the understanding of RPC development since Rx is one of the first eye field transcription factors expressed in the anterior neural plate as RPCs are specified. The identification of the FBE within the Rx2A promoter led to further investigation of the involvement of the forkhead family of transcription factors in vertebrate eye development. We present a discussion of current data regarding the expression and function of this family of transcription factors during eye development, and we present the expression pattern of 5 forkhead transcription factors (FoxG1, FoxN2, FoxN4, FoxM1 and FoxP1) in the maturing X. laevis retina. These forkhead gene products have not been previously described in the maturing retina of X. laevis . We chose to pursue studies of FoxO3 and FoxM1 in the developing neural retina to further the understanding of RPC regulation by transcription factors of the forkhead family. These two factors were chosen for co-current studies for the following reasons: (1) neither gene product had been ascribed a role in developing RPCs, (2) their known functions suggested they act in opposing ways with regards to the cell cycle, and (3) due to their expression patterns both FoxO3 and FoxM1 serve as candidate factors to regulate the Rx2A promoter through the FBE. To define the role of FoxO3 during vertebrate eye development, we overexpressed FoxO3 RNA in the anterior neural plate of X. leavis embryos. FoxO3 overexpression results in embryos with small eyes. The small eye phenotype is a result of decreased proliferation, induction of apoptosis, and changes in RPCs gene expression. The phenotype can be exacerbated by introducing a threonine to alanine mutation at a conserved PI3K phosphorylation site, which produces a constitutively nuclear form of FoxO3. The changes in gene expression suggest that FoxO3 can function to delay the differentiation of RPCs, although they are properly specified. The data supports our original hypothesis regarding FoxO3 as cell cycle antagonist with the ability to alter the differentiation capacity of RPCs. To investigate the function of FoxM1 in developing RPCs, we performed loss-of-function studies using morpholino oligonucleotides (MO). FoxM1 knockdown in the anterior neural region of X. laevis results in embryos with slightly smaller eyes and clear defects in retinal lamination. Our data reveals that FoxM1 is not necessary for the specification of RPCs, and suggests that differentiation into the Muller glia, and rod photoreceptor lineages is possible with reduced levels of FoxM1. The studies presented concern the transcriptional regulation of the Rx gene product, Rx2A, and a role of FoxO3 and FoxM1 during RPC development. Collectively, they represent an advancement in the knowledge regarding two important intrinsic mechanisms governing RPC development: transcriptional regulation and transcription factor function.