THE ROLE OF THE RETINOBLASTOMA PROTEIN

IN mTINAL DEVELOPMENT

Irina D. Burcescu

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

O Copyright by Irina D. Burcescu, 2001

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THE ROLE OF THE RETINOBLASTOMA PROTEIN IN RETINAL DEVELOPMENT

Irina D. Burcescu

Master of Science, 2001

Department of Laboratory Medicine and Pathobiology, University of Toronto

ABSTRACT

Although RB1 was the first turnor suppressor gene identified, its role during retinal

development is poorly understood. We first attempted to determine this by performing in

vivo misexpression studies in the post-natal rat retina. Retroviral lineage analysis revealed

that clone size in pRB-infected eyes was significantly reduced when compared to control

eyes. This suggested that pRB has a role in regulating progenitor ce11 cycle in the developing

retina. The results fiom in vivo misexpression studies also suggested a possible pRB

involvement in ce11 fate determination ancUor re-specification, but more expenments must be

performed before concluding this. In vitro rescue experiments with the RB-'- retina is the

second expenmental approach that rnay help us clai@ the role of pRB in retinal

development. To this end, we established a retinal culturing protocol wherein development

itz vitro mimics that in vivo, thus laying the groundwork for future rescue expenments in the

RB" retina. This study presents significant findings that may help us elucidate the role pRB

plays during retinal development.

Acknowledgements

1 would like to thank Rod Bremner for his guidance throughout this work. Rod's dedication and love for his work are inspiring. And despite the occasional "differences in opinion" 0, 1 wouldn't have traded this expenence for anything else. 1 have truly learned more than just the mysteries of RB throughout the past 2 years. Many thanks also to Brenda Gallie; her support was greatly appreciated. Brenda's enthusiasm for anything RB is simply contagious. The past and present members of the Bremner lab also deserve to be mentioned, especially Bpbobech, Sam and Kim. You guys are awesome and have actually made this f h . And no, 1 don't have THAT many pairs of shoes!

1 also wish to thank Judy Trogadis. I didn't think it was possible, but you made performing a protocol like immuno fun! Go figure! And then again, we're Gemini.. . we can make anything we put our mind to fun, right?

A few other people deserve credit for keeping me focused. Mark, 1 still can't believe how much fun we had going for a simple drive. "Urnrnrnm.. . tum nght" led to more adventures than 1 can recall. You helped me relax, and in some way, kept reminding me what was really important, what 1 was really striving for. 1 will always cherish our fkiendship. And Danny, words can't describe how much 1 appreciate the little things you did to help me get through this. Your advice was most helpful. You've been a pillar of support for me throughout the past few months. XXX And my bro: dude, you cool; love ya!

My parents: Va iubesc mult!

Contents

Absîrac t

Acknowledgements

Table of contents

List of Tables

List of Figures

CHAPTER 1 : INTRODUCTION

. . 11

... 111

i v

vii

... V l l l

1

.................................................................................. 1.1 Retinoblstoma 1

..................................................................... 1.1.1 The disease - 1

...................................................................... 1.1.2 The protein 2

.................................................................. 1.1 -3 Mouse models 3

......................................................... 1.1.4 pRB and the ce11 cycle 5

............................................................. 1.1.5 pRB and apoptosis 6

....................................................... 1.1.6 pRB and differentiation 7

1 -2 The retina ................................................................................... -9

......................................................... 1.2.1 Structure of the retina 9

......................................................... 1.2.2 Retinal development 10

............................................................. 1.3 pRB expression in the retina 12

................................................. 1.4 The role of pRB in retinal development 13

...................................................... 1.5 The retinal explant culture system 15

............................................................... 1.6 Gene transfer to the retina -16

...................................................... 1.6.1 Retroviral gene transfer 16

....................................... 1.6.2 Alphavirus gene expression system -16

iv

............................................................................... 1 . 7 The question - 1 7

CHAPTER 2: MATERLALS AND METHODS 19

....................................................................................... CeUs -19

Generation of recombinant retroviral particles ....................................... 19

............................................... 2.2.1 Titenng of retroviral particles 20

Generation of recombinant Semliki Forest Virus (SFV) particles .................. 20

.......................................... 2.3.1 Titering of recombinant particles 21

.................................................................................. Westerns -21

. . . .............................................................. Neonatal rat eye injections 22

........................................................................... Retinal explants 23

..................... 2.6.1 Infection of retinal explants with retroviral vectors 24

............ 2.6.2 Infection of retinal explants with recombinant SFV vectors 25

................................................................... Immunohistochemistry 25

........................................................................ Photomicroscopy -26

....................................................................... Statistical methods 27

CKAPTER 3: RESULTS 28

...................................... In vivo misexpression of pRB in the rat retina -28

....................................... 3.1.1 Retroviral vector protein expression 28

... 3.1.2 Misexpression of AK11 affects clone size in the neonatal rat retina 30

..................... Characterization of an in vitro retinal explant culture system 32

+/+ ...................................... 3.2.1 RB retinal explant characterization 32

3.2.2 RB-'- retinal explant characterization ....................................... 33

3.3 Two virus-based systems may be used for gene delivery to the post-natal munne ....................................................................................... retina -34

3.3.1 Retroviruses may be used for gene delivery to the retina .............. -34

3.3.2 A novel alphavirus may be used for gene delivery to the retina ........ 34

CHAPTER 4: DISCUSSION 36

4.1 In vivo misexpression of pRB in the rat retina ...................................... -36

....................................... 4.1.1 Retroviral vector protein expression 36

4.1.2 Misexpression of Ml 1 reduces clone size in the neonatal rat retina: .................................... implications for ce11 fate specification 38

4.1.3 Can pRB reprograrn post-natal progenitors so that they become cone ............................................................... photoreceptors? 42

4.2 Characterization of an in vitro retinal explant culture system ...................... 44

.......................................... 4.3 Viral systems for gene delivery to the retina 45

................................................................................... 4.4 Summary 46

CHAPTER 5: REFERENCES 48

Tables

Table 1

Table 2

Previous Page

........................ Bicistronic gene expression in retroviral vectors tested.. 28

........................ Summary of in vitro retinal explant culture protocols.. .33

vii

Figures

Previous Page

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

The retinoblastoma protein and cell cycle.. ........................................ .2

Structure of the retina.. ................................................................ ..9

......................................... Retinal development and differentiation.. .10

.......................................................... Retroviral lineage analysis. -29

............................. pRB drives mitotic progenitors out of the ce11 cycle.. 30

Charactenzation of embryonic retinal explants via irnmunohistochemical . . ................................................................................. stalning -32

...... Producing, testing, and using viruses to infect retinal explants in vitro.. 34

Does pRB re-speciQ cone photoreceptor celi fate in post-natal retinal ........................................................................... progenitors? .42

1.1 Retinoblastoma

1.1.1 The Disease

The first literary evidence of fungus haematodes came in 1597, when Petrus Pawius

of Amsterdam described a leA eye tumor in a 3 year-old child that was "filled with a

substance similar to brain tissue mixed with thick blood and like crushed stone" (Albert,

1987). In 1926 the American Ophthalmological Society adopted the name retinoblastoma to

describe the malignant pediatric retinal tumor (Verhoeff, 1926). Retinoblastoma tumors

occur in 1 out of 20,000 live births; this incidence does not Vary with ethnicity, geography or

level of industrialization. The retinoblastoma gene (MI) was initially identified as a genetic

locus associated with the development of the inhented eye tumor (Friend et al., 1986;

Knudson, 197 1). The retinoblastoma locus is located on chromosome 13q 14 (Sparkes et al.,

1980). The occurrence of this disease has been correlated with the inactivation of both

alleles of RB1 @um et al., i989). About 40% of children with retinoblastoma carry a

gemline mutation in one of the RB1 alleles and most of these children develop multifocal

bilateral tumors. The other 60% of affected children do not carry a germline mutation in RB1

and are generally affected only unifocally. While gemline mutations at the RB1 locus

predominantly manifest as retinoblastoma at a very early age, children canying these

mutations are also predisposed to osteosarcornas and sofi tissue sarcomas later in life

(reviewed in Hamel et al., 1990). The retinoblastoma tumor suppressor protein (pRB) is

inactivated in over 60% of studied human turnors (Sherr, 1996) either through mutations in

RB], or through defects in other proteins that affect the fùnction of pRB in the cell. For

instance, RB1 loss of heterozygosity (LOH) has been associated with the progression of

carcinomas such as breast (Lee et al., 1988), bladder (Horowitz et al., 1989), prostate

(Bookstein et al., 1990) and small ce11 lung carcinoma (Harbour et al., 1988). Thus, it is

worthwhile to study the retinoblastoma protein given its prevalence in disease.

1.1.2 The Proteia

Although RB1 was first identified for its function in the retina, it is expressed in most

tissues. The retinoblastoma gene product is a phosphoprotein that is important in cellular

proliferation, differentiation and death. Two normal copies of RB1 are present in most

human cells, and generally, the function of the protein they encode is to limit growth of the

cell. Only one normal copy of the gene is needed to accomplish this function. Human RB

contains 27 exons within 180 kilobases (kb) of genomic DNA (McGee et al., 1989). The 4.8

kb human RB1 rnRNA encodes a 928-amino acid protein (921 aa in the mouse). pRB

contains three distinctive domains: the N-terminus, the central A and B domains separated

by a linker region (these make up the A/B "pocket"), and the C-terminus (Fig. 1). The

structural integrity of the A/B domains is required for the interaction of pRB with most of its

associated proteins (Morris and Dyson, 2001). Several cellular and viral pRB-binding

proteins harbor an LXCXE motif (where L is leucine, C is cysteine, E is glutamine and X is

any amino acid). The LXCXE motif was originally identified as being crucial for viral

oncoproteins such as the EIA protein of adenovirus (Whyte et al., 1988), the large-T antigen

of SV40 (DeCapno et al.. l988), and the E7 protein of the human papilloma virus @yson et

Minimum binding to E2F on DNA

O 1 3 4 6

CDK sites 2 5 (AKI 1)

t

Cyclin DlCDK4

Cyclin N C D K 2

Fig. 1 : The retinoblastoma protein and ce11 cycle. A. plU3 domains: sites for protein binding, phosphorylation and mutation. The nmbers on top of the pRB schematic indicate human residues. NLS = nuclear localization signal. Two low penetrance (lp) alleles are indicated. Numbering of CDK sites is based on Brown et al (1999); human pRB lacks site 5 found in mouse pRB, but includes site 0, which mouse pRB lacks. The CDK sites marked in red represent the sites mutated in AK11. Binding sites for E2F on DNA and for proteins containing an LXCXE motif are indicated. B. Different CyclidCDK complexes regulate pRB phosphorylation throughout the ce11 cycle. The schematic diagram illustrates what Cyclin/CDK complexes phosphorylate pRB during certain phases of the ce11 cycle.

al., 1989) to interact with pRB. The A and B domains along with a portion of the C-terminus

of pRB are required for binding to members of the E2F farnily of transcription factors on

DNA (reviewed in Dyson, 1998) (see Fig. 1). The activity of pRB is regulated by

phosphorylation. Hypophosphorylated, active pRB binds E2F and thus prevents ce11 division

(see below).

Two other members of the pRB gene farnily have been identified, p l O7 and p130.

RB1 and p107/p130 most closely resemble each other in the pocket region. p 107 and pl 30

share some biochemical properties with pRB, and they are also believed to regulate ce11

growth (Lipinski and Jacks, 1999). Mouse knockout modeis suggest that the three pocket

proteins have some compensatory functions during development (see below).

Genes that cause cancer have been typically classified into two broad categones:

oncogenes, and tumor suppressor genes. Oncogenes contribute to malignancy in a positive

manner, following activation by qualitative (eg point mutations, translocations) or

quantitative (eg overexpression) means (Weinberg, 1985). In contrast, tumor suppressors

block tumorigenesis. pRB was the first tumor suppressor identified (Cavenee et al., 1983;

Godbout et al., 1983) and it must be functionally inactivated to permit tumor progression.

1.1.3 Mouse Models

Homologues of RB1 have been found in many vertebrates including mice, cats,

chickens and sharks (Lee et al., 1987). The mouse protein shows over 90% identity to the

human protein (Bernards et al., 1989). RB-'- mice die in utero around embryonic days 13-15

(E13-13, due to defects in erythropoiesis and neurogenesis (Jacks et al., 1992; Lee et al.,

1992). Partial rescue of RB-'- mice to birth by an RB1 transgene that drives low expression of

pRB showed that muscle differentiation also fails (Zacksenhaus et al., 1996). RB+/- mice are

viable and do not develop retinoblastoma tumors; instead, they show tumors in the

intermediate lobe of the pituitary gland (Hu et al., 1994; Jacks et al., 1992). However, loss of

both pRB and p 107 does predispose chimeric animals generated using embryonic stem (ES)

cells deficient for both pRB and pl07 to retinoblastoma (Robanus-Maandag et al., 1998).

This result suggests that in mice, pl07 may be able to compensate for loss of pRB.

Retinoblastoma-like tumors have been observed in transgenic mice expressing SV40 TAg

under the control of the luteinizing hormone B subunit promoter (Windle et al., 1990) or the

interstitial retinal binding protein (IRBP) promoter (Al-Ubaidi et al., 1992). TAg causes

transformation through functional inactivation of al1 pocket proteins @RB, p 107 and p 130)

and p53 (DeCaprio et al., 1988) in the subsets of retinal ceils in which the promoters used are

active (rod and cone photoreceptors for the IRBP promoter). This result suggests that in

murine retinas, oncogenes (such as Tag) dnven by promoters that are active in subsets of

retinal cells may induce tumors when pS3 is also inactivated.

Although the genetic requirements for induction of retinoblastoma in mice appear to

be different than those in humans, the retinal tumors that occur in both species are similar in

histology and antigenic profile (Bernstein et al., 1994; Howes et al., 1994; Robanus-Maandag

et al., 1998). Also, the overall development of the eye is highly similar arnong vertebrates.

Given these similarities, using a rodent mode1 to study the role of pRB in retinal development

is justifiable in the study of the human cancer.

1.1.4 pRB and the Cell CycIe

pRB is a ce11 cycle regulator. It inhibits cell cycle progression by binding to and

repressing several members of the E2F family of transcription factors. E2F1-5 contain

highly conserved domains necessary for pRB binding, gene promoter binding, and

transcriptional activation of genes required for DNA synthesis (Harbour and Dean, 2000).

E2F proteins are found as dimers in combination with the DP family of proteins (DP1-3).

This association increases the ability of E2F to transactivate genes and stabilize binding to

pRB (Bandara et al., 1993).

The ce11 cycle consists of four phases: the S phase @NA synthesis), the M phase

(mitosis), and two separating gap intervals, G1 and G2. The interaction between the E2F

family of transcription factors and pRB depends on the phosphorylation state of pRB

throughout the ce11 cycle. When pRB is phosphorylated, it releases E2F which may then go

on to transactivate genes necessary for ce11 cycle progression. pRB contains 16 cyclin

dependent kinase (CDK) phosphorylation recognition sites (S/TP), seven of which are

located within the C-terminus (Fig. 1). The active hypophosphorylated state wherein pRB is

bound to E2F!DP heterodimers occurs during GO and through most of G1. pRB is then

gradually phosphorylated, or inactivated, near the Gl/S boundary and remains so through

most of M phase. Different cyclin-CDK complexes regulate the phosphorylation state of

pRB throughout the ce11 cycle. In mid to late G1, pRB phosphorylation is mediated by

cyclins Dl, D2, and D3 complexed with CDKs 4 or 6, in mid G1 to eariy S phase by the

cyclin E-CDK2 complex, and throughout S phase by the cyclin A-CDK2 complex (reviewed

in DiCiommo et al., 2000). The complex regulation of pRB activity by the cyclin/CDK

complexes hint at the critical role this protein plays in a cell7s lifespan.

1.1.5 pRB and Apoptosis

Apoptosis or programmed ce11 death is a genetically controlled mechanism allowing a

ce11 to commit suicide. A universal feature of differentiated tissue, such as the adult retina, is

decreased apoptosis. Recent studies have suggested that pRB c m block apoptosis (reviewed

in Wang, 1997). Nonnally, in order for a ce11 to differentiate, active, hypophosphorylated

pRB blocks entry into the S phase of the ceIl cycle. Factors that support S phase and

proli feration are then withdrawn, and factors that promote di fferentiation are activated.

Thus, if pRB is inactivated through mutations, cells enter S phase unsupported by

proliferative factors, and apoptosis will be initiated. The evidence that pRB is involved in

apoptosis protection came initially from RB knockout mice: these mice exhibited excessive

ce11 death in neuronal, hematopoietic and lens tissues (Jacks et al., 1992; Morgenbesser et al.,

1994). Later, Zacksenhaus et al. (1996) engineered RB~OX;RB-'- transgenic mice which

expressed low levels of pRB in the RB" background. These mice survived to birth, but their

muscle cells showed highly increased apoptosis, indicating once again that pRB may be

involved in blocking ce11 death. More importantly, concomitant inactivation of pocket

proteins and p53 by overexpression of HPV-6 E7 (in a p53 nul1 background) or SV40 Tag

led to development of retinoblastoma (Windle et al., 1990; Al-Ubaidi et al., 1992; Howes et

al., 1994). These findings indicated that the munne retina rnay be better protected against

tumongenesis than the human retina, since, unlike the human retina, in subsets of murine

retinal cells, p53-dependent apoptosis must also be disnipted in order for tumors to be

observed. Simply put, pRB appears to have a role in protecting some cells from death by

apoptosis.

1 J.6 pRB and Differentiation

RB1 and the homologous genes p l 0 7 and pl30 are widely expressed in the

developing nervous system (Jiang et al., 1997). Zn situ hybridization studies determined that

in the central nervous systern (CNS), R B I was expressed in areas of both proliferating and

differentiating cetls, whereas pl07 expression was restricted to proliferating cells

surrounding the ventricles. Expression o f p l 0 7 overlapped with that of RBI in the liver and

the CNS. pl30 was expressed at low levels in the nervous system throughout

embryogenesis. Members of the E2F family are also present in developing neurons, with

maximal expression in the ventricular zone @agnino et al., 1997). These expression patterns

suggest that the RB farnily might play an important role in regulating proliferation of

neuronal precursors in the CNS. Studies on RB-'- mice support this notion (Jacks et al., 1992;

Lee et al., 1992). As mentioned previously, these mice die between E 13- 15 due to defects in

hematopoietic, neural and lens development. The RB" mice exhibit abnonnal patterns of

ce11 division in the central and peripheral nervous systems (Lee et al., 1992; Morgenbesser et

al., 1994). Dividing cells in the central nervous system (CNS) are normally restricted to the

ventncular zone, but in RB mutants, cells outside this region divide. Increased ce11 death of

proliferating cells in the hindbrain, spinal cord and sensory ganglia is also observed in the

RB"- mice.

Other sîudies on RB" mice revealed that pRB appears to be required at least

temporarily dunng neuronal development. Slack et al. (1998) used RB*" mice expressing P-

galactosidase from the early panneuronal Ta1 a-tubulin promoter (Ta l :nlacZ). This study

found that Ta1 a-tubulin expression was present in RB-'- cortical neurons, indicating that

pRB was not required for the induction of early neuronal gene expression during

differentiation. However, while in E 12.5 RB-'- embryos the Ta 1 :nlacZ transgene was

strongly expressed throughout the developing nervous system, by E14.5 there were

perturbations in Ta1 :nlacZ expression throughout the nervous system, including deficits in

the forebrain and retina, consistent with an alrnost cornplete loss of neurons between El 2.5

and E 14.5; this indicated that differentiating neurons are dying in the absence of functional

pRB. Further, when a mutant E l A adenovirus was used to inactivate the pRB gene family in

post-mitotic cortical neurons, it was found that pRB was not required for their survival (Slack

et al., 1998). Together, these studies demonstrated that pRB was essential f ~ r determined

cortical neurons to exit the ce11 cycle and survive, but was not necessary for the induction of

neuronal gene expression or for the maintenance of post-mitotic neurons.

Other studies showed that RB*/- neurons exhibit differentiation defects such as

aberrant DNA synthesis and apoptosis (Slack et al., 1998; Lee et al., 1994), suggesting a role

for pRB dunng differentiation. Devlin et al. (2001) showed a major defect in ganglion ce11

differentiation in the retina of RB~OX;RB-'- rnice: ganglion cells continued to divide at

abnormal time points during developrnent, and they showed increased death by apoptosis.

pRB was also shown to bind and trigger transcriptional activators that are required for the

differentiation of muscle, adipocytes, monocytes and keratinocytes (Zacksenhaus et al., 1996;

Gu et al., 1993; Chen et al., 1996; Bergh et al., 1997). Overall, these results indicate that

pRI3 may be essential not only for proliferation of neuronal precursors, but also for survival

and differentiation of some post-mitotic neurons.

1.2 The Retina

1.2.1 Structure of the Retina

The retina is a highly ordered array of neurons and glia that lines the back of the eye.

Neurons in the adult retina are organized in three distinct nuclear layers, separated by two

layers of synaptic connections, the imer plexiform and the outer plexiform layers (Fig. 2).

The first neuronal layer, the Ganglion Ce11 Layer (GCL), is comprised mostly of ganglion

cells. The second layer, the Inner Nuclear Layer (INL), contains amacrine, bipolar,

horizontal, rare interplexiform neurons and Müller glia, and the third layer, Outer Nuclear

Layer (ONL), is composed of rod and cone photoreceptors (Gilbert, 1994). The neurons in

the adult retina specialize in sensing, transducing and transmitting the visual information to

the brain. Thus, in the adult retina, light stimulates rod and cone photoreceptor cells.

Photoreceptors synapse with two types of intemeurons, bipolar and horizontal cells. Further

information is extracted through synapses between bipolar cells and amacrine cells. Finally,

retinal ganglion cells, the output neurons o f the retina, transmit the result of al1 of the

information processing to various target locations within the brain via the optic nerve.

Müller glia have a role in maintaining the structure of the retina and in providing

neurotrophic factors (Dowling, 1987; Amaratunga et al., 1996; Edwards et al., 1992).

Canelion CeU Laver ((;CL) ganglion cells

Inner Plexifom Laver fïPL)

lnner Nuclear Laver (INL) amac rine bipolar

horizon ta1 Müller glia

Outer Plexiforma Laver flPL)

Outer Nuclear Laver (ONL) cone photoreceptors r d photoreceptors

Outer Limiting Membrane

Retinal Pigment Epithelium @PE)

5 months old hurnan retina

Fig. 2: Structure of the retina. Shown is a schematic diagram of the structure of a 5 month old human retina. The GCL contains ganglion cells (G), the ML contains amacrine (Am), bipolar (B), horizontal (H), interplexi fom (1) and Muller (M) cells, and the ONL contains rod (R) and çone (C) photoreceptors.

1.2.2 Retinal Development

The retina arises fiom a thin sheet of undifferentiated tissue where ce11 genesis and

differentiation spread horizontally frorn the center to the periphery. Early in embryogenesis,

a region evaginates from the rostral neural tube to form a pouch called the optic vesicle. As

the optic vesicle grows and comes into contact with the overlying ectoderm (which will give

nse to the lens), it fonns a concave structure known as the optic cup. Neuroepithelial cells in

the optic cup initially undergo syrnmetric divisions to generate a large pool of plunpotent

newoblast precursors. These precursors then begin to divide asyrnmetrically, producing the

neurons and the glia that make up the Iayers of the retina (Turner and Cepko, 1987; Holt et

al., 1988; Wetts and Fraser, 1988; Turner et al., 1990). The various neurons are 'born' (the

day during which they undergo their 1 s t S phase) or exit the ce11 cycle at the outer edge of

the proliferating neuroblastic layer P L ) in a well-defined temporal pattern. Post-mitotic

neurons then migrate fiom the NBL to their final position, where they terminally

differentiate. Each retinal ce11 type is bom within a time frame characteristic of that cell. In

rodents, ganglion, arnacnne, horizontal and cone photoreceptor cells are born prenatally,

whereas bipolar and Müller glia are bom mainly postnatally. Rod phgtoreceptor cells are

bom both pre- and postnatally, with the peak of terminal mitosis occuning shortly after birth

(reviewed in Cepko et al., 1996). For most retinal cells, terminal mitosis and terminal

differentiation are usually separated by a few days. For ganglion cells, however, these two

events occur in a very short period of time (reviewed in Fig. 3).

Retinal development is influenced by the microenvironment, which provides

important cues (such as growth factors) that have a significant role in detennining or altering

Ganglion 1 I

Amacrine - Bipolar

I I

Horizontal

Müller glia

Cone

Rods

Fig. 3: Retinal development and differentiation. A. Cells are bom sequentially in the rodent retina. The majonty of cells differentiate as rod photoreceptors. (Adapted from Young, 1985). G.C. = ganglion celis, H.C. = horizontal ceiis. B. Timing of murine retinal development. Grey bars indicate pRB expression (the question marks indicate that to date, it is unknown when pRB is first expressed in the ceIl types indicated) (Devlin et al., 201) . The solid black lines represent the interval for phenotypic differentiation. The vertical red dotted line indicates the time point for retroviral injections during misexpression studies, and the ce11 type names highlighted in red represent the cells whose progenitors are usually targeted by retroviral PO injections. E 1 O = ernbryonic day 10, P 1 = pst-natal day I .

the fate of multipotent retinal progenitors (Lillien, 1995; Ezzeddine et al., 1997; Patel and

McFarlane, 2000; reviewed in Cepko et al., 1996; Edlund and Jessell, 1999; Livesey and

Cepko, 2001). For example, Patel and McFarlane (2000) showed that when fibroblast

growth factor-2 (FGF-2) is overexpressed in the Xenupus retina, it influences ce11 fate

decisions such that more rod photoreceptors are produced at the expense of cone

photoreceptors, and more ganglion cells but fewer Müller glia are produced. Transcription

factor contro! of signaling events within and between cells is also essential for the regdation

of eye development. Thus, throughout development, the importance of transcription factors

such as the homeobox proteins (e.g. Pax6, ChxlO, POU homeodomain genes), the basic

helix-loop-helix (bHLH) proteins (e-g. Hes, NeuroD), the zinc finger proteins (e.g. GLI,

retinoic acid), or the Notch pathway proteins (e.g. Notch) has been proven repeatedly

(reviewed in Freund et al., 1996; Bao and Cepko, 1997). Specifically, in mice, Pax6

homozygous mutants lack eyes and nasal structures (Hill et al., 1991; Stuart and Gruss,

1995). ChxlO homozygous mutants are characterized by microphthalmia, a thin hypocellular

retina and a lack of differentiated bipolar cells (Burmeister et al., 1996). Mutation of the

POU homeodomain gene Pou4/2 is associated with loss of ganglion cells (Gan et al., 1996).

Of the bHLH family, Hesl mutants exhibit premature retinal differentiation with an increase

in bipolar ce11 death and an increase in the number of rod and amacrine cells (Tomita et al.,

1996); NeuroD overexpression induces neurogenesis at the expense of Müller glia, and it

appears to have a role in amacrine ce11 differentiation (Morrow et al., 1999). Mutations in

the GIi3 gene (member of the zinc finger family of transcription factors) are associated with

poorly developed eyes (Hui and Joyner, 1993), and disruption of retinoic acid levels results

in a reduction of retinal size (reviewed in Kastner et al., 1995). Finally, loss of Notch results

in an increase in neuron production in the retina early in retinal development (reviewed in

Bao and Cepko, 1997). It is clear therefore, that both intrinsic and extrinsic cues contribute

to the differentiation and maturation of cells in the retina.

1.3 pRB Expression in the Retiaa

In order to fully understand the role played by pRB in retinal development, it is

important to determine its expression pattern in the retina. To date there is still controversy

as to what the ceIl of origin of retinoblastoma is. The ce11 of origin has often been assumed

to be the photoreceptor, since the characteristic morphological feature of retinoblastomas, the

Flexner-Wintersteiner rosette, resembles a sphemle of photoreceptor cells (Flexner, 1981).

Cone photoreceptor gene expression and features of Müller glia are evident in

retinoblastomas (Bogenmann et al., 1988; Gonzalez-Fernandez et al., 1992; Nork et al.,

1995). However, retinoblastomas found in chimenc RB-' ' ;~~ 07~'- mice showed arnacrine ce11

differentiation markers (Robanus-Maandag et al., 1998). Furthemore, Devlin et al. (2001)

have shown that Flexner-Wintersteiner rosettes occur in small tumors that clearly arise in the

INL of a human eye. Therefore, based on these latest findings, it appears that the ce11 of

origin of retinoblastoma may be a ce11 (or cells) committed to the INL.

A first step in answering the question of origin is deterrnining what retinal neurons

express pRB. Devlin et al. (2001) have shown that pRB is expressed early in rodent ganglion

ce11 development, since it is present in migrating ganglion cells as early as El 1.5. pRB

continues to be expressed in the GCL throughout development and adulthood. Around birth,

as ïNL cells begin to terminally differentiate, pRB expression in the NBL is widespread. In

the adult human retina, bipolar, amacrine, most horizontal and rod photoreceptor cells do not

express pRB, suggesting that pRB rnay not have a direct role in their differentiation. On the

other hand, ganglion, cone photoreceptors, Muller glia and a subset of horizontal cells

express pRB. nius, since pRB expression in the NBL is widespread around birth, this rnay

suggest that pRB rnay have a general role in the terminal mitosis of many (if not all) retinal

progenitors. However, since in the adult human retina pRB is only seen in a subset of retinal

cells. this rnay indicate that in these particular cells, pRB has an additional role in

differentiation. These possibilities remain to be tested.

1.4 The Role of pRB in Retinal Development

The pediatric cancer indicates that pRB is essential for proper eye development.

Dunng retinal development, there are several potential points of origin for retinoblastoma.

First, loss of the second RB allele must occur before a retinal neuroblast undergoes its last

division. This would ensure that the initial RB" ce11 would continue to proliferate, thus

allowing the turnor to form. Second, pRB rnay be required temporarily during the transition

fiom retinal neuroblast to post-mitotic progenitor. Slack et al. (1998) have already shown

pRB to have such a role in cortical neurogenesis (discussed above). Thus, retinoblastoma

tumors would arise fiom the subset of retinal progenitors that normally would require pRB to

exit the ceIl cycle. Third, pRB rnay be required for terminal differentiation of one or more

retinal ce11 types. pRB has already been show to positively regulate the activity of

transcription factors involved in muscle, adipocyte, and epithelial ceIl differentiation (see

above). We now know that loss of pRB by mitotic retinal neuroblasts is an unlikely point of

ongin for retinoblastoma. Our group (Devlin et al., 2001) has shown that ChxlO, a

homeodomain protein expressed in normal developing retinal neuroblasts, was absent fiom

retinoblastoma tumors and retinoblastoma ceIl lines (these lack functional pRB). Moreover,

pRB is not expressed in retinal neuroblasts, so these cells are unlikely to give rise to

retinoblastoma in the retina. Again, it remains to be tested whether retinoblastoma arises

fiom retinal progenitors that require pRB to exit the ce11 cycle or fiom retinal cells that

require pRB for terminal differentiation.

One way to study the precise role played by pRB in retinal development is to perform

misexpression studies in rodents. Such an approach has been used successfûlly with other

retinal transcription factors (Morrow et al., 1999; Dyer and Cepko, 2001). With this method,

the gene of interest is introduced into the retina of rat pups while progenitor cells are still

dividing. The pups are allowed to grow to retinal maturity (P21), and the retina is then

analyzed for phenotypic changes that might result from the misexpressed gene. The

retroviral vectors typically used cany a marker gene (such as enhanced green fluorescent

protein or EGFP, human placenta1 alkaline phosphatase or IiPLAP, or ZacZ) that is inert in

the ce11 (has no effect on cellular functions), and the gene of interest. Retrovimses require

ce11 division to enter the nucleus (Papadopoulos et al., 2000), so in the retina, at the time of

injection such vectors will only infect dividing progenitors. For example, at PO virus will

infect progenitors that are fated to become rod photoreceptors, arnacnne cells, Müller glia or

bipolar cells (Fig. 3). Previous studies have already established that retinal progenitors

infected at PO with a control retrovirus (one that only carries the marker gene) will give nse

to "clones" (a group of cells that arise fiom one (infected) retinal progenitor) that may be

composed of a combination of four different cell types (rod photoreceptor, arnacnne, Müller

glia, bipolar) (Turner and Cepko, 1987; Fields-Bemy et al., 1992; Cepko et al., 1996). When

a retrovirus canying the gene of interest is used, the retina is analyzed for any changes in

clona1 size or composition; changes observed may be indicative of the gene of interest having

roles in ce11 cycle or cell fate, respectively. In this project, I took advantage of these facts in

order to study the role of pRB in retinal development by misexpressing pRB in neonatal rat

retinas.

1.5 The Retinal Expfant Culture System

Since RB" mice die in utero between El 3-1 5 and since retinal development is

completed post-natally, not much is known about the presurnable retinal developmental

defects in the RB" retina. Having a system that enables us to study retinal development in

RB1- mice beyond El5 would be ideal. An in vitro retinal system has already been

developed and used to study genes essential for retinal development (Tomita et al., 1996;

Morrow et al., 1999). With this system, the retina cm be explanted as an intact tissue and

grown in culture for more than two weeks. During this time, the correct ce11 types continue

to be born and differentiate, mimicking il1 vivo development with respect to layer lamination,

cellular organization and timing (Caffe et al., 1989; Adler, 1990; Sparrow et al., 1990;

Morrow et al., 1999). Such a system would enable us to characterize the developmental

defects associated with the absence of pRB in the munne retina. Once characterized, such a

retinal explant system will enable us to rescue expression of pRB in the RB knockout retina.

With this approach, pRB is transduced into the RB-'- retina via a viral vector, and rescue of

developmental defects is assessed. The success of such a procedure depends to a great extent

on having an appropriate method of gene delivery to the retina. For this purpose, 1

investigated retroviral and alphaviral gene delivery systems.

1.6 Gene Transfer to the Retina

1.6.1 Retroviral Gene Transfer

Retroviral gene transfer is a potent technique for the stable introduction of genetic

material into actively dividing marnmalian cells, and has been used successfully to study the

effects of misexpressing a gene on retinal progenitor ceIl behavior (Tomita et al., 1996;

Furukawa et al., 1997). Replication-incompetent retrovirus vectors may be used to infect

retinal progenitors in vivo during misexpression studies, or in vitro during rescue-of-function

experiments. Basically, the retroviral vector containing the gene of interest and a marker

gene is transfected into a helper cell line that constitutively expresses the viral envelope and

capsid proteins in order to obtain infectious virions. These particles c m then be used for

subsequent in vivo or in vitro infections.

1.6.2 Alphavirus Gene Expression System

Our group has developed a Semliki Forest Virus or SFV (an alphavirus) DNA-based

gene expression system that exploits the powerfiil SFV replicase to produce high arnounts of

a protein of interest (DiCiornrno and Bremner, 1998). The system employs two vectors: one

encodes the genes necessary for viral replication alongside a reporter gene (in this case,

l a d ) , and the other vector encodes the structural proteins necessary for the packaging of the

virus particles. Once the two vectors are CO-transfected into a suitable ce11 line, infectious

particles capable of infecting a wide anay of hcst cells at any point during their ce11 cycle are

produced (Strauss and Strauss, 1994). These virions may then be concentrated and used for

subsequent infections following activation (Berglund et al., 1993). Though not previously

used in the retina, recombinant SFV vectors have been successfully used to infect

hippocampal slice cultures in vitro (Ehrengniber et al., 1999).

1.7 The Question

RBI was the first human tumor suppressor gene identified (Cavenee et al., 1983;

Godbout et al., 1983), and has since been found to regulate ce11 cycle division, transcription,

differentiation and apoptosis (reviewed in DiCiomrno et al., 2000). The retinoblastoma

disease illustrates the importance of the protein in human retinal development, since the

retina is uniquely sensitive to tumor formation once pRB fails to fùncticn properly. To date,

we do not know much about the function of R B I in the developing retina, and the question of

why the retina is so exquisitely sensitive to RB loss compared to other tissues remains

unanswered. In this thesis, 1 investigate the role of pRB in retinal developrnent. Based on

our knowledge of pRB function, 1 hypothesize that pRB has a role in ce11 cycle regulation

during the developrnent of the retina. As well, since pRB is expressed in a subset o f retinal

cells throughout adulthood and since it positively regulates the differentiation of some cells,

pRB may also be involved in ce11 fate specification in the retina. One way to answer to

detemine the exact role(s) of pRB during retinal development is to perform misexpression

studies on rat retinas by delivering pRB to retinal precursors that may not normally express

it. A second approach that rnay be used is to perform rescue experiments with pRB on RB-'-

retinas. We already know that in the RB-'- retina ganglion cells fail to exit the ce11 cycle

properly (Devlin et al., 2001). Other retinal cells may be similatly affected by loss of pRB.

Thus, pnor to performing rescue experirnents in the RB-/- retina, we must characterize the

developmental defects of the RB knockout retinas. in this report, 1 present my findings

regarding possible roles played by pRB during retinal development. Overall, this project

opens the door to exciting studies that may lead to our full understanding of the induction of

cancer in the absence of functional pRB in the retina; subsequently, this will lead to the

development of more effective and preventative treatments for retinoblastoma and potentially

other tumors.

CHAPTER 2: MATERIALS AND METHODS

2.1 Cells

Human embryo kidney 293 cells (Microbix Biosystems inc, lot number LP001A1)

were passaged in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10Y0

Heat Inactivated Fetal Bovine Serum (HI-FBS) (Gibco-BRL). 293-derived Phoenix-eco

(obtained fiom William Harbour) were passaged in Dulbecco's Medium (DMEM) H21

supplemented with 10% HI-FBS. NM3T3 cells were cultured in DMEM supplemented with

10% FBS. Mouse embryonic fibroblasts (MEF) (derived fiom embryonic day 13.5 mice by

Samantha Pattenden) were grown in a-MEM supplemented with antibiotics and 10% FBS.

2.2 Generation of Recombinant Retroviral Particles

Phoenix-eco cells (293T ecotrophic producer ce11 line obtained fiom Gary Nolan; see

Cepko et al., 1998) were plated at least 12 hours pnor to transfection ont0 6 cm plates

(Starstedt). When 60-70% confluent, DNA was introduced into the cells via the calcium

phosphate-mediated transfection protocol (in Ctrrrent Protocols in Molealai- Biology, 1996,

1 :9.1.1-9.1- 1 1). Prior to adding the DNNtransfection mix to the plate, the cells were washed

briefly with Phosphate Buffered Saline (PBS), and fiesh media containing 50 pM

chloroquine was added. Generally, 10 pg of the retroviral vector, and 6 pg of a retroviral

helper vector (Mike Dyer) encoding the structural proteins needed for retroviral packaging

were transfected per 6 cm plate. Twenty-four hours later, the transfected cells were washed

with PBS, and fiesh media was added. Forty-eight hours post transfection, the supernatant

containing the retroviral particles was collected, frozen on dry ice in aliquots, and stored at -

80°C.

2.2.1 Titering of Retroviral Particles

NM3T3 cells were plated at 1 xlo5 cells per well of a 24-well plate. Approxirnately 4

hours later, they were infected with varying amounts of retrovirus stock in growth media

containing 8 pglml hexadimethrine bromide (polybrene) and incubated at 37OC / 5% COz.

Twenty-four hours later, the cells were washed with PBS, and fiesh media was added.

Twenty-four hours later still, the cells were analyzed for expression of the respective marker

protein (EGFP or hPLAP). EGFP expression was detected using a fluorescent filter (see

Photomicroscopy). Staining for the reporter alkaline phosphatase protein was perfonned as

described previously (Cepko et al., 1998).

2.3 Generation of Recombinant Semliki Forest Virus (SFV) Particles

One million 293 cells were plated pet- 6 cm plate. Twenty-four hours later, the cells,

at about 45% confluence, were transfected via the calcium phosphate rnethod with pSCAP

(2.5 pg16 cm plate) and pSCAHelper (1.5 pg/6 cm plate). pSCAP encodes the P-

galactosidase reporter marker, whereas pSCAHelper encodes the structural proteins

necessary for viral packaging. Forty-eight hours later the supernatant containing the

recombinant viral particles was collected and the virions were concentrated using a sucrose

gradient as described previously (Liljestrom and Garoff, 1994). The viral particles were

fiozen on dry ice in aliquots and stored at -80°C. Pnor to use, the viral stocks were quickly

thawed in a 37°C water bath and were activated with a-chymotrypsin (as described

previously in DiCiommo and Bremner, 1998).

2.3.1 Titering of SFV Recombinant Particles

The virus was titered on Baby Hamster Kidney cells (BHK-21), and reporter B-

galactosidase activity was detected via 5-bromo-4-chloro-3-indolyl-PD-galactop y i d e

(X-gal) assays performed as descnbed previously (DiCiommo and Bremner, 1998).

2.4 Westerns

Expression of the protein of interest fiom retroviral vectors was assayed via Western blot

assays. Approximately 100 pl of 10~40 ' W/ml retroviral stock were used to infect 50%

confluent RB-'"' MEFS monolayer cultures. Two days later, the protein was extracted from

the retrovirally-infected RB-'- MEFS cultures using Reporter Lysis Buffer (Promega), as per

the manufacturer's instructions. The 100 pl suspension was then pulse-sonicated once,

centrifuged for 10 min at 4OC, and the supernatant containing the protein of interest was

removed. Pnor to loading on gels, samples containing 5 pl of the supernatant were boiled for

5 min with loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 15% Ficoll type

400, 50 mM EDTA in ddHtO). Denaturing gel electrophoresis was conducted at 26-34 mA

on 6% polyacrylarnide gels until the blue loading dye ran off the gel. For al1 blots, gels,

Whatman paper and nitrocellulose membranes were soaked in transfer buffer (25 mM Tris,

192 mM glycine, 20% methanol in ddHzO) and transfer was accompiished using a Trans-

Blot Electrophoretic Trans fer Cell (Bio-Rad). Nitrocellulose blots were then blocked in 5%

powder milk in PBS ovemight at 4OC. Immunoblots were washed in PBS plus 0.1% Tween-

20 (PBS-T) once for 15 min, and twice for 5 min each. The blots were then incubated with

mouse 14001A a-Rb (Phanningen) pnmary antibody (1 pgml in PBS) for 1 hour. The wash

cycle was repeated as before, and the goat anti-mouse HRP secondary antibody (15000,

Jackson Laboratories) was incubated with the blots for 30 min. The blots were then washed

and cherniluminescence was detected with the BM Cherniluminescence Blotting Substrate

(POD) kit (Boehringer).

2.5 Neonatal rat eye injections

In order to study misexpression of the gene of interest via a retroviral vector, untimed

pregnant CD rats (Charles River Laboratories) were used. On the first day of birth (post-

natal day 0, or PO), the neonates were anesthetized on ice for 3-5 min. The eyelids were

gently pried open, a small incision was made in the comea with a fine knife, and up to 1.5 pl

of retrovirus stock were injected into the sub-retinal space (though the exact titer of the

viruses used for injection in these misexpression experiments was unknown, we usually

generate retroviral stocks at 1 06- 10' IU/ml). Typically, the control retroviral vector was

injected into the arbitrarily chosen nght eye, and the vector carrying the gene of interest was

injected into the lefi eye of the sarne rat pup. The rats were allowed to grow to retinal

maturity (P21) when they were euthanized via cervical dislocation, and the eyes were

removed. The cornea was nicked with a fine knife to allow penetration of the fixative, the

eyes were immersed in 4% paraformaldehyde (pH 7.2-7.4) for 1 hour at room temperature,

followed by 30% sucrose in PBS for 3 hours-overnight at 4OC, and embedded in OCT for

cryosectioning. 20 Pm thick sections were then placed on siaiinated slides, and stored at -

80°C until needed.

2.6 Retinal Explants

To characterize embryonic retinal growth iri vitro, timed pregnant CD-1 (Charles

River), or C57BLI6 mouse strains were used. RB'" embryos were denved from breeding

RB+/- heterozygotes (kindly provided by Tyler Jacks) and were genotyped by Polymerase

Chain Reaction (PCR) of tail DNA as described (Jacks et al, 1992). Timed pregnancies were

determined by vaginal plug observation, with midday time of plug observation counted as

embryonic day 0.5 (E0.5). Pregnant fernales were sacrificed by cervical dislocation. Many

different retinal culture protocols were tested before finding one that allowed proper retinal

development in vitro. With this protocol, the embryonic retinas with lens intact were

removed in prewarmed retinal explant culture medium (45% HAMS F-12 and 45% DMEM

media, 10% FBS, 5 &ml insulin, 2 mM L-glutamine, 10 pg/ml Streptomyciflenicillin, IO

pglml HEPES). The retinas were placed on polycarbonate filters (1 pm pore size,

CorninglNucleopore) in 1 ml pre-warmed explant culture medium in a 12 well dish

(Corning). The retinas were cultured for at least 3 or 4 days, and each day 20 pl of

conditioned media was added to each explant. (The conditioned media was prepared as

follows: E14.S CD rat retinas with lens intact were explanted in the same explant culture

medium as descnbed above, and cultured for 4 days. During each culture day, 50-100 pl of

the medium was added to each of 4 explants fiom the well containing the explants. On the

fourth day, the explants were removed, the cultured supernatant was diluted twofold with

fresh explant culture medium, filtered though a 0.22 Fm Millex-GV filter, and stored at -

80°C.) AAer 4 days, the retinas were removed from the filters they were grown on, fixed in

fieshly made 4% parafomaldehyde (pH 7.2-7.4) at 4OC for 1 hour, briefly i-insed in PBS,

incubated in 30% sucrose in PBS ovemight, placed in OCT and stored at -80°C for

cryosectioning. The retinas were cut in 20 Fm sections and used for subsequent expenments.

Retinas dissected at PO were also used for a number of experiments. These were

explanted as described above, and grown in the explant culture media already mentioned.

The post-natal retinas were not supplemented with rat conditioned media, but the culture

media was replaced every 2-3 days. These retinas were grown in culture for up to 8 days.

2.6.1 Infection of Retinal Explants with Retroviral Vectors

Twenty-forty pl of 10~40' IUlml retroviral stock were diluted in growth media

containing 100 pg/ml polybrene and pipetted ont0 the embryonic retinal explants. The

infected explants were cultured for up to 4 days at 37OC 1 5% COz. Similarly infected post-

natal retinal explants were grown in culture for up to 8 days.

2.6.2 Infection of Retinal Explants with Recombinant SFV Vectors

Fifiy-one hundred pl of 104 W/ml recombinant SFV stock were activated and added

to the E12.5 or PO C57BL/6 mouse retinal explants. The infected explants were cultured for

2-5 days at 37°C / 5% COz.

2.7 Immunobistochemistry

Imrnunohistochemistry staining was performed on cryosections. Slides were thawed

at room temperature for 1-4 hours. The general protocol used was as follows: the slides

were rehydrated for 10-20 min in PBS, biocked (1% Bovine Semm Albumin, 0.45% Triton-

X, 2% serum in PBS) for 40 minutes at room temperature, incubated for 1 hour at room

temperature then placed at 4°C overnight with the pnmary antibody (primary antibody

diluted in the 1% BSA, 0.15% Triton-X, 2% serum in PBS). The slides were then rinsed in

PBS 3 times for 10 min, incubated for 1 hour at room temperature, dark, with the secondary

fluorescent antibody (diluted in PBS), rinsed in PBS twice for 10 min. The slides were

incubated last in 4',6-Diamidino-2-phenylindole Dihydrochloride (DAPI) (0.1 pg/ml in

ddH20) for 25 minutes, or with propidium iodide (PI) (0.05 p d m l in PBS) for 10 minutes at

room temperature, in the dark. They were then rinsed in PBS and mounted with mowiol

containing an antibleaching agent, DABCO (1,4-Diazabicyclo-[2,2,2]0ctane, Sigma).

Primary antibodies were ItAP (1 : 1000, mouse monoclonal, Sigma), Brn3b (1 : 100, goat

polyclonal, Santa Cruz Biotechnology), B-tubulin (clone TUB2.1, 1 : 1000, mouse

monoclonal, Sigma), CRALBP (1 : 1000, rabbit polyclonal, Dr. J. Saari, University of

Washington), GAP-43 (150, mouse monoclonal, Sigma), GFP (1:1000, rabbit polyclonal,

Molecular Probes), olf-1 (1:400, rabbit polyclonal, Dr. R. Reed, John Hopkins University

School of Medicine), syntaxin (clone HPC- 1, 1 :2000, mouse monoclonal, Sigma), 14001 A

anti-pRB (15 pg/rnl, mouse monoclonal, Pharmingen). Secondary antibodies used were

Alexa 488 or 586-conjugated goat anti-mouse or goat anti-rabbit IgG (1 : 1000). Secondary

antibodies used were Alexa 488 or 586-conjugated goat anti-mouse or goat anti-rabbit IgG

(1 : 1000, Molecular Probes), or FITC conjugated rabbit anti-goat IgG (1 5 0 , Sigma).

2.8 Photomicroscopy

Immunostained sections were analyzed with a Nikon Optiphot microscope with

epifluorescence attachent, or with a Zeiss Axioplan 2 haging microscope with

epifluorescence attachent, using 16x, 20x, 40x or 60x fluor objectives. The Zeiss

AxioVision 3.0 software was used for image analysis. Morphology and location within the

retina were the criteria used to identify the type of neurons that were EGFP-labeled in the

retrovirus-injected rat eyes. For this purpose, the Zeiss Axioplan 2 haging microscope was

used. Thus, labeled cells were considered to be photoreceptors if their ce11 body was found

in the ONL of the retina, if they had axons extending to the OPL and toward the outer

limiting membrane, and if they had an outer segment. The thick processes of Muller glia

spanned the entire retina, fiom the vitreous surface to the outer limiting membrane; the ce11

body was located in the INL. Arnacrine cells had a ce11 body in the lower third of the INL

(closer to the GCL), and (an array of) processes extending toward the GCL. Bipolar neurons

had a ce11 body in the upper third of the INL (toward the ONL), and both upward and

downward processes.

2.9 Statistical Methods

To evaluate the significance o f differences in the proportion of cell types between the

MXIE-AKll and the MXIE-injected eyes, a student's t-test o f equal variance was performed.

Al1 P values are one-sided.

CHAPTER 3: RESULTS

3.1 1' Yivo Misexpression of pRB in the Rat Retina

3.1 .1 Retroviral Vector Protein Expression

One woy to study the precise role played by pRB in retinal developrnent is to perform

in vivo misexpression studies using neonatal rat retinas. The first step in this process is to

generate a retroviral construct that expresses the RB gene together with a marker gene that

can be used to easily identify infected cells. Once cloned, infecting RB-" MEFS with the

retrovirus, and then performing a Western Blot assay with an anti-pFü3 antibody would

enable us to test expression of pRB. In tum, expression of the marker gene would be tested

by infecting NIH3T3 cells with the retrovirus and then either perfoming an

immunohistochemical assay to detect activity fiom the marker protein (in the case of proteins

such as hwnan placenta1 alkaline phosphatase (hPLAP) or P-galactosidase), or simply by

looking for fluorescent activity of the marker protein (in the case of Enhanced Green

Fluorescent Protein (EGFP)). Thus, I cloned human RB 1, mouse AKI 1, and two human low

penetrance mutants of MI, A24/25 (Bremner et al., 1997) and C7 12R (Yilmaz et al., 1 W8),

into the retroviral vectors PLIA-E a d pNIN-E (Table 1). PLIA-E expresses the marker gene

hPLAP fiom an interna1 ribosome entry site (IRES) whereas pNIN-E expresses the IacZ gene

fiom an IRES (Bao and Cepko, 1997; Dyer and Cepko, 2000). Mouse AK11 is a

constitutively active, hypophosphorylated mutant form of pRB, since I l of the 16 possible

phosphorylation sites of pRB are mutated (Brown et al., 1999) (Fig. 1). "Low penetrance

Table 1: Bicistronic gene expression in retroviral vectors tested. At least 5 différent retroviral vectors were used for cloning of the genes indicated. However, infection and Western blot assayF indicate that not al1 retmviruses were succasfùl in expressing the marker gene and the gene of interest, respectively. MCS = multiple cloning site, IRES = intemal nbosome entry site, LTR = long terminal repeats, hPLAP = human placental alkaline phosphatase, EGFP = enhanced green fluorescent protein. EcoRI = restriction endonuclease site used for cloning. Note that oniy a minimal schematic representation of each retroviral vector is shown.

Retroviral Backbone

I pMIC-RB: IRES -LTR1.LTR -

I LTR I R E S LTR- - - hlCS

Cloned Gene X

Marker Expression?

(NIH3T3 hfecticrn)

Yes Yes Yes Yes

Yes Yes Y es Yes

Yes

Yes Y es

-

Protein X Expression? (Western Mot

'-9')

Yes

Yes (weak) Yes

pRB mutants" carry mutations in various domains of pRB leading to functional inactivation

of the protein. Aî4/25 represents a large deletion in the C-terminus of the RB protein,

whereas C712R represents an amino acid substitution in the pocket region (Fig. 1).

Following testing of the retroviral constructs built with PLIA-E or pN[N-E, 1 found that

while they al1 expressed the marker proteins hPLAP or P-galactosidase, respectively, none of

them expressed the pRB protein of interest. We then obtained the retroviral vector MIG-RB,

which encodesRBl and the marker gene EGFP (Tyler Jacks, Massachusetts Institute of

Technology). However, while this vector expressed pRB, it did not express the marker

EGFP. Thus, because this retrovirus offered no working marker, we were unable to use it in

our studies, as we would have been unable to identify the cells it would have infected. We

obtained two more retroviral vectors that expressed the marker protein EGFP fiorn IRESs,

pCLIG (R. Kageyama, Kyoto University) and pMXIE (D. van der Kooy, University of

Toronto). 1 cloned MC1 1 into pCLIG, but when tested, 1 found that although the vector

expressed the marker protein EGFP, it did not express AK11. Finally, 1 cioned AK11 and

human RB into the retroviral vector pMXIE. When tested for bicistronic expression, 1 found

that MXIE-hRB and M m - M l 1 both expressed the marker protein EGFP. 1 also found that

both vectors expressed the cloned gene of interest. However, while MXIE-AKl 1 showed

strong expression of AK11, MXIE-hRB only showed weak expression of hRB (Fig. 4). 1

therefore proceeded to use the MXIE-AKl 1 retrovirus for the in vivo misexpression studies.

MXIE-Ml1 RE/- MEFS MXIE-hRb

Fig. 4: Retroviral lineage analysis. A. Retroviral vector gene expression. Western blot irnmunoassays on infected RB-/- MEFS demonstrate that the pMXIE vectors express the proteins of interest (though MXIE-hRB expresses hFU3 at low levels), whereas the pCLIG-ml1 vector does not express the M l 1 protein. Al1 vectors expressed EGFP as assayed in infection experiments of NM3T3 cells (data not shown). BE. Retinal progenitors infected with MME- AKl 1 gave rise to small clones, composed predorninantly of photoreceptors (retrovims-labeled cells are shown in rd ) . Also shown are clones wntaining Muller glia and bipolar cells. F-G. Retinal progenitors infected with a conho1 retrovinis, MXIE, gave rise to larger clones composed of various ceIl type combinations (retrovims-labeled cells are shown in green). DAPI staining (blue) was used to demarcate al1 retinal nuclei. Though the images captured here may not show al1 individual cells clearly, focusing on different planes during lineage analysis does confirm the identity of each ceIl within a clone. ( 160x-600x magnification). ONL = outer nuclear layer, M L = inner nuclear layer, GCL = ganglion ce11 layer, PR = photoreceptor.

3.1.2 Misexpression of A K 1 1 Affects Clone Sue in the Neoaatal Rat Retina

For the studies on misexpression of pRB in the retina, 1 injected the eyes of PO rat

pups with the control vector MXIE, or with the MXIE-AICI 1 retrovirus. At PZ 1,1 performed

lineage analysis on the infected retinas by investigating the nurnber and the composition of

the EGFP-labeled ce11 clones. The cells infected by the retrovirus were detected by

performing immunohistochemical analysis with an anti-EGFP antibody. The results are

summarized in Figure 5 . One of the most strîking results was that clone size was

substantially reduced in the eyes injected with MXIE-MC11 relative to those injected with the

control vector MXIE. The overall average clone s i x in MXIE-infected eyes was 2 2 0 . 1

cells/clone (4 eyes total were analyzed fiom 2 rat Mers), whereas in the MXIE-AKI 1-

infected eyes it was 1.5+0.0 cells/clone (5 eyes total were analyzed fiom 2 rat litters). The

results were similar when only littermates were compared, indicating that there were no

naturally occumng differences associated with retroviral infections of different rat litters.

This same trend was observed when the size of clones containing only photoreceptors was

compared. There were more clones containing one photoreceptor cell, for exarnple, in the

MXIE-AK11-infected eyes than in the control MXIE-infected eyes (64.1*4.5% and

3 1 .SI 16.4% of clones counted, respectively). Altematively, there were significantly fewer

clones that contained three photoreceptors, for instance, in the MXE-AK11-infected eyes

than in the control (7.4*0.9% O versus 10.5*0.3%, respectively; -0.002) (Fig. 5). Overall,

the average photoreceptor clone size was smaller in the MXIE-AKI 1-infected eyes at 1.4*0.0

celVclone than in the ME-infected control eyes, which had an average of 1.7k0.1

photoreceptor cells/clone.

Total PR ' unclear

BcpR Total AmtPR

Clone composition and size

Fig. 5: pRB drives mitotic progenitors out of the ce11 cycle. Over 2620 clones (5 eyes) were scored for the MXIE-AK 1 1 -infected eyes, but only 178 (4 eyes) were scoreci for the MXIE-infected eyes (total nurnbers from two independent experiments). The clone size distribution data as well as clonal composition are shown. PR = photoreceptor cells, M = Muller glia, B = bipolar cells, Am = macrine cells. While performing lineage analysis, 1 was unable to use morphological criteria to identifi a few retrovirus- labeled cells, and these are tabulated in the "Unclear" category. Each bar with standard deviation is the average of four and five different eye samples in MXIE and MXIE- AK 1 1, respectively.

The M l 1 misexpression studies also indicated that there were significantly more

clones containing only photoreceptors in the MXIE-AK11-infected eyes than in MXIE-

infected eyes (96.7*l.4% and 74.6*2.7%, respectively; P=0.0002). Figure 5 also shows that

there were significantly fewer clones containing photoreceptors and Müller glia in the MXIE-

AK1 1-infected eyes than in the MXIE-infected eyes (2.6*0.1% and 14.3*0.3%, respectively;

P=0.000003). As well, there were hardly any labeled clones containing bipolar cells in the

MXIE-AK11-infected eyes (0.4*0.8%) compared to the control MXIE-infected eyes

(4.8&3.7%). Moreover, no labeled amacrine neurons were observed in the MXIE-AKll-

infected eyes, but there were 2.W1.7% clones with amacrine neurons in the MXIE-infected

control eyes. It must be noted that while 2621 total clones were scored for MXIE-AKI 1-

infected eyes, only 178 clones were scored for the MXIE control eyes. This fact was

reflected in the larger standard deviations observed for the MXIE clones, and it may be due

to a lower titer of the MXIE retrovirus stock. More MXIE clone counts are required to

reduce the standard deviation.

Collectively, these results indicate that AKll reduces clone size in retrovirally-

infected retinas. As well, it appears that in MXIE-AKI 1-infected retinas more photoreceptor

cells are produced at the apparent expense of Müller glia, bipolar, and amacrine neurons.

3.2 Characterizatioa of an In Ktro Retinal Explant Culture System

3.2.1 RB+'+ Retinal Explant Characterization

The retinal explant culture system is an itr vitro approach that may be used to study

the effects of pRB on retinal development. Such an explant system is necessary since RB-'-

embryos die in utero around E 1 3 - 1 5, well before retinal di fferentiation is completed.

However, before rescue expenments are performed with retinal explants, the developmental

defects of the RB knockout retinas as well as growth of these retinas in vitro must first be

fùlly charactenzed. We started by establishing the conditions necessary for proper retinal

development in vitro. 1 tested the proper growth in vitro of embryonic or post-natal mouse

retinas under different explantation procedures, culture conditions and substrates

(surnmarized in Table 2). Protocol 3 gave the most reliable results. imrnunohistochemical

staining with six different markers showed that development in vitro mimicked that in vivo

for age-matched control retinas (Fig. 6). in Protocol 3, the age-matched control eyes used

were CD-1 mouse eyes that had been removed and prepared for immunohistochemical

experimentation at El6 or E17, as needed. The immunohistochemical markers used were

Bm3b (early marker of ganglion ce11 differentiation) (Xiang et al., 1993), Gap43 (late marker

of ganglion ce11 differentiation; labels axonal outgrowths and synapses) (Capone et al.,

199 1), B-tubulin (found in neuronal axons) (Sullivan, 1988). HPC- 1 (labels syntaxin in

arnacrine cells) (Barnstable et al., 1985), olf-1 (labels neurons in the GCL) (Walters et al.,

1996), and pRB (labels the nuclei of post-mitotic cells) (Devlin et al., 2001). To date, similar

staining patterns for Bm3b, Gap43, B-tubulin, HPC-1 and olf-1 have been observed with

Explanted retinas Age-Matchd Control Eyes

E13+4 explanted rethas EL7 control eye

Fig. 6: Characterization of embryonic retinal explants via immunohistochemical staining. The panels on the lefi show immunostaining of retinas explanted at E 13 and grown in culture for 3 or 4 days as indicateà. The right side panel shows staining for the same antibodies in retinas explanted at E 16 or El 7 as indicated, and fixed immediately. The lamination and cellular organization of the cultured explants closely mimics that of the control retinas. A. Anti-Bm3b (ganglion), B. anti-GAP43 (neuronal axons, synapses), C. anti-phibulin (neuronal axons), Do anti-olf- 1 (cells in the GCL), E. HPC- 1 (amacrine neurons), and F-H. anti-pRi3 (some pst-mitotic cells) antibodies weie used. The arrows demonstrate that the pRB staining (green in 8.) observed is nuclear, as assessed by double imrnunostaining with the nuclear marker propidium iodide (red in 8.). GCL = ganglion cell layer, NBL = neuroblastic layer.

many other explants grown separately, indicating that the explant culture system that 1 have

been working with is reproducible and reliable.

3.2.2 RB" Retinal Explant Characterization

Since 1 have been successful in growing wild type CD-1 retinal explants in vitro, 1

proceeded to characterize the RB-'- retinal explants in a similar fashion, using the sarne six

markers: Bm3b, Gap43, P-tubulin, W C - 1, olf-1 and pRB. While the staining appeared to

have worked somewhat with the Gap43 and HPC-1 antibodies (data not shown), none of the

other antibodies produced any satisfactory results. Specifically, I was unable to obtain any

positive staining in the RB" or, sutprisingly, in the RB"' retinal explants that 1 had used as a

positive control for the immunohistochemical staining expenments. The

imrnunohistochemical staining protocols were repeated at least three different times, and

every time the sarne unsatisfactory results were obtained. We do not know why the

immunohistochemical staining protocol stopped yielding results in the RB+'" retinal explants.

It appears that the staining protocol might need re-optimization.

Therefore, immunohistochemical analyses suggest that development in a 4-day RB"'

retinal explant culture system closely follows that in vivo for at least six markers tested.

However, while attempting to characterize the growth in vitro of RB" retinal explants, 1

found that the imrnunohistochemical protocol that 1 had used previously had stopped

working.

Table 2: Summary of in vitro retinal explant culture protocols.

Pro- toc01 No.

Explan -tation

Age (Strain)

E12.5 or PO, Pl, P2, P3

(C57BL /6 mice)

El4

(C57BL 16 or CD- 1 mice)

E 13

(CD- 1 mice)

Days Crown In Vitro

Explant Make-up

Retina alone

Retina + lem Or Retina + lens + RPE

Retina + lens

Culture Media (Notes)

HAM'S F 12 media (Gibco BRL) supplemented with 1 Ocig/d StreptomycinPenicill in, 3 m M taurine, 2mM gluatamine, 5% Fetal Bovine Serum (FBS) (Media was replenished every 2-3 days.1

HAM'S F 12 media (Gibco BRL) supplemented with 1 O c i l m l Streptomycin/Penicill in, 3 m M taurine, 2mM gluatamine, 5% Fetaf Bovine Semm ( F W (Media was replenished every 2-3 day S.)

45% HAM'S F 12 media and 45% DME media supplemented with lOpg/rnl Streptomycin/Penicill in, Spdml insulin, 2mM gluatamine, 10pg/d HEPES, 10% FBS (Explants were fed with rat conditioned media (see Materials and Methods) daily.)

Culture Substrate

0.2pm Millipore Millicell-CM filter, 30mm diameter

ACLAR film (Sparrow et al, 1990)

ACLAR film coated with 3 3 cig/ml laminin/O. 1 mg/ ml poly-D- lysine (Wang et al, 2001)

Nucleopore track-etch membrane (Corning ), 1 pm pore size

Results

No ganglion cells detected via anti-Bm3b antibody immunohistochemical staining

Anti-RetP 1 (rod photoreceptor), anti- syntaxin (amacrine), anti- CRALBP (Muller glia) and anti-PKC (bipolar) immunohistochemical staining revealed improper lamination

In al1 cases: GangIion cells were

detected (using an anti- GAP-43 antibody), but the staining was very irregular and sparse; no ganglion cells were detected with the anti-Bm3b antibody

P-tubulin immunohistochemical staining (neuronal axons) revealed improper lamination

Immunohistochemical staining with anti-B-tubut in, GAP43, B d b , Olf- 1 (neurons in the ganglion ce11 layer), syntaxin, and 1400 1 A anti-RB antibodies revealed that retinal drvelopment in vitro mimicked that in vivo for an age matched retina

3.3 Two Virus-Based Systems May Be Used for Gene Delivery to the Post- Natal Murine Retina

Two important conditions must be satisfied before we may proceed with the pRB

rescue experiments in the RB"' retinal explants. First, we must have a reproducible, fùlly

characterized retinal explant culture system, and second, we must have an appropriate means

of delivering pRB to the retinal explants. We have already begun the RB" retinal explant

charactenzation, as already mentioned. For the purpose of gene delivery to the retina, 1 have

explored two different viral systems: a retrovirus, and an alphavirus.

3.3.1 Retroviruses M a y Be Used for Cene Delivery to the Retina

1 attempted to infect embryonic and post-natal retinal explants with retroviral vectors

as descnbed in the Materials and Methods chapter. Figure 7 shows that retrovinises do

successfblly inject post-natal retinal explants. The efficiency of embryonic explant infection

is very low (data not shown). Thus, the system still needs to be refined for use in embryonic

explants.

3.3.2 A Novel Alphavirus May Be Used for Gene Delivery to the Retina

In a novel approach, 1 have used recombinant SFV particles to infect embryonic and

postnatal murine retinal explants, which were then grown in culture for up to 5 days. As with

the retroviral system, the SFV vectors were inefficient at infecting embryonic retinas (data

Are the marker -O asnd protein of

f NI83T3 ceUs interest expressed? Retroviral vect0rtranSfecl

carrying

Retinal Ex plant

B Are the marker Alphaviral \ and protein of

vector /1 BEK-21 eells interest expressed? carrying infect

gene of interest + 293 cells

Helper vector carrying

packaging genes --

Retinal Explant

Fig. 7: Producing, testing, and using viruses to infect retinal explants in vitro. A. The retrovinis gene expression systern. With this system, a retroviral vector canying the gene of interest is tnuisfected into a helper ce11 line in order to produce infectious particles. Bicistronic retrovirus expression may be tested by infecting NIH3T3 cells and assaying for expression of the marker gene and the gene of interest. The retroviruses may be then used to infect pst-natal retinal explants in vitra The white arrows point to retinal progenitors that have been infected with the LIA retrovirus vector (this retrovirus carries the hPLAP marker gene as descnbed by Bao and Cepko, 1997). B. The Semliki Forest Virus (SFV) gene expression system. With this system two vectors carrying the protein of interest and the genes necessary for viral packaging are CO-transfected to produce infectious virions. These may then be assayed for expression of both, marker gene and gene of interest. Such SFV virions may be used to infect pst-natal retinal explants in vitro. The white arrow points io retinal cells that have been infected with SFV virions carrying the marker gene lucZ

not shown), but infected post-natal retinas very well (Fig. 7). We do not currently know

what retinal cells are infected by SFV.

CHAPTER 4: DISCUSSION

Development of neural tissues is a complex process involving ceIl proliferation, ceIl

type specification and differentiation, and ce11 death. As an initial step in understanding the

fùnction of the transcription factor pRB in the development of the neural retina, we have

conducted misexpression analyses in this tissue, and have laid the groundwork for rescue

experiments in RB-'- retinal explants. My data demonstrates that pRB is involved in ce11

cycle regulation in retinal progenitors.

4.1 I n Kvo Misexpression of pRB in the Rat Retina

4.1.1 Retroviral Vector Protein Expression

Experiments were designed to test whether pRB bas a role in ce11 cycle regulation

ancilor differentiation in the developing post-natal rodent retina, and for this we needed an

appropriate retroviral vector. 1 buiit and tested a few different retroviral vectors for

expression of a marker protein and the RB protein of interest until 1 was able to find one that

worked. Thou& the reason is not always well understood, bicistronic retrovimses that

express both proteins can be difficult to obtain (Mike Dyer, personal communication). We

found that one of our vectors, MXIE-AKl 1, expressed both the marker protein EGFP and the

constitutively active pRB mouse variant, & I l . 1 also built a bicistronic retroviral vector

that expressed human pEU3 (MXIE-W), albeit at apparently low levels. We do not

currently know why the expression level of human pRB was so much lower than that of

AK11. One possibility is that the hurnan RB cDNA has cryptic splice sites, which, if

introduced into some retroviral vectors, are activated and prevent the production of a fully

fiuictional protein. Since 1 was able to obtain a fiinctional bicistronic vector when a mutant

form of mouse pRB was used (AK1 l), presumably, mouse cDNA does not contain such

putative cryptic splice sites. The human and mouse RB proteins show 91% identity in their

arnino acid sequence (Bernards et al., 1989). To venfy whether only human cDNA is

defective when expressed in the retroviral vectors tested, we will build a retroviral vector that

encodes mouse pRB (MXIE-mRB) and test its expression. Strong expression of wild type

mouse pRB from such a retroviral vector would indicate that there may be something toxic

about human RB cDNA. One question that Our group will address in functional pRB studies

involves the identification of the specific pRB domains required to rescue retinal

development. For this purpose, a series of RB retroviral constructs that carry domain

mutations that ablate various fùnctions of the protein would be used. Conveniently, several

such low penetrance pRB mutants have already been identified from hurnan retinoblastoma

tumors (Bremner et al., 1997). Thus, in order to obtain bicistronic retrovirat vectors that

expressed such human pRB domain mutations, we would build mouse pRB hybrids that

contained the human R B mutations.

MXIE-mRB would also provide a particularly significant control for the MXIE-AKI 1

experiments, because we want to ensure that the in vivo results obtained with MXIE-AKll

were not mere artifactual effects of the mutant AK11, but were indeed due to the intnnsic

action of pRB on retinal progenitors. At the same time, in vivo experiments with M m -

mRB may prove that using a 1 1 instead of pRB is preferable to determine the role of pRB

in retinal development. Numerous studies have already shown that when wild type pRB is

introduced into some ce11 types it is largely inactivated by the endogenous activity of

cycIin1CDK complexes within those cells (Bookstein et al., 1990; Muncaster et al., 1992;

Knudsen et al., 1999). Should this happen to be true in the infected rat retinal progenitors,

using pRB would be ineffective since the protein would be inactivated and we would be

unable to observe any effects on retinal development. Thus, the constitutively active,

hypophosphorylated AKI 1 would be the preferred experimental approach to dissecting the

role of pRB in the retina. 1 anticipate that the results obtained with constitutively

hypophosphorylated ml1 mutant are not due to aberrant activity of AKl 1, but that they are

indicative of intrinsic pRB activity in the retina. Numerous studies have Iooked at pRB CDK

phosphorylation sites by using many different pRB phosphorylation site mutants (Harnel et

al., 1990; Knudsen and Wang, 1997; Knudsen et al., 1998; Knudsen et al., 1999; Brown et

al., 1999). None of these studies reported aberrant activities of the mutated RB proteins.

Hence, iî appears that ablation of CDK phosphorylation sites in RB proteins serves simply to

block phosphorylation of these protein by cyclidCDK complexes, and does not have

aberrant effects on other pRB functions.

4.1.2 Misexpression of AK11 Reduces Clone Size in the Neonatal Rat Retina: Implications for Cell Fate Specification

We misexpressed the constitutively active AK11 in neonatal rat eyes in order to

determine what role pRB has in retinal development. 1 found that the overall average clone

size was smaller in the MXIE-AM t -infected eyes than in the control MXIE-infected eyes.

As well, when only photoreceptor clones were analyzed, there were more clones containing

only one photoreceptor cell in the MXIE-AK1 1-infected eyes than in the MXIE-infected

eyes. I also observed that, when compared to the MXIE-infected eyes, the MXIE-AKll-

infected eyes contained fewer clones with Müller glia and hardly any clones with bipolar

neurons. Similarly, unlike the MXIE-infected control eyes, eyes injected with the MXIE-

Ml1 retrovirus showed no clones containing amacnne neurons. These observations give

nse to at least two models that may help explain the role played by pRB in retinal

development. First, pRB may be involved exclusively in ce11 cycle regulation in the

developing retina. The peak pet-iod of rod photoreceptor genesis occurs just before the peaks

for bipolar and Müller glial cells (Fig. 3) (Cepko et al., 1996). Thus, it would not be

surprising that premature cell cycle exit mediated by pRB would lead to an increase in the

proportion of clones containing photoreceptors. This phenornenon has been observed

previously with two other inhibitors of the ce11 cycle, the cyclin kinase inhibitors p27Kip' and

p57Kip2 (Dyer and Cepko, 2000; Dyer and Cepko, 2001). This model is fùrther supported by

observations that in the mouse, pRE3 is expressed in a large number of retinal progenitors

(found in the NBL) around birth (Devlin et al., 2001). Thus, pRI3 is likely involved in

stopping the division of progenitors so that they may differentiate. The second model

proposes that besides a role in regulating the ce11 cycle, pRB is also involved in ce11 fate

specification in the developing retina. Thus, according to this model, pRB actually drives the

differentiation of photoreceptors at the expense of the other ce11 types. There are several

experiments that we may perforrn in order to differentiate between the two models.

First, in order to confirm that pRB has a role in blocking cellular proliferation, we

rnight take an immunohistochemical staining approach. Thus, following the PO injection

time point, every few days infected progenitors in the rat retins would be evaluated for

proliferation using antibodies against the retroviral marker EGFP and a known cellular

proliferation marker such as Ki67 (Satow et al., 2001). If staining with the two markers is

mutually exclusive, then this would be evidence that pRB does indeed block proliferation in

retinal progenitors.

Nurnerous studies have already dernonstrated that intrinsic and extnnsic cues

contribute to differentiation of retinal progenitors (reviewed in Livesey and Cepko, 2001).

Thus, if pRB forces a progenitor to exit the ce11 cycle before certain extrinsic cues are

available or before certain genes are activated, the progenitor may divide along a different

lineage. This could be tested using an approach descnbed previously by Belliveau and

Cepko (1999). With this system, PO retinal explants would be infected with the MXIE-AIS1 1

retrovirus and the growth media would be supplemented with retinal extract fiom a different

developmental time point, such as E l 6 or P4. Such retinal extracts would contain different

growth factors than at PO (Belliveau and Cepko, 1999), since different proportions of

different retinal cells are found at these time points. Hence, if pRB only has a role in ce11

cycle regdation and is not involved in ce11 fate specification, adding the El6 or P4 retinal

extracts should yield a different proportion of retinal cells than were obtained at PO, while

maintaining a reduced clone size. If, however, pRB directly prornotes differentiation of a

certain ce11 type, adding the retinal extracts would have little or no effect on the number of

cells observed for that particular ce11 type.

Another approach would be to compare the effect of KI1 and another cell cycle

inhibitor, such as a dominant negative mutant of DPl (dnDP1) (Wu et al., 1996) in neonatal

rat misexpression studies. The dnDPl mutant binds E2F and prevents binding of the

heterodimer to DNA, thus blocking the ce11 cycle at the Gl /S boundary. If pRB has an

indirect role in stimulating differentiation through its inhibitory effect on the ce11 cycle, then

dnDPl should have the same effect when misexpressed in proliferating retinal progenitors.

However, if pRB has a direct role in promoting differentiation (by binding and activating

transcription factors involved in retinal differentiation, for instance) then dnDPl would be

ineffective at promoting differentiation in the retina.

The second mode1 stipulates that besides its role as a ce11 cycle regulator, pRB may

have a direct influence on the development of some retinal cells. Müller and bipolar cells are

among the last retinal ce11 types to differentiate, and at P4 there are significantly more Müller

and bipolar ce11 progenitors that go through their last mitosis than at PO (Fig. 3) (Cepko et al.,

1996). In order to test whether pRB is implicated in the differentiation of Müller or bipolar

cells, one approach would be to inject MXIE-AKl 1 in the retinas of P4 rat eyes. If as a result

of injecting AKl 1 at this time point hardly any Müller or bipolar neurons are observed, as

was the case with PO injections, then this would support the idea that pRB has an additional

role in ce11 fate detennination in the retina. The alternative is that the AK11 would reduce

clone size, but that the proportion of rods, Müller and bipolar cells would be typical of cells

"born" on P4. This would indicate that pRB does not inhibit Müller or bipolar ce11 fate

determination in the retina. In the case of amacrine neurons, similar iri vivo injection

expenments would be performed in utero at E18, when a higher proportion of arnacrine ce11

progenitors are dividing (Cepko et al., 1996). The proportion of clones containing arnacrine

neuroris would then indicate whether pRB has a role in amacrine ce11 fate determination or

not.

One final explanation of the data presented that is not included in the models

discussed above is that progenitors infected with the MXIE-AKl1 retrovirus died by

apoptosis. More experiments need to be performed in order to determine if such was the

case. Thus, following the PO retroviral injection, infected retinas would be removed and

analyzed every few days by using an immunohistochemical approach for the marker EGFP

concomitantly with TUNEL analysis to identify apoptotic cells. If EGFP-labeled cells are

not labeled with the TUNEL approach, then this would confirm that the ratios of cells we

observed were obtained as a result of AK11 action in the retina, and were not due to early

death.

4.1.3 Can pRB Reprogram Post-natal Progenitors so that They Become Cone Photoreceptors?

While performing lineage analysis on the MXIE-AK1 1-infected eyes, 1 noticed that

for some clones the size of the photoreceptors' inner and outer segments was slightly

different, as shown in Figure 8. Analysis of the preceding and following eye sections

revealed that the differences noted were not due to the section plane. The photoreceptors in

question were compared against putative rod photoreceptors, whose outer segments extended

fûrther away from the outer limiting membrane, al1 the way to the Retinal Pigment

Epithelium (RPE) (Fig. 2). Closer examination of the photoreceptors in question revealed

that, beside shorter imer segments they also had ce11 bodies close to the outer limiting

membrane and pedicles located in the outer plexiform layer. This characterization fits the

rnorphological criteria that have been applied previously to identify cone photorecepton

(Chiu and Nathans, 1994). The observation was surprising because, at the time of retroviral

labeling (PO), cone photoreceptor progenitors have already exited the ce11 cycle (Fig. 3)

(Cepko et al., 1996). Previous experiments with retroviral labeling of post-natal progenitors

have never yielded clones containing cone photoreceptors (Cepko et al., 1996), indicating

Fig. 8: Does pRB re-specify cone photoreceptor ce11 fate in pst-natal retinal progenitors? A-D. Immunohistochemical studies with an anti-EGFP antiboày identifies photoreceptor clones in the rat retina (labeled cells are shown in r d ; DAPI staining, blue, identifies nuclei in the retina), in different planes of focus. B. Careful lineage analysis through al1 planes of focus led to the observation that there may be two types of photoreceptors (rods and cones?) labeled by pst-natal retroviluses carrying pRB. The schematic diagram simplifies our view of the photoreceptor clone. RPE = retinal pigment epithelium, ONL = outer nuclear layer, OPL =

outer plexi form layer.

that cone photoreceptor progenitors could not have been labeled by the MXIE-Ml1

retrovirus. Could it be then, that misexpression of pRB in post-natal retinal progenitors led

to the development of cone photoreceptors?

Before we consider the implications of this finding, we must first confirm whether my

observations of morphology are tmly indicative of a cone photoreceptor phenotype. This can

be done by performing double irnmunohistochemical staining expenments with anti-EGFP

and anti-cone photoreceptor markers antibodies. Should the two stains CO-localize, we would

have strong evidence that in the developing retina, pRB really does have a role in altering

ce11 fate and not just ce11 cycle. Such a finding would challenge current models on retinal

development. We know that post-mitotic neurons are produced from a pool of cycling

progenitors in an orderly fashion during development. It has been well established that this

process of neural cell-fate determination is regulated by a combination of extrinsic and

intrinsic influences. Recently, Livesey and Cepko (2001) proposed a competence model of

retinal development wherein progenitors pass through a series of competence states. Dunng

each state, the progenitors are competent to produce a subset of retinal ce11 types. The

available data indicate that competence states are intnnsically determined in progenitors at

the level of gene and protein expression, but, the production of a particular ce11 fate from a

ce11 that is within a competence state might be regulated to a large degree by extrinsic

signaling (Alexiades and Cepko, 1997; reviewed in Livesey and Cepko, 2001). Basically,

this model suggests that extrinsic cues can alter pre-programmed progenitor ce11 proportions;

however, once a progenitor has passed the competency state, it cannot be reprogrammed

along a different ce11 fate. Thus, if immunohistochemical staining expenments can confinn

that retrovirally-delivered pRB really is expressed in cone photoreceptors, this would

represent the fxrst ever example of a transcription factor reprogramming differentiation in

such a drarnatic way.

Therefore, preliminary in vivo misexpression studies in the rat retina have shown that

pRB is involved in ce11 cycle regulation during retinal development. An interesting question

that stems from these studies is whether pRB has an additional role in ce11 fate specification

in the developing retina. Further experiments need to be performed before we can answer

this.

4.2 Characterization of an In Viiro Retinal Explant Culture System

A complimentary approach to in vivo misexpression studies that we may use in order

to determine the role of pRB in retinal development is to perform in vitro rescue experiments

with RB knockout mice. Before proceeding with in vitro rescue expenments, we must (1)

establish proper culturing conditions for the retinal explants, and (2) charactenze the putative

developmental defects of the RE'' retinal explants. In order to satisfy the first condition, I

tested at least three different retinal explantation protocols. Only one of these protocols

yielded satisfactory results (Protocol 3, Table 2). With Protocol 3, tissue lamination and

development in vitro for CD-1 mouse retinas mimicked that in vivo as assayed by

imrnunohistochemical staining for six different markers. Having a reliable retinal culture

protocol offers an added advantage. Similarly to the in vivo post-natal experiments discussed

above, these retinal explants could be used to misexpress pRB in progenitors of ganglion,

horizontal and cone photoreceptor neurons, al1 of which are bom before the in vivo injections

at PO are perfonned. Such an experiment would greatly improve Our understanding of the

role played by pRB throughout embryonic and post-natal retinal development.

Once I established a reliable retinal explantation protocol, 1 proceeded to characterize

the growth in vitro of RB-/- retinas. However, 1 was unsuccessfûl in applying the

immwiohistochemicaI staining protocol that 1 had used previously with the CD-1 retinas to

the analysis of the RB-'- retinal explants. Specifically, in three different tries, for most of the

antibodies tested, I was unable to obtain any staining in either RB-'- or control RB"'

(C57BL/6 mouse strain) retinal explants. This means that the immunohistochemical staining

protocol that 1 had used previously may have to be re-optimized until positive staining is

obtained.

4.3 Viral Systems for Gene Delivery to the Retina

In these studies we investigated the use of a retroviral and a novel alphaviral system

for gene delivery to the retina. While both viruses infected post-natal retinas, they did not

infect embryonic retinas efficiently. This was an unexpected result, especially for the

retroviral gene delivery system. Retroviruses will typically infect mitotic c e k At E13,

more than 90% of retinal cells are dividing in the rodent eye (Alexiades and Cepko, 1996)

and hence the expected efficiency of infection should be a lot higher than observed. To

obtain a higher infection efficiency, it may be necessary to use more virus. As well, we

might have to add the retrovirus to the retinal explants constantly over the span of a few

hours (rather than only once, as was the case here). Another possibility is that retinal

progeniton may not have the appropriate receptors that will mediate successful retroviral

uptake. This problem could be solved by pseudotyping the retroviral envelope with the

Vesicular Stomatitis Virus G-protein (VSV-G), which has already been shown to have a very

wide range of host cell receptors (Chen et al., 1999). Not using enough SFV viral particles

may be the reason why the alphavirus was unsuccessfiil at infecting embryonic retinal

explants. Recently, David DiCiornrno has used SFV particles at a very hi& titer to obtain

efficient infection of mouse embryonic explants (David DiCiommo, personal

communication). SFV vectors provide short-term, transient expression of the gene of interest,

and that may not be appropriate for our lineage analysis studies which require long-term,

constitutive gene expression. Nevertheless, for some short term in vitro studies on post-natal

retinas, the SFV system may be the preferred approach for gene delivery to post-mitotic cells,

since SFV vectors infect cells at any point during their ce11 cycle. This alphaviral system

represents a novel finding not reported previously, and it may be a useful alternative gene

delivery system to the retina.

4.4 Summary

In this study 1 examined the effects of misexpressing pRB on the development of

post-natal rat retina in vivo. Lineage analysis using the constitutively hypophosphorylated

protein AK11 indicates that pRB drives mitotic progenitors out of the ce11 cycle. pRB may

also have a role in ce11 fate detemination or re-specification, but more expenments need to

be performed in order to confirm this. 1 have also laid the groundwork for pRB rescue

studies in RB'" retinal explants by devising a protocol wlierein RB+'+ retinal explants may be

grown in vitro for at l e s t 4 days. But before any rescue experiments are attempted, in vitro

RB" retinal explant development must be completely charactenzed. The results presented in

this study pave the way for exciting research opportunities and represent a signifiant

advance toward Our understanding of the human cancer, retinoblastoma.

CHAPTER 5: REFERENCES

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