Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

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The decision to exit the cell cycle during retinal development must be carefully coordinated with intrinsic changes in retinal progenitor competence to ensure that the correct proportion of each cell type is generated. The Rb family of proteins—Rb, p107, and p130—are at the heart of the cell cycle machinery that executes the decision to exit the cell cycle in retinal progenitor cells (RPCs). The individual Rb family members are expressed in a dynamic pattern during retinogenesis, and genetic studies have revealed that intrinsic genetic compensation and redundancy helps to prevent deregulated proliferation when individual family members are absent. Interestingly, the expression of the Rb family during retinal development and their compensatory mechanisms are different in mouse and human retinas. These differences are important because they provide an explanation for the unique susceptibility of humans to retinoblastoma following RB1 gene inactivation. Beyond its role in RPC proliferation, Rb is also required for rod photoreceptor development in mice. This role is unique to Rb and illustrates how a single protein can contribute to the coordination of cell cycle exit and cell fate specification in the developing retina. More recent studies have extended these data by using mice with mosaic inactivation of Rb in their developing retina to study synaptogenesis between rods, bipolar cells, and horizontal cells. These examples highlight how studying tumor suppressor genes in the developing retina can contribute to our understanding of RPC proliferation, retinoblastoma formation, neuronal cell fate specification, and synaptogenesis.

The regulation of proliferation during development is often associated with the regulation of tissue size. In the developing retina, defects in proliferation can result in microphthalmia, retinal degeneration, and partial or complete loss of vision (Burmeister et al., 1996; Ma et al., 1998). Alternatively, ectopic proliferation during retinal development can lead to retinoblastoma, which is fatal if left untreated and often results in compromised vision when treatment is successful (Dyer, 2004; Dyer et al., 2005; Dyer and Harbour, 2006). Beyond the regulation of tissue size, the decision to exit the cell cycle must also be precisely coordinated with intrinsic changes in RPC competence to ensure that each neuronal cell type is generated in the correct

proportion (Dyer and Cepko, 2001a). Because retinal neurons are believed to process visual information as functional clusters (Jeon et al., 1998), generation of the correct proportion of each neuronal cell type is critical for normal visual signal processing. Precisely how the decision to exit the cell cycle is coordinated with the changing competence of RPCs remains a significant challenge in the field of retinal development today (Donovan and Dyer, 2005).

Efforts to better understand the regulation of RPC proliferation during development have benefited from advances in our understanding of the regulation of changes in RPC competence (Cepko et al., 1996) and of the proteins that regulate cell cycle progression (Dyer and Cepko, 2001c). Over the past decade, several laboratories have focused on the intrinsic factors that regulate RPC proliferation during development. These studies have focused on the cell cycle proteins themselves, including cyclin D1, p27, p57, Rb, p107, and p130 (Ma et al., 1998; Dyer and Cepko, 2000a, 2000b, 2001a, 2001b, 2001c; Donovan and Dyer, 2004; Zhang et al., 2004a, 2004b; Donovan et al., 2006; Johnson et al., 2006; Laurie et al., 2006; Sun et al., 2006), and the transcription factors that regulate proliferation in RPCs, such as Prox1, Chx10, and Six3 (Zhu et al., 2002; Dyer, 2003; Dyer et al., 2003). In addition to intrinsic regulators of RPC proliferation, extrinsic factors such as sonic hedgehog (Dakubo et al., 2003) and glutamate (Martins et al., 2006) provide mitogenic cues in the developing retina. Therefore, both proliferation and cell fate specification are regulated by carefully orchestrated signaling cascades between extrinsic and intrinsic factors. However, unlike the aforementioned studies on RPC competence, less is known about how signals from growth factors are balanced by the intrinsic ability of RPCs to respond to those cues. For example, how are postmitotic cells kept from reentering the cell cycle? Newly born cells are often adjacent to proliferating RPCs, and it is not known why they no longer respond to the surrounding growth factors that drive their neighboring RPCs to proliferate. As a first step toward improving our understanding of these issues, we must understand better how each of the intrinsic factors regulates RPC proliferation, and then explore how the intrinsic programs are coordinated with extrinsic signals.

There are three major reasons for studying the coordination of cell cycle exit with cell fate specification. First, we hope to learn how the ratios of different cell types are generated during retinogenesis from multipotent RPCs. A better understanding of this process is needed to develop cell-based therapies for retinal degeneration involving retinal stem cells (Tropepe et al., 2000; Coles et al., 2004) or RPCs (MacLaren et al., 2006). Second, a thorough understanding of intrinsic genetic compensation and redundancy has facilitated the development of the first knockout mouse models of retinoblastoma (Chen et al., 2004; MacPherson et al., 2004; Zhang et al., 2004b). These and other preclinical models have been used to test new therapies for the treatment of retinoblastoma in clinical trials (Laurie et al., 2005, 2006). Third, studies on cell cycle proteins have led to some unexpected insights into processes not traditionally associated with proliferation control, such as photoreceptor development and synaptogenesis (Johnson et al., 2006). In the absence of Rb, rod photoreceptors fail to form, and this has allowed researchers to study the response of their synaptic partners (bipolar cells and horizontal cells) in the absence of rod inputs. These different lines of inquiry demonstrate how

genetic, molecular, cellular, and neuroanatomical studies of tumor suppressor knockout mice can contribute to our understanding of retinal development and disease.

Intrinsic genetic compensation and redundancy

For most researchers using mouse genetics to study retinal development and disease, separating the cell-autonomous and non-cell-autonomous roles of a given gene is of fundamental importance for interpreting the retinal phenotype. Once the cell-autonomous and non-cell-autonomous effects of genetic changes have been elucidated, intrinsic genetic compensation and redundancy must also be addressed (figure 25.1). This is of particular importance for the cell cycle genes in the developing retina, because most genetic changes in this pathway lead to some form of compensation or there is redundant gene expression (discussed in Dyer and Cepko, 2001c). Intrinsic genetic redundancy occurs in a cell that expresses two or more gene family members that have overlapping functions. As a result, inactivation of more than one gene is required to see changes in proliferation, apoptosis, or differentiation. In this example, the different gene

Figure 25.1 Intrinsic genetic redundancy and compensation among the Rb family of proteins. A, The Rb family members (Rb, p107, and p130) are expressed in a dynamic pattern during mouse retinal development. Shown is a summary of expression data from real-time RT-PCR, in situ hybridization, immunofluorescence, and immunoblotting: p107 is expressed at high levels during embryonic retinal development in the proliferating retinal progenitor cells (RPCs). Rb is expressed in postnatal RPCs and newly postmitotic neurons and glia. In the adult retina, virtually all cell types express Rb, and p130 is expressed in the inner nuclear layer

and ganglion cell layer redundantly. B, In the absence of p107, Rb is upregulated in embryonic RPCs to prevent deregulated proliferation and retinoblastoma. C, In the absence of Rb, p107 is upregulated in postnatal RPCs to prevent deregulated proliferation and retinoblastoma. D, Intrinsic genetic compensation is distinct from redundancy. In the case of postnatal RPCs, they normally express Rb and little p107. However, when Rb is absent, the cells sense this imbalance and upregulate p107 in a compensatory manner to prevent deregulated proliferation. Only when both Rb and p107 are inactivated does retinoblastoma form.

family members do not necessarily have the same molecular function or partners, but they can effect the same eventual outcome in a given cell at a particular time during development.

Intrinsic genetic compensation can occur in a number of different ways when the expression of one gene family member is lost. For example, another gene family member that was not previously expressed may become upregulated (see figure 25.1). Alternatively, compensation may occur through unrelated proteins that lead to the induction of downstream events by an alternative pathway (discussed in Dyer and Cepko, 2001c).

In the developing mouse retina, p107 is expressed in embryonic RPCs and Rb is expressed in postnatal RPCs (Donovan et al., 2006). When Rb is deleted, p107 compensation prevents deregulated proliferation of postnatal RPCs, and when p107 is deleted, Rb compensation prevents deregulated proliferation of embryonic RPCs (Zhang et al., 2004a; Dyer and Bremner, 2005; Donovan et al., 2006). The p107 promoter has E2F binding sites, and there is evidence to suggest that induction of p107 in the absence of Rb is mediated through these regulatory elements (Aslanian et al., 2004). Specifically, when Rb is present, it binds E2F/DP at the p107 promoter and prevents its transcription. When Rb is deleted, repression is lost and the p107 gene is upregulated in a compensatory manner. It is not known how Rb compensation is induced in the embryonic retina when p107 is deleted. The functional significance of p107 compensation was revealed when both Rb and p107 were deleted in the developing mouse retina. Retinoblastoma does not form in Rb-deficient or p107-deficient retinas. However, when both Rb and p107 are deleted, retinoblastoma forms (RobanusMaandag et al., 1998; Chen et al., 2004; MacPherson et al., 2004; Zhang et al., 2004a), which indicates that inactivation of these two family members is sufficient to initiate tumorigenesis.

There is no compensation by p130 when either Rb or p107 are deleted. However, Rb and p130 are redundantly expressed in the inner nuclear layer (INL) of the developing mouse retina (Donovan et al., 2006). It is possible that redundant expression of Rb and p130 in these cells may prevent tumorigenesis because Rb;p130-deficient mice develop retinoblastoma (MacPherson et al., 2004). It will be interesting to test whether p107 is upregulated in a compensatory manner in the developing INL cells of Rb;p130- deficient retinas and whether p107 compensation is sufficient for normal development of INL neurons and glia in these mice.

Species-specific differences in Rb family compensation

Children who inherit a defective copy of the RB1 gene have an increased susceptibility to develop retinoblastoma through

inactivation of the remaining wild-type allele (Knudson, 1971; Friend et al., 1986). In contrast, mice with one defective copy of the Rb gene have normal retinas and never develop retinoblastoma (Clarke et al., 1992; Jacks et al., 1992; Lees et al., 1992). As mentioned earlier, mice with both copies of the Rb gene inactivated in the developing retina also fail to develop retinoblastoma (Robanus-Maandag et al., 1998).

The first clue to this species-specific difference in the susceptibility to retinoblastoma came from chimeric mouse studies using Rb;p107-deficient embryonic stem (ES) cells (Robanus-Maandag et al., 1998). Chimeric mice made by mixing Rb;p107-deficient and wild-type ES cells developed retinoblastoma (Robanus-Maandag et al., 1998). Analysis of the expression of the Rb family during retinal development and their redundant and compensatory roles provided the key mechanistic insight into these data and the mouse retinoblastoma paradox. Rb-deficient and p107-deficient mice do not develop retinoblastoma because of reciprocal compensation by other family members in the developing retina (Donovan et al., 2006). However, simultaneous inactivation of both genes results in deregulated proliferation and retinoblastoma (Chen et al., 2004; MacPherson et al., 2004; Zhang et al., 2004a).

The expression of the Rb protein family during human retinal development is different from that reported for mice (Donovan et al., 2006). There is very little if any p107 expression in the developing human retina, and RB1 is the primary family member expressed throughout retinogenesis (Donovan et al., 2006). To test whether p107 can compensate for loss of RB1 in human retinas, the RB1 gene was inactivated in human fetal week 14 retinas by square wave electroporation of a plasmid expressing an RB1 siRNA (Donovan et al., 2006). There was no increase in p107 expression following RB1 gene inactivation in human fetal RPCs using this experimental approach (Donovan et al., 2006). Therefore, it is possible that the difference between mouse and human susceptibility to retinoblastoma following RB1 gene inactivation can be explained by these differences in p107 expression and compensation.

The role of Rb in rod development

In addition to the role of Rb as a tumor suppressor, it regulates rod photoreceptor development in mice (Donovan and Dyer, 2004; MacPherson et al., 2004; Zhang et al., 2004a; MacPherson and Dyer, 2007). Because traditional Rb-knockout mice die in utero, for these studies Rb was inactivated by mating RbLox mice (Marino et al., 2000) with the Chx10-Cre transgenic mouse line (Rowan and Cepko, 2004), which targets Cre recombinase to RPCs and bipolar cells. In the Chx10-Cre;RbLox/− retina, Cre is expressed in a mosaic pattern (Donovan and Dyer, 2004; Rowan and

Cepko, 2004) such that the Rb-deficient retina is chimeric, that is, apical-basal stripes of retina in which Rb has been inactivated are flanked by wild-type stripes of retina (Donovan and Dyer, 2004; Rowan and Cepko, 2004; Zhang et al., 2004a). This model provides an internal control with which to differentiate direct, cell-autonomous effects and non-cell-autonomous effects within a single retina. The genetic mosaic feature of the Chx10-Cre;RbLox/− retinas has suggested that Rb is cell autonomous for rod development (Zhang et al., 2004a). These data are consistent with data from cultured retinas from Rb-deficient E13.5 embryos, as well as Mox-Cre;RbLox/Lox mice (Zhang et al., 2004a). As further evidence of a cell-autonomous role of Rb in rod development, lineage analysis using a replicationincompetent retrovirus that expresses Cre-recombinase and alkaline phosphatase (LIA-Cre) showed that rod photoreceptors fail to form in the absence of Rb (Zhang et al., 2004a). Subsequent studies using square wave electroporation to acutely inactivate Rb in the developing retina with a plasmid expressing Cre-recombinase confirmed the finding from the retroviral lineage studies (Donovan et al., 2006). Interestingly, p107 cannot take the place of Rb in rod development in the mouse retina (Zhang et al., 2004a, 2004b; Donovan et al., 2006). This shows that some roles for the Rb family, such as proliferation control, are overlapping for Rb and p107, but other roles, such as rod development, are not.

An alternative to the hypothesis that Rb regulates rod development in the mouse retina is that Rb is required to keep newly postmitotic rod photoreceptors from reentering the cell cycle. According to this alternative hypothesis, when Rb is deleted, rods are formed normally but reenter the cell cycle and undergo apoptosis, leading to retinal degeneration. In support of this hypothesis, it has been shown previously that ectopic expression of cyclin D1 or other oncogenes in rod photoreceptors leads to ectopic cell cycle exit and cell death (Howes et al., 1994; Skapek et al., 2001). However, it is unlikely that this is the primary mechanism of photoreceptor cell loss in Rb-deficient retinas. The reason is that there is limited ectopic proliferation of Rb-deficient ONL cells during the period of rod commitment and differentiation, and this cannot account for their death (Zhang et al., 1993; Donovan et al., 2006). Moreover, this does not account for the presence of immature cells that express RPC markers and have morphological features of RPCs in the ONL of

Chx10-Cre;RbLox/Lox and Pax6-Cre;RbLox/Lox retinas (Donovan and Dyer, 2004; Donovan et al., 2006; Johnson et al., 2006). It is more plausible that rods fail to mature properly in the absence of Rb and the presence of immature cells in the ONL leads to neuronal stress and cell death of neighboring rod photoreceptors. Of course, a combination of the two models is also a reasonable possibility, as discussed later in the chapter.

One important consideration that has come from these and other genetic studies is the timing of Rb inactivation during rod genesis. Previous analysis of IRBP-Cre;RbLox/Lox mice revealed that rods developed normally when Rb was inactivated in cells already committed to the rod fate (Vooijs et al., 2002). These data, combined with the data from

Chx10-Cre;RbLox/Lox, Pax6-Cre;RbLox/Lox, and Nestin-Cre;RbLox/Lox mice (Chen et al., 2004; MacPherson et al., 2004; Zhang et al., 2004a), suggest that Rb is required during the early stages of rod development but once they commit to the rod fate, Rb is no longer required. The LIA-Cre lineage studies are consistent with these data. Specifically, when LIA-Cre is injected into the eyes of newborn mice, the retrovirus integrates into the genome of proliferating RPCs. However, at P0, most RPCs are undergoing terminal cell cycle exit or one to two rounds of cell division. Studies in mouse embryonic fibroblasts have shown that it takes 36–48 hours for LIA-Cre to recombine two copies of a floxed gene (Schweers and Dyer, 2005). Therefore, LIA-Cre inactivates RbLox around the time that cells are exiting the cell cycle and committing to the rod fate, and, depending on the number of rounds of cell division following retroviral integration, some cells may inactivate Rb after they have committed to the rod fate. The prediction based on these data is that some clones would contain normal rods and some clones would contain cells that failed to commit to the rod fate because of the timing of Rb inactivation. This is exactly what has been reported (Zhang et al., 2004a; Donovan et al., 2006). Similarly, if the retroviral injection is performed 12–24 hours later, the prediction is that the proportion of normal rods in clones would be increased over that seen following injection on P0. Preliminary studies suggest this is also true.

Another consideration is the selection of the reporter gene used in the retroviral lineage studies. The advantage of alkaline phosphatase in LIA-Cre is that the enzyme reporter gene provides excellent sensitivity because of the enzymatic amplification of the signal. Moreover, alkaline phosphatase is membrane associated, so it provides exquisite detail on the morphology of the infected cells using light microscopy and transmission electron microscopy (TEM) (Zhang et al., 2004a; Donovan et al., 2006; Johnson et al., 2006). In contrast, green fluorescent protein (GFP) is not suitable for most retroviral lineage studies. The reason is that the expression of GFP is often too low for the fluorescence to be directly detected. Some researchers rely on immunofluorescent detection of GFP from replication-incompetent retroviruses expressing GFP, but these approaches suffer from similar limitations in reporter gene detection. Of particular importance is the reduction in reporter gene expression from retroviruses when genes are ectopically expressed or when cells fail to develop normally. For example, when Rb is inactivated in RbLox/Lox retinas in vivo with the use of LIACre, the Rb-deficient cells that fail to develop into rods

express lower levels of the reporter gene than do their normal rod counterparts or control littermates. When a GFPreporter gene is used instead of alkaline phosphatase in RbLox/Lox mice, the Rb-deficient cells that fail to develop as rods are below the level of detection, leading to a very different interpretation of the data. These examples demonstrate how important it is to use multiple independent approaches to elucidate the role of cell cycle proteins such as Rb in the developing retina, and the importance of using well-characterized genetic tools.

Caution must also be taken when distinguishing the cellautonomous and non-cell-autonomous roles of Rb in rod development. Even though there is considerable evidence that Rb plays a cell-autonomous role in regulating rod development, this does not eliminate the possibility of a non-cell- autonomous role for Rb in rod maturation or survival. Indeed, rod photoreceptors are very sensitive to their microenvironment, and it is reasonable to propose that the failure of some Rb-deficient cells to differentiate into rods may have a profound effect on their normal neighboring cells that retain their Rb locus. Consistent with this hypothesis, all the genetic studies using conditional inactivation of Rb during retinal development have reported extensive rod photoreceptor degeneration in addition to the failure of rods to differentiate normally (Chen et al., 2004; MacPherson et al., 2004; Zhang et al., 2004a). These non- cell-autonomous effects on normal Rb+ rods can make the interpretation of studies directed toward elucidating the cell-autonomous role of Rb difficult, especially if the analysis is performed at late stages of development. Moreover, broad inactivation of the Rb gene in the developing retina using Pax6-Cre or Nestin-Cre can amplify these non-cell- autonomous effects and further complicate data interpretation. To minimize these effects, comprehensive analysis of several early developmental stages is required, as well as a Cre transgenic line that has a mosaic pattern of expression, such as Chx10-Cre (figure 25.2).

In addition, genetic mosaic analysis is required at the level of individual cells. To achieve this, it is possible to perform EM analysis of lead citrate–stained Chx10-Cre;RbLox/Lox;Z/AP retinas. The Z/AP transgene expresses alkaline phosphatase in every cell that expressed Cre at some time during its development (Lobe et al., 1999). Lead citrate staining of samples processed for TEM facilitates the detection of APexpressing cells and provides enough resolution to study individual processes and synapses (Gustincich et al., 1997; Johnson et al., 2006).

Bipolar cells are also affected in the Rb-deficient retinas. Analysis of early-stage retinas lacking Rb showed no defect in bipolar cell development, yet there was some reduction in later stages of development (Zhang et al., 2004a). This suggests that Rb does not play a role in bipolar cell formation but may be important for their survival. Further

Figure 25.2 Mosaic Rb inactivation in the Chx10-Cre;RbLox/Lox retina. A key question that must be addressed to distinguish between primary and secondary effects of gene inactivation is whether the changes are cell autonomous or non-cell autonomous. Specifically, is the change caused by loss of that gene in the cell (i.e., a cellautonomous effect), or is it caused by the loss of that gene in a neighboring cell (i.e., non-cell-autonomous effect)? If the effect is cell autonomous, then changes in that cell are more likely to reflect a primary role of that gene in that cell. On the other hand, if the effect is non-cell autonomous, then changes are more likely to be a secondary effect. Traditionally, these effects have been distinguished by genetic mosaic analysis. Mosaic patches of genetically altered cells are generated on a background of normal tissue. By analyzing the cells at the boundaries of mutant and wild-type mosaic patches and comparing their phenotypes with those of cells surrounded by mutant cells, one can distinguish between cellautonomous and non-cell-autonomous effects. For example, if loss of a gene leads to a particular cellular phenotype, and if mosaic analysis shows that this phenotype is reverted when mutant cells contact wild-type cells, then the phenotype is probably non-cell autonomous. A key feature of performing genetic mosaic analysis is labeling the mutant cells and wild-type cells unambiguously. For synaptogenesis studies this is particularly important. The Chx10Cre;RbLox/Lox mice are ideal for such studies because there are alternating stripes of cells that are wild type and Rb-deficient. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

evidence came from TEM analysis of Chx10-Cre;RbLox/Lox retinas stained with lead citrate (Johnson et al., 2006). In addition to Cre, the Chx10 promoter also drives alkaline phosphatase expression in a subset of bipolar cells of mature Chx10-Cre;RbLox/Lox retinas. These studies have shown that some bipolar cells can develop normally in the absence of Rb and form functional synapses with rods and horizontal cells ( Johnson et al., 2006). It remains a formal possibility that a subtype of bipolar cells that do not express Chx10 are susceptible to dying in the absence of Rb.

Nonetheless, despite these important considerations in interpreting genetic data on the role of Rb in retinal development, there is now broad consensus in the field that Rb is

required for rod development. Indeed, independent laboratories using distinct genetic approaches have all shown that Rb is required for rod photoreceptor development (reviewed in MacPherson and Dyer, 2007). Current efforts are focused on elucidating the mechanism underlying the regulation of rod development by Rb.

The first clue regarding the mechanism for the regulation of rod development by Rb came from studies on p107 compensation in the mouse retina. As mentioned earlier, p107 can compensate for Rb loss in RPCs, but it cannot take the place of Rb in differentiating rod photoreceptors. The other Rb family member, p130, does not compensate for loss of Rb, p107, or the combination of the two (Donovan et al., 2006). It has been well established that different Rb family members bind to different E2F/DP heterodimers, and the first evidence pertaining to specific targets of Rb in rod development came from analysis of an exon-specific Rb knock-in allele (Rb654) with a single amino acid substitution at position 654 of the mouse Rb protein (Sun et al., 2006). The Rb654 mutant protein has reduced binding to E2F1 and E2F3, and rods fail to form in Chx10-Cre;Rb654/Lox retinas (Sun et al., 2006). This suggests that Rb regulates rod develop-

ment through E2F1 or E2F3, or both. Specifically, it has been proposed that there is a putative rod repressor gene that is regulated by Rb/E2F1 and/or Rb/E2F3 (figure 25.3) (Sun et al., 2006). The prediction from this model is that Rb;E2F1-deficient retinas will have normal rod development, and preliminary data have confirmed this hypothesis.

Another possible mechanism for the regulation of rod development by Rb is through the genes that are important for early rod development, such as Nrl, Crx, and Nr2e3 (Furukawa et al., 1997; Haider et al., 2001; Mears et al., 2001). The evidence for this has come from preliminary studies showing that these genes are downregulated in Rb-deficient retinas at all stages of development. Preliminary chromatin immunoprecipitation experiments have shown that Rb can bind to the promoters of these genes (Chen and Dyer, 2008), suggesting that Rb may modulate the rod commitment and differentiation pathway more directly. It will be important to perform genetic epistasis analysis to determine if Rb lies functionally upstream of Nrl, Crx, and Nr2e3 and to confirm Rb protein binding at the promoters of these genes.

Figure 25.3 The regulation of rod development by Rb/E2F interactions. One hypothesis for the regulation of rod development by Rb is that there is a putative rod repressor gene that is an E2Fregulated gene. A, In proliferating RPCs, the rod repressor is expressed to prevent premature differentiation. This is achieved by phosphorylation of Rb by cyclin D/CDK4/6 and cyclin E/CDK2, which releases it from binding to activator E2Fs such as E2F1 and E2F3. B, In newly postmitotic cells that are competent to become rods, the putative rod repressor is silenced because Rb is no longer phosphorylated by cyclin/CDK and it can bind and repress the activity of the activator E2Fs. This allows these cells to become rods in response to intrinsic and extrinsic cues but is not sufficient for the rod fate. C, In Rb-deficient cells the rod repressor is constitu-

tively expressed in newly postmitotic cells, thereby preventing the rod fate. These cells do not adopt another fate and remain partially quiescent in the ONL. D, In the Rb654 mutant, Rb cannot bind to the activator E2Fs (E2F1 and E2F3), and this leads to the same phenotype as the Rb null, suggesting that these E2Fs are those responsible for regulating the putative rod repressor. E, In the absence of E2F1/3, there would be no rod repressor expressed, and rod development would proceed normally. F, Similarly, simultaneous deletion of Rb and E2F1/3 would result in rescue of the rod phenotype using the same rationale. Examples of such hypothetical rod repressors are Pax6 and Chx10. Both of these genes are expressed in RPCs, both are induced in the absence of Rb, and both can inhibit the rod fate.

A third possible mechanism for the role of Rb in regulating rod development is through chromatin organization. It is well established that Rb can recruit histone-modifying enzymes such as histone deacetylases to DNA (Zhang et al., 2000). Among the unique hallmarks of differentiated rod photoreceptors are their condensed chromatin structure and small nuclei. It is not known why rods condense their chromatin in this manner, but one possibility is that rod photoreceptors silence much of their genome through condensation, in order to efficiently express high levels of genes required for phototransduction. Owing to the rapid turnover of rod outer segments, these cells are unique in their metabolic requirements, and an efficiently organized genome may help to ensure that gene transcription is sufficient to maintain proper outer segment homeostasis. It is also possible that rod photoreceptors must condense their chromatin for more efficient packing in the ONL. In either case, it is reasonable to propose that Rb may play a role in this process because Rb-deficient cells in the ONL have an open chromatin conformation (Donovan et al., 2006). Current efforts are focused on determining whether Rb is required for chromatin condensation, and this in turn is required for normal rod differentiation, or whether Rb is required for normal rod differentiation, and this in turn leads to condensation of rod chromatin.

Defects in synaptogenesis in Rb-deficient retinas

Synaptogenesis in the inner plexiform layer (IPL) and outer plexiform layer (OPL) proceeds somewhat independently (McArdle et al., 1977; Hinds and Hinds, 1979; Redburn and Madtes, 1986; Sharma et al., 2003). In the IPL of the mouse retina, ganglion cells and their accompanying interneurons, the amacrine cells, elaborate their processes and form conventional presynaptic and postsynaptic connections before birth. In the OPL, horizontal cells first appear 7–10 days before birth. During the first week after birth, cone axons and then rod axons make synaptic contacts with horizontal cells. Rod synaptic terminals, called spherules, have distinctive features, including a single mitochondrion, and usually a single active zone marked by a synaptic ribbon. In contrast, cone synaptic terminals, called pedicles, are much larger and have multiple mitochondria and multiple active zones, each having a synaptic ribbon. Synaptogenesis of bipolar cells serves to link the two plexiform layers; thus, once the bipolar dendrites join horizontal cell presynaptic and postsynaptic contacts with spherules and pedicles in the OPL and the bipolar axons form ribbon-containing synapses with ganglion cells and amacrine cells in the IPL, the basic wiring of the primary visual pathway in the retina is established for later activity-dependent pruning and other synaptic modifications.

In Chx10-Cre;RbLox/− retinas, rods failed to differentiate in the patches of retinas lacking Rb. Immunostaining, real-time RT-PCR, and morphological analyses have shown that the cells that would normally differentiate into rod photoreceptors remained as immature cells for the first few weeks after birth (Donovan and Dyer, 2004; Zhang et al., 2004a; Donovan et al., 2006). On TEM those cells exhibited nuclear morphology consistent with immature cells, that is, their chromatin failed to condense, which is a characteristic of rod differentiation (Johnson et al., 2006). Therefore, the Chx10Cre;RbLox/− retina represented an ideal model in which to study horizontal cell and bipolar cell synaptogenesis in the absence of mature rods and their inputs.

Immunostaining analysis indicated that calbindin+, neurofilament+, and syntaxin+ horizontal cell processes extended as far as the outer limiting membrane (OLM) in P12 Chx10Cre;RbLox/− retinas and persisted to adult stages (Donovan and Dyer, 2004; Johnson et al., 2006). The total number of horizontal cells in Chx10-Cre;RbLox/− retinas was no different from that in control (Chx10-Cre;RbLox/−) retinas, nor was the horizontal cell density, as measured by flat-mount immunostaining and nearest-neighbor analysis, different (Johnson et al., 2006). TEM analysis of the Chx10-Cre;RbLox/− retinas showed that horizontal cells extend processes apically (beginning at P7) rather than laterally, as seen in control Chx10-Cre;RbLox/+ retinas (Johnson et al., 2006). Frequently, ectopic horizontal cell processes reached the OLM and formed ectopic synapses with rod spherules (Johnson et al., 2006). Using antibodies that label bipolar cells (anti-PKCα, -mGluR6, and -Goα), there was no evidence of ectopic bipolar cell dendrites associated with the ectopic rod–horizontal cell synapses (Johnson et al., 2006). This was confirmed by EM analysis (Johnson et al., 2006). The unique feature of the horizontal cell–rod photoreceptor synaptic dyads in Chx10Cre;RbLox/− retinas is that they are ectopically formed deep in the ONL, and in some cases reach the OLM (figure 25.4).

Retinal degeneration or other cellular stressors can lead to reorganization of synaptic connections in the retina (Jones et al., 2003; Marc and Jones, 2003; Marc et al., 2003). One unanswered question is whether Rb deficiency causes reorganization of previously normal horizontal cell projections or whether normal projections were never formed. Horizontal cells are born early along the apical surface of the retina and then migrate inward to establish the location of the developing OPL. Previous immunostaining analysis showed that normal horizontal cells extend apical processes during migration and then reorganize their processes into the lateral configuration in the postnatal retina. P4 is a critical stage of development when this reorganization begins. P4 horizontal cells in Chx10-Cre;RbLox/− and control retinas migrated to the same positions and displayed similar morphology. Even in Rb-deficient areas, little difference was observed in horizontal cell bodies in Rb-deficient and control retinas. However,