Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors

Seminars in Cell & Developmental Biology 15 (2004) 63–74

Cell-intrinsic regulators of proliferation in vertebrate retinal progenitors Edward M. Levine∗ , Eric S. Green Departments of Ophthalmology & Visual Sciences and Neurobiology & Anatomy, Eccles Institute of Human Genetics, University of Utah, 15 North 2030 East, Salt Lake City, UT 84112, USA

Abstract The proliferative expansion of retinal progenitor cells (RPCs) is a fundamental mechanism of growth during vertebrate retinal development. Over the past couple of years, significant progress has been made in identifying genes expressed in RPCs that are essential for their proliferation, and the molecular mechanisms are beginning to be resolved. In this review, we highlight recent studies that have identified regulatory components of the RPC cell cycle machinery and implicate a set of homeobox genes as key regulators of proliferative expansion in the retina. © 2003 Elsevier Ltd. All rights reserved. Keywords: Chx10; Six6; Rx; Cyclin D1; p27Kip1

1. Introduction The vertebrate neural retina is a complex sensory tissue whose function depends on the correct formation of its laminar cytoarchitecture, which depends on the production of a sufficient number of cells of each cell type. All of the neural cell types (except astrocytes) arise from a common precursor, the retinal progenitor cell (RPC), and do so with two important characteristics: each cell type is generated during a limited period, and the number of cells that are born varies significantly across cell types. In rodents, the early born cell types (ganglion cells, cone photoreceptors, and horizontal cells) are relatively few in number, whereas middle and late born cell types (amacrine cells, rod photoreceptors, bipolar cells, and the Muller glia) make up the majority of cells in the retina. Because the interval in which the complete complement of retinal cells is produced overlaps with the interval in which RPCs exit the cell cycle and differentiate into mature retinal cells, RPCs have to balance two opposing forces: they must proliferate extensively and at an appropriate rate to produce a sufficient number of cells for generating late cell types, but must also stop proliferating to produce the cell types that are born early (Fig. 1). Over the past several years, there has been much effort to identify the molecules that coordinate exit from the cell cycle with the onset of differentiation. As part of this effort, ∗ Corresponding

author. Tel.: +1-801-587-9537; fax: +1-801-585-3501. E-mail addresses: [email protected] (E.M. Levine), [email protected] (E.S. Green). 1084-9521/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2003.09.001

several cell cycle proteins that participate in this process are now known (described below) and basic helix–loop–helix transcription factors have been implicated as coordinators of cell cycle exit and differentiation [1]. However, an equally important question in regard to the regulation of proliferation is what are the molecular mechanisms that drive the proliferative expansion of the retinal neuroepithelium? In this review, we describe recent results that identify genes with demonstrated functions in the G1 phase of the cell cycle as important targets of regulation for RPC proliferation and summarize observations that point to a set of homeobox genes expressed in RPCs as candidate regulators of proliferative expansion during retinal development.

2. The cell cycle: the fundamental cell-intrinsic mechanism of proliferation control Factors that regulate proliferation must ultimately exert their effects on the cell cycle, which is divided into four phases based on cellular activity. In S phase, the genome is replicated by DNA synthesis. In G2 phase, cells ensure that they have no DNA replication errors and prepare for mitosis. Mitotic cell division occurs in M. G1 phase is the primary interval for cell growth, but it is also the phase in which growth-promoting and -inhibiting signals have a direct impact on whether a progenitor cell will exit from or continue progressing through the cell cycle. The G1 phase is divided into two intervals based on a cell’s dependence on mitogen signals for sustained cell cycle

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Fig. 1. Cell number accumulation during mammalian retinal development. Over a limited period of time, there is a significant expansion of total retinal cell number that coincides with the generation of postmitotic differentiating cells. Shown are all retinal cells (black line), postmitotic cells (red line), and mitotic cells (green line) as a fraction of the final number of retinal cells produced. The accumulation of postmitotic cells is progressive, whereas mitotic cell number at any stage (RPCs) is dependent on the parameters of proliferation. The earliest generated cell types contribute only a small fraction of the total retinal cell number whereas the later generated cell types are much more abundant, which is correlated with proliferative expansion. Altering the parameters of proliferation (i.e. rate or mode of proliferation) can have profound impacts on retinal size and/or cell type distribution. (Data is for the rat retina and derived from [124].)

progression (Fig. 2A). In early G1, proliferation-competent cells require mitogenic signals to keep progressing through the cell cycle. Once a cell progresses past the restriction point (R) and into late G1, it is no longer dependent on

mitogens and is intrinsically committed to progress through M [2–4]. Molecules belonging to the fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), insulin-like growth factor (IGF), Hedgehog, mitogens

(A)

G0 (exit)

CycD

G1 rly Ea

R

G1 te La

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mitogen signal

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M G2

KIP

CycD:Cdk4/6 CycE

S

CycE:Cdk2

RB:E2F p

E2F + RB:E2F p

p

E2F + p RB p

S-phase entry (proliferation)

p

Fig. 2. The eukaryotic cell cycle and G1 progression. (A) The cell cycle consists of four phases. Passage through the R point in G1 phase commits the cell to another round of proliferation. Cells exit the cell cycle (G0) in G1 before R. (B) Model of G1 progression. Mitogens upregulate D-cyclins which activate Cdk4/6 resulting in hypophosphorylation (p) of retinoblastoma proteins (RB), partial activation of E2Fs as well as inhibition of KIP proteins such as Kip1. This ultimately leads to Cdk2 activation by increasing the expression of Cyclin E and by removing KIP inhibition on CycE:Cdk2. CycE:Cdk2 hyperphosphorylates RB causing complete inactivation of RB, release and activation of E2Fs, which results in a CycE positive feedback loop and ultimately transcriptional activation of genes necessary for S-phase.

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and Wnt families have been shown to act as mitogens for CNS progenitor cells including RPCs [5–8]. Although G1 progression is initiated by mitogen signaling, it is driven by cyclin-dependent kinase (CDK) activity (Fig. 2B). G1 CDKs require the binding of D- and E-cyclins for activation and are inhibited by the cyclin-dependent kinase inhibitor proteins (CDKI) such as the KIP proteins. Mitogens upregulate the expression of D-cyclins, resulting in two positive effects on G1 progression: (i) the activation of Cdk4 and/or Cdk6 (Cdk4/6), and (ii) the repression of KIP-mediated inhibition of CycE:Cdk2 complexes. Activated Cdk4/6 hypophosphorylates the retinoblastoma proteins (RB), resulting in two primary effects: (i) an increase in CycE expression by the partial release of E2F transcription factors from RB-mediated inhibition, and (ii) priming of the RB proteins for hyperphosphorylation by Cdk2. The combined increase in CycE and decrease in KIP inhibition activates Cdk2. Activated Cdk2 hyperphosphorylates the RB proteins, resulting in: (i) activation of a CycE positive feedback loop, and (ii) progression into S phase.

3. G1 phase proteins are essential for vertebrate retinal development The model described above and in Fig. 2B is generalized from studies done in diverse systems. Therefore, it must not be assumed that this is the mechanism employed by RPCs. However, this working model has been useful for studies done in the Drosophila eye [9–18] and vertebrate retina to identify G1 components important for retinal development (see below). 3.1. RB proteins RB proteins are thought to be central mediators of G1 phase progression, in that their phosphorylation state acts as a convergence point for proliferation and differentiation signals. In humans, mutations in the pRb gene cause retinoblastoma and other tumors [19]. pRb-null mice die between E12 and E15 [20–22], but chimeric mice containing pRb-null cells can be evaluated throughout development, and show ectopic mitoses and increased cell death in the retina at E16.5 [23]. These chimeric mice do not have retinal tumors, but chimeras with a double knockout of pRb and p107 (another RB protein) develop severe retinal dysplasia at E17.5 [24]. These and other studies strongly suggest that tumor formation associated with Rb inactivation is sensitive to secondary genetic mutations, modifiers, and compensation [25,26]. From a developmental perspective, however, these data indicate both that RB proteins have a critical growth inhibitory role during the mouse RPC proliferative period, and that the exact utilization of these family members differs between mice and humans. What is still not clear is whether the RB proteins are required for RPC proliferation, or if their primary function

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is in ensuring that differentiated cells properly exit the cell cycle. 3.2. E2Fs The E2F family of proteins in mammals is divided into three subclasses [27]. One of these consists of three proteins, E2F1–3, which act as transcriptional activators and are essential for the transition from G1 phase to S phase in many proliferating cells. Over 100 transcriptional targets of these factors have been identified in a large variety of cells, including prototypical S-phase genes like subunits of DNA polymerase-alpha. The other two subclasses (E2F4/5 and E2F6) are normally repressors, and often act to facilitate cell cycle exit. Mouse embryonic fibroblasts (MEFs) that have the combined inactivation of E2F1–3 fail to enter S-phase, in contrast to MEFs with inactivations of one or two E2Fs [28]. This experiment highlights the essential but redundant role of these E2Fs in G1 to S phase transition. In the CNS, mice lacking E2F1 show reduced neurogenesis, but this is most pronounced in the postnatal period, when E2F1 is the primary E2F expressed [29]. In the retina, E2F1 and E2F2 are expressed in the neuroblast layer [30], and evidence for the role of E2Fs in RPC proliferation comes from studies in the quail, where E2F1 is regulated by its interaction with pRb, in a manner consistent with the model in Fig. 2B [31,32]. Also, ectopic expression of E2F1 driven by the IRBP promoter in transgenic mice promotes the proliferation of differentiating photoreceptors, which suggests that E2F1 activity must be downregulated at the onset of differentiation to ensure cell cycle exit [33]. E2F4 and E2F5 are expressed in the neuroblast layer, but their role in RPC proliferation has not been reported [30]. 3.3. D-cyclins Cyclin D1 (CycD1) is the predominant D-type cyclin expressed in RPCs and newborn CycD1-null mice have hypocellular retinas due to reduced proliferation ([34–36] and unpublished observations). Analyses of mutant mouse strains, including a knock-in of CycE into the CycD1 locus [37], and the CycD1,p27Kip1 -double null mouse [38,39], demonstrate that CycD1 is genetically upstream of CycE and p27Kip1 (Kip1) function. Furthermore, CycD1 mRNA and protein levels are downregulated in the hypocellular Chx10-null retina, and in individual cells, the loss of CycD1 correlates with an increase in the levels of Kip1 [40] (see below). It therefore seems likely that CycD1 promotes G1 phase progression both by activation of Cdk4, and by preventing Kip1 accumulation in RPCs. The mechanism by which CycD1 accomplishes this latter task is not known. It could be a consequence of Cdk4 activation, or it could depend on the ability of CycD1 to sequester Kip1 away from Cdk2, which may facilitate Kip1 degradation.

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Interestingly, proliferation still occurs in the CycD1-null retina and this could be due to the persistent expression of CycD3 or to a D-cyclin-independent mechanism. It appears that both may be responsible, since the combined inactivation of CycD1 and CycD3 further reduces RPC proliferation, but not completely [41]. What proliferation remains appears to be D-cyclin independent since CycD2 is not expressed. This suggests that other mechanisms may exist to support at least a basal level of G1 progression in RPCs. 3.4. Kip1 Through its ability to inhibit Cdk2, Kip1 has two potential functions in tissue development. First, it could modulate the rate of the cell cycle of progenitors [42]. Second, it could be part of the program to ensure that RPCs exit the cell cycle at the onset of differentiation. Kip1-null mice exhibit organomegaly and focal retinal dysplasia, which could be consistent with either function [43–45]. Kip1 accumulates progressively in mouse CNS oligodendrocyte precursors during successive cell cycles, and at a threshold expression level, forces these cells out of the cell cycle [46]. The Xenopus Kip1 homologue p27Xic1 is expressed in RPCs and cell cycle exit appears to depend on a similar mechanism [47]. Kip1-null mice show BrdU incorporation well beyond the normal end of the retinal proliferative period [48], which is consistent with a requirement for Kip1 in RPC cell cycle exit. However, Kip1 protein is not expressed at detectable levels in mouse RPCs. Rather, Kip1 protein may be upregulated in the last cell cycle before exit [49] and is abundantly expressed in postmitotic, differentiating retinal neurons [40,48,49]. Although the Kip1 function in cell cycle exit appears to be conserved, its regulation during retinal development appears to vary across vertebrates. Interestingly, p27Xic1 is both necessary and sufficient for the promotion of the Muller glial fate choice in the Xenopus retina and this function is independent of its inhibition of CDK activity [47]. In contrast, modulating Kip1 levels in the mouse retina does not affect glial fate, but its inactivation is associated with Muller glia reactivity and cell cycle re-entry [48–50]. Since Kip1 is expressed in mature Muller glia, this may reflect a cell autonomous role for Kip1 in reactivity, but this has not yet been addressed. 3.5. p57Kip2 (Kip2) Kip2 is expressed by a subset of embryonic RPCs as they exit the cell cycle [49,51]. Within this population, Kip2 is both necessary and sufficient for appropriate cell cycle exit, as demonstrated by the knockout phenotype and overexpression experiments. Kip2 is not expressed in exiting RPCs during the postnatal period. However, starting at postnatal day 2 (P2), it is expressed in a subset of amacrine cells, where it may participate in their maturation [51].

3.6. p19Ink4d (Ink4d) Another class of CDKI proteins, in addition to KIP, is the INK4 protein family. INK4 proteins bind and inhibit Cdk4 and Cdk6. In general, the expression of the family member Ink4d in the developing CNS is similar to that of Kip1 [52]. In the retina though, where Kip1 protein is present in postmitotic cells, Ink4d is present in RPCs. However, in a subset of cells, Ink4d and Kip1 are co-expressed in a pattern correlated with RPCs undergoing cell cycle exit [53]. Like Kip1-null RPCs, Ink4d-null RPCs proliferate beyond the normal period. However, the Ink4d phenotype is less severe than that of Kip1. Interestingly, there is a synergistic increase in ectopic proliferation in the Ink4d,Kip1-double null retina compared to the sum of the ectopic amounts seen in the single null retinas [53]. Furthermore, retinal neurons either re-enter or remain in the cell cycle in the Ink4d,Kip1-double null retina, suggesting that cell cycle exit is actively regulated for some time after the onset of differentiation. These observations demonstrate that Ink4d is a negative regulator of RPC proliferation and cooperates with Kip1 to ensure timely cell cycle exit. Interestingly, more horizontal cells are seen in the Ink4d,Kip1-double null retina suggesting that these proteins may cooperate to prevent overproduction of this cell type [53]. Are Ink4d,Kip1-double null retinas larger than wild type? Although cell number quantification and volumetrics have not been done, these retinas have up to five times as many apoptotic cells at late stages of retinal development as wild type [53]. Cell death is only partially alleviated by genetic inactivation of the apoptosis-inducing protein p53, suggesting that multiple cell death pathways are activated by the defects in Ink4d,Kip1-double null retinas [53]. It could be that upon co-deletion of these two CDKI genes, there is an extensive activation of cell death pathways arising from a major uncoupling of the cell cycle and differentiation mechanisms.

4. Homeobox genes as regulators of proliferation in the developing retina The rates and extent of proliferation vary in different regions of the neuroepithelium, and this is nowhere more apparent than in the optic vesicle. The domains from which the neural retina and retinal pigmented epithelium (RPE) arise are similar in cell number at the onset, but by the end of development their cell numbers vary by orders of magnitude, due primarily to differences in proliferation. Although differences may exist in the cell cycle machinery utilized by RPE progenitors and RPCs, it is unlikely that this alone can explain the differences in proliferation, especially since some of the same cell cycle proteins are expressed in both tissues [54] (unpublished observations). How then is proliferation regulated in specific regions of the CNS such as the retina? One possibility is that there

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Fig. 3. Expression of homeobox genes implicated in regulating RPC proliferation. Generalized expression patterns of the Rx, Pax6, Six3, Six6/Optx2, Chx10, and Prox1 genes during optic vesicle patterning and early retinal development. All of the genes except for Chx10 and Prox1 are expressed broadly at first, and then become restricted to the RPC population during the period of RPC proliferative expansion. Another exception is Pax6, which is expressed in RPE progenitors. Within RPCs, Six6/Optx2 is only expressed during the early stages of retinal development, and Prox1 is expressed in a subset of RPCs.

are region- or tissue-specific regulators of proliferation that interact with the cell cycle machinery and/or with mitogen pathways that drive G1 progression. Recent studies suggest that the transcription factors BF-1, Pax6, Emx2, and Otx1 may fulfill this role in specific domains of the rostral CNS [55–61]. In the retina, several homeobox genes that are expressed in RPCs (Fig. 3) may also serve as important regulators of proliferation by controlling cell number expansion on several levels, from setting the correct rate of proliferation to promoting cell cycle exit. For the remainder of this section, we describe what is currently known about the regulation of RPC proliferation by six homeobox genes: Rx/Rax, Pax6, Six3, Optx2/Six6, Chx10/Vsx2/Alx1, and Prox1. 4.1. Rx/Rax The Rx genes are paired-like homeobox genes and at least two genes have been found in all vertebrate species except for mouse, which only has one known Rx gene [62–68]. The expression of individual Rx genes is variable but the common theme is that at least one Rx gene per species is expressed in the ventral diencephalon and is among the earliest genes expressed in the presumptive eye field. Expression persists through retinal development in RPCs and is downregulated in differentiated cells, with the exception of expression in photoreceptors and a subset of cells in the inner nuclear layer. The function of the Rx genes has been analyzed extensively by inactivation and overexpression approaches. Overexpression of Rx in the anterior neural tube leads to neural tube hyperplasia, a dramatic expansion of retinal tissue and in some cases, formation of ectopic retina [62,69,70]. Conversely, genetic inactivation of Rx in mice [62,71] and

medaka [72,73] causes a failure of eye development prior to the completion of optic vesicle formation. Because of the early and widespread expression of Rx genes, these experiments likely reflect the importance of Rx in the specification of the eye field in the anterior neural tube rather than demonstrating a direct and exclusive role in regulating RPC proliferation. However, in the eyeless mutation of medaka, which is a temperature-sensitive mutation of Rx3, Vsx2 (a Chx10 orthologue) expression is absent, suggesting a possible genetic hierarchy between Rx and Chx10 genes [72,73]. In Xenopus embryos injected with Xrx1 mRNA, CycD1 expression was significantly increased in the retina compared to controls [74]. Further evidence of a direct proliferative effect was demonstrated by increased clone size of RPCs that were transfected with Xrx1 [74]. This effect was similar to that observed in transfections of CycA/Cdk2 with one important difference; the cell type distribution in Xrx1 transfected clones matched those in control clones whereas the CycA/Cdk2 transfected clones had a distribution biased towards later generated cell types. These results suggest that in addition to stimulating proliferation, Xrx1 may coordinate the timing of cell-type generation with proliferation, whereas CycA/Cdk2 simply delays cell cycle exit to a later stage of retinal development. What remains unknown for the function of Rx is whether it is required to regulate the RPC proliferation rate and if so, if Rx is a direct regulator of cell cycle control. 4.2. Pax6 The majority of studies of Pax6 in retinal development have focused on its roles in eye field specification and proximodistal patterning of the optic vesicle. Since Pax6 loss of function phenotypes affect these early processes as well

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as lens development [75], identifying potential functions of Pax6 in RPCs has been difficult. However, Marquardt et al. [76] elegantly dealt with this problem by producing a retinal specific knockout in which Pax6 was inactivated (Pax6-floxed) after optic vesicle patterning was completed. In the regions of the retina where Pax6 was inactivated, the retina was hypocellular and this was correlated with fewer BrdU labeled cells. The expression of Rx, Six3, Six6, and Hes1 in Pax6-floxed RPCs were unaffected suggesting that the specific function of Pax6 in RPC proliferation is downstream or independent of these homeobox genes. Although there is currently no evidence for Pax6 being a direct regulator of the cell cycle machinery, its importance in proliferation is further supported by the identification of the putative cytoplasmic calcium binding protein, necab, as a direct transcriptional target whose overexpression induces Chx10 expression [77] (see below). Furthermore Pax6-null cortical progenitors and radial glia have multiple cell cycle defects [56,60,78] and BrdU labeling indices are altered in the Pax6-null diencephalon [79]. 4.3. Six3 Six3 was cloned based on sequence conservation to the Drosophila sine oculis gene, but is more similar in sequence to Drosophila Optix. Six3, like Rx, is initially expressed in the anterior neuroectoderm, and later in retinal development its expression is restricted to RPCs [80] and references therein; see also [81,82]. The majority of Six3 functional analyses with respect to retinal development have been done using overexpression strategies in several vertebrate models including medaka [83], zebrafish [84], Xenopus [85], and mouse [86]. In all of these models, Six3 overexpression induced ectopic optic vesicles in the region of the neural tube that gives rise to the midbrain and hindbrain, suggesting an important and early role in optic vesicle induction. Six3 overexpression in medaka also caused an expansion of the normal optic vesicle in a manner suggestive of Six3 having a role in promoting proliferation of the early optic neuroepithelium. Six3 overexpression in zebrafish expands the rostral forebrain, suggesting that this region of the neuroepithelium is sensitive to the ability of Six3 to promote proliferation. Recently, Wittbrodt and colleagues [87] addressed the functional requirements of Six3 by morpholino knock-down experiments in medaka and observed a general loss of forebrain structures including the retina due to apoptosis when the morpholino was used at high concentrations. At lower morpholino concentrations, however, the forebrain defects were minimized and the most significant defects observed were in the proximo-distal patterning of the optic vesicle. The range of phenotypes observed in these experiments is remarkably similar to the range in defects associated with Six3 mutations in humans [88]. Because of the prevalence of anterior neuroectoderm and patterning defects associated with modulation of Six3 function, the specific requirements of Six3 in RPC proliferation are still not resolved.

4.4. Six6/Optx2 Six6 and its Xenopus orthologue Optx2, like Six3, are closely related in sequence structure to Drosophila Optix. In contrast to Six3, Six6 is expressed in a more restricted manner and is detected in the ventral diencephalon, optic stalk, and retina, and its expression initiates after Rx, Pax6 and Six3 [82,89–93]. Optx2 overexpression in Xenopus is sufficient to induce ectopic retinal tissue and expand the normal optic vesicle and rostral neuroepithelium in a manner similar to Six3 [85,92]. Because of the structural and biochemical similarities between Six3 and Six6/Optx2 [94–96], overexpressing Six6/Optx2 prior to its normal expression may simply be a phenocopy of Six3 overexpression. To more directly address whether Optx2 can promote RPC proliferation, Harris and colleagues [92] determined that the clone sizes of RPCs directly transfected with Optx2 were larger than RPCs transfected with control constructs. Furthermore, embryos injected with Optx2 mRNA and treated with the S-phase inhibitor hydroxyurea failed to undergo retinal expansion, which suggests that proliferation, not patterning, is responsible for the Optx2-induced expansion. Inactivation of Six6 in mouse caused retinal hypoplasia that was correlated with reduced BrdU labeling indices and premature cell cycle exit [97]. The proliferation defect was detectable only at early stages of retinal development (prior to E15.5), which is consistent with the Six6 expression pattern: Six6 is expressed in RPCs early in retinal development and is downregulated in most RPCs by E15.5. While the data described above point to an important role of Six6/Optx2 in regulating RPC proliferation, they do not provide evidence that Six6/Optx2 directly regulates the cell cycle. To address this, Rosenfeld and colleagues [97] examined the transcriptional properties of the mouse Six6 protein. They found that Six6 directly interacts with the co-repressor proteins Dach1 and Dach2, and that Six6:Dach complexes function as potent transcriptional repressors in heterologous reporter assays. Interestingly, they also found that Six6 binds to the Kip1 promoter in retinal cells and that several co-repressors including Dach2 were associated with this binding, suggesting that Six6 may be a direct repressor of Kip1 transcription in RPCs. Consistent with this, Kip1 mRNA and protein is upregulated in the Six6-null retina, providing compelling evidence for a direct molecular interaction between mouse Six6 and the cell cycle. 4.5. Chx10/Vsx2/Alx1 Chx10 and its vertebrate orthologues, Vsx2 and Alx1, are members of the paired-like homeodomain/CVC domain class of homeobox genes [98]. Chx10 is among the most specific markers of vertebrate RPCs [99–106]. Chx10 expression begins during optic cup formation and its ocular expression pattern is restricted to the RPCs of the neural retina and adjacent ciliary margin. It is expressed in all RPCs throughout retinal development. Chx10 is not expressed in

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postmitotic retinal cells with the exception of a subset of bipolar interneurons. In contrast to the genes described above, the majority of studies addressing Chx10 function have been done in a loss of function context. The reason for this is that natural mouse mutations of Chx10 have been present for several decades and were originally identified as fully penetrant, autosomal recessive mutations named ocular retardation [107,108]. McInnes and colleagues [109] identified the mutation in ocular retardation as a premature stop codon in the Chx10 homeodomain, resulting in a null. Two human families with congenital microphthalmia have null mutations in Chx10 [110] and in zebrafish, inactivation of Alx1 by antisense RNA injection caused a failure of embryonic eye formation [103]. The phenotypic similarities across species suggest that the function of Chx10 is conserved through vertebrate evolution. In ocular retardation mice (referred to previously and hereafter as Chx10-null), the initial patterning of the optic vesicle patterning appears normal, but the neural retina is hypoplastic by E13 and there is a severe reduction in retinal cell number by P0 [40,109]. Increased cell death was excluded as a cause of this phenotype [111,112]. However, tritiated thymidine and BrdU labeling indices revealed a profound defect in RPC proliferation [109,113,114]. Mitotic index measurements in E10 retinas showed that Chx10-null RPCs have a longer cell cycle time than wild type RPCs and this increase is correlated with a longer G1 phase [113]. We have analyzed the Chx10-null retinal phenotype at P0 and found similar changes: a lengthening of the cell cycle rate and an aberrant accumulation of cells in G1 (unpublished observations). We also analyzed the ratios of RPCs and differentiated cells and found that although total cell number is significantly lower, the ratios of progenitors and differentiated cells are the same as in wild type [40]. These observations suggest that Chx10 inactivation does not cause premature cell cycle exit with respect to the age of the embryo, although Chx10-null RPCs probably do exit after fewer cell cycles. More importantly, Chx10 inactivation does not deplete the RPC population and does not diminish the multipotential character of RPCs. This is in sharp contrast to the Hes1-null retinal phenotype in which hypocellularity is accompanied by accelerated differentiation of RPCs (i.e. premature cell cycle exit) [115] and to the Pax6-floxed retinal phenotype in which reduced proliferation is accompanied by the inability of RPCs to generate multiple cell types (i.e. loss of multipotentiality) [76]. Rather, the primary defect in Chx10-null RPCs appears to be due, specifically, to a change in the rate of proliferation. Is Chx10 then, like Six6, a direct regulator of the cell cycle? To begin to address this question, we recently examined the genetic relationship between Chx10 and Kip1 [40]. We found that Kip1 protein is expressed in significantly more cells in the Chx10-null retina than in wild type. To determine if the excess Kip1 expression was contributing to the Chx10-null retinal phenotype, we generated

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Chx10,Kip1-double-null mice. Inactivation of Kip1 caused a significant improvement in retinal cell number, the cytoarchitectural defects of the retina were greatly diminished and the severity of the microphthalmic phenotype due to Chx10 inactivation was alleviated (Fig. 4). The ratios of the RPC, differentiated cell, and apoptotic cell populations at P0 were unchanged in the double null compared to the Chx10-null retina (and wild type) suggesting that the restoration was not due to compensatory changes in cell death or in the timing of cell cycle exit, but rather to a correction in the rate of RPC proliferation. Interestingly, Chx10 is also required for bipolar cell development, and inactivation of Kip1 did not rescue the bipolar cell population, strongly suggesting that Chx10 utilizes distinct molecular mechanisms in regulating RPC proliferation and bipolar cell differentiation. The antagonistic interaction between Chx10 and Kip1 suggests that Chx10, like Six6, may function as a direct transcriptional repressor of the Kip1 gene. We found, however, that although the Chx10 and Kip1 proteins are expressed in distinct cell populations, RPCs and postmitotic cells respectively, the mRNAs for both genes are expressed in the same cell population: the RPCs (Fig. 5). Furthermore, Kip1 mRNA levels are unchanged in the Chx10-null retina compared to wild type. These results rule out direct transcriptional repression of Kip1 by Chx10 as an important regulatory mechanism and suggest that Kip1 is regulated post-transcriptionally. Interestingly, we found that CycD1 is a candidate mediator of the interaction between Chx10 and Kip1; CycD1 is required to prevent Chx10 and Kip1 proteins from being co-expressed in RPCs (Fig. 5), and CycD1 mRNA and protein expression are reduced in the Chx10-null retina [40]. It remains to be determined, however, if Chx10 is a direct transcriptional regulator of CycD1. 4.6. Prox1 Prox1 is the vertebrate orthologue of prospero, which has an important role in asymmetric cell division [116] and in cell cycle exit [117] in Drosophila. It has been known for some time that Prox1 is expressed in RPCs and horizontal cells [104,118–120], but its function in retinal development, and specifically in RPC proliferation has only recently been examined. Oliver and colleagues [121] observed that a restricted subset of embryonic RPCs express Prox1 with kinetics that suggest that Prox1 expression oscillates with cell cycle progression. Prox1-null explants cultured for 10 days had more cells than wild type explants as well as a shift in the ratios of differentiated cells towards later cell types, suggesting that Prox1 is required for cell cycle exit in a subset of embryonic RPCs. Interestingly, horizontal cells are not observed in the Prox1-null retina, and clonal overexpression of Prox1 results in an excess of one cell clones with a significant increase in horizontal cells than control virus. It remains to be determined if this reflects Prox1 function in promoting cell cycle exit, horizontal cell genesis, or both.

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Fig. 4. Genetic inactivation of Kip1 in the Chx10-null retina restores cell number. (A–D) The different sizes of the mature retinas from each strain are readily apparent. (E–H) Radial cross sections through the retinas shown in A–D, showing that the Chx10,Kip1-double null retina has near normal lamination and many more cells than the Chx10-null retina; from [40].

5. Concluding remarks and future directions The factors used by RPCs to regulate proliferation during retinal development are beginning to be identified. However,

we still lack a basic understanding of how proliferative expansion is regulated during retinal development. In this review, we have described the genes and proteins in the cell cycle that are likely to be key downstream effector molecules

Fig. 5. CycD1 mediates the interaction between Chx10 and p27Kip1. Summary of expression profiles of Chx10, CycD1, and Kip1 in RPCs from wild type, Chx10-null, and CycD1-null retinas. In wild type RPCs, Chx10 and CycD1 are expressed, but Kip1 protein is not (although the mRNA is present). As RPCs exit the cell cycle, Chx10 and CycD1 are downregulated and Kip1 protein is upregulated. In mature cells, none of these genes are expressed with the noted exceptions. In Chx10-null RPCs, CycD1 is expressed but in fewer cells. In Chx10-null RPCs that express CycD1, Kip1 protein is not expressed. Kip1 is expressed in cells that do not express CycD1. It is unclear if these two populations are spatially or temporally distinct. In CycD1-null RPCs, Kip1 is co-expressed in most RPCs that express Chx10 (see [40] for more details).

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important for proliferative expansion of RPCs, and we have described findings that suggest that a set of homeobox genes expressed in RPCs are the upstream factors that directly or indirectly regulate their expression and activity. We do not suggest that these are the only factors, but rather that they are good candidates given their effects on retinal development when overexpressed or inactivated. Our understanding of how the cell cycle is regulated in RPCs has improved significantly with the identification and functional analysis of several components of the cell cycle machinery. One of the more interesting findings is that several of these genes also have functions in cell fate and differentiation. It is important to note, however, that the actual cell cycle mechanism in RPCs, especially with respect to G1 progression, is still not sufficiently resolved. For example, the possibility that the D-cyclins may not be absolutely required for RPC proliferation suggests that our current model of G1 progression is probably not complete. Furthermore since parameters like the period of histogenesis, the rate of RPC proliferation, the timing of cell cycle exit, and the total retinal cell number output vary across species, the study of retinal development should continue to include identification of cell cycle proteins (both positive and negative regulators), especially those that are rate-limiting for cell cycle progression. It will be important to develop a more accurate picture of how G1 progression occurs in RPCs, especially with respect to input from regulatory pathways that impinge on the cell cycle, since this will contribute to our understanding of how proliferative expansion is regulated in the retina. With respect to homeobox genes, although the data is strong that the genes described here are good candidates for regulating RPC proliferation, there is a complicating issue that needs to be considered for Rx, Pax6, Six3, and Six6. These genes are all expressed prior to the period of RPC proliferative expansion, and have demonstrated roles in eye field specification and/or optic vesicle patterning. Because alterations in these processes can profoundly affect the number of RPCs independently of RPC-specific proliferation, experiments must be designed that account for, or better yet, bypass functions in field specification and patterning. This has been accomplished in some studies and these approaches should be exploited further. There are several open questions that if addressed, will greatly increase our understanding of how proliferation is regulated in RPCs. For example, how direct is the regulation of proliferation by these homeobox genes? Is there a hierarchy of homeobox genes that converges onto one or two critical genes, such as Chx10, that is analogous to that proposed for eye field specification [122,123]? Or does each homeobox gene contribute independently to ensuring the appropriate rate and extent of proliferation? For those genes with direct roles, do they regulate the cell cycle machinery, mitogen signal transduction pathways, or do they regulate proliferation at both of these levels? To begin to address these questions, it will be important to determine exactly how proliferation is altered in mutants and in overexpres-

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sion paradigms. For example, quantitative measurements of histogenesis and cell cycle kinetics will provide much more detail into alterations of proliferation than can be achieved with simple single pulse BrdU labeling measurements. Second, identifying direct downstream transcriptional targets of these homeobox genes will provide direct insight into molecular mechanisms. From this information, we should be able to assemble a comprehensive picture of the regulation of RPC proliferation that encompasses molecular pathways from mitogen to cell cycle and cellular pathways from RPCs to retinal neuron and glia. Acknowledgements The authors’ work has been supported by a Career Development Award from Research to Prevent Blindness (E.M.L.), the Foundation Fighting Blindness, the Knights-Templar Eye Foundation, and the National Institutes of Health (NEI R01-EY013760 (E.M.L.) and NEI F32-EY13922 (E.S.G.)). The authors also thank Richard Dorsky, Sabine Fuhrmann, and Monica Vetter for reading and providing insightful critiques during the preparation of this manuscript. References [1] Ohnuma S, Philpott A, Harris WA. Cell cycle and cell fate in the nervous system. Curr Opin Neurobiol 2001;11:66–73. [2] Pardee AB. A restriction point for control of normal animal cell proliferation. Proc Natl Acad Sci USA 1974;71:1286–90. [3] Pardee AB. G1 events and regulation of cell proliferation. Science 1989;246:603–8. [4] Planas-Silva MD, Weinberg RA. The restriction point and control of cell proliferation. Curr Opin Cell Biol 1997;9:768–72. [5] Reh TA, Levine EM. Multipotential stem cells and progenitors in the vertebrate retina. J Neurobiol 1998;36:206–20. [6] Cameron HA, Hazel TG, McKay RD. Regulation of neurogenesis by growth factors and neurotransmitters. J Neurobiol 1998;36:287– 306. [7] Levine EM, Fuhrmann S, Reh TA. Soluble factors and the development of rod photoreceptors. Cell Mol Life Sci 2000;57:224–34. [8] Megason SG, McMahon AP. A mitogen gradient of dorsal midline Wnts organizes growth in the CNS. Development 2002;129:2087– 98. [9] Knoblich JA, Sauer K, Jones L, Richardson H, Saint R, Lehner CF. Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 1994;77:107–20. [10] Lane ME, Sauer K, Wallace K, Jan YN, Lehner CF, Vaessin H. Dacapo, a cyclin-dependent kinase inhibitor, stops cell proliferation during Drosophila development. Cell 1996;87:1225–35. [11] Brook A, Xie JE, Du W, Dyson N. Requirements for dE2F function in proliferating cells and in post-mitotic differentiating cells. EMBO J 1996;15:3676–83. [12] de Nooij JC, Letendre MA, Hariharan IK. A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis. Cell 1996;87:1237–47. [13] Du W, Vidal M, Xie JE, Dyson N. RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila. Genes Dev 1996;10:1206–18.

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