It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle–independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors.
The simplicity of the retina makes it an ideal tissue to study neurogenesis. Its development proceeds through three overlapping steps starting with retinal progenitor cell (RPC) proliferation, followed by birth of post-mitotic retinal transition cells (RTCs, also referred to as precursors), and ending with terminal differentiation of seven major cell types (Figure 1A) [1]. RPCs are multipotent and exit the cell cycle to generate different RTCs at specific time periods in development [1]. This process of RTC “birth” requires coupling of differentiation and cell cycle exit. Once born, post-mitotic RTCs migrate and form different retinal layers. Rods and cones make up the outer nuclear layer (ONL); horizontal, bipolar, and amacrine cells, as well as Müller glia cell bodies, reside in the inner nuclear layer (INL); and ganglion and displaced amacrine cells form the ganglion cell layer (GCL) (Figure 1A). The outer plexiform layer (OPL) and inner plexiform layer (IPL) house synaptic connections separating the ONL/INL and INL/GCL, respectively. The retinoblastoma protein (Rb) is critical for cell cycle exit during retinal transition cell birth. Rb knockout (KO) RTCs continue to proliferate inappropriately and some (rod, ganglion, and bipolar cells) die by apoptosis [2,3]. Rb controls the cell cycle by binding and inhibiting E2f transcription factors (E2fs) (Figure 1B), first defined as transcription factors that bind adenoviral E2 regulatory elements and subsequently shown to be critical cell cycle regulators [4,5]. E2fs bind to DNA as heterodimers with proteins of the related Tfdp family. E2f1, E2f2, and E2f3a are “activating E2fs” that are required for fibroblast division. They are strong transcriptional activators that can drive G0 fibroblasts into cycle, and are inhibited when bound to Rb [4,5]. Ectopic division in Rb KO embryos can be rescued to various extents in different tissues by knocking out E2f1, E2f2, or E2f3 [6–9], but which member (s) drive division in Rb KO RTCs is unknown. Other members of the family, such as E2f4 and E2f5, are known as “repressive E2fs” because they are weak activators and appear to be primarily involved in gene silencing in quiescent or differentiated cells. Activating E2fs may also promote apoptosis in the Rb KO retina (Figure 1B). Originally, E2f1 was considered the primary pro-apoptotic member of the family [10]. However, this view was reevaluated when it was shown that either E2f1 or E2f3 deletion rescues apoptosis in the developing central nervous system (CNS) of Rb KO embryos [6,11]. Subsequently, CNS apoptosis was shown to be an indirect result of placental defects and probable hypoxia [12–14]. Indeed, E2f3-induced apoptosis in fibroblasts has recently been shown to require E2f1 [15]. Thus, it is controversial whether E2f3 is required for apoptosis of any Rb KO cell type. Determining which activating E2fs promote death in distinct Rb KO tissues requires conditional rather than germ line models of Rb deletion to avoid secondary indirect effects (such as hypoxia). E2f family diversity is expanded by E2f3 isoforms. Alternative promoters generate two forms (a and b) that are identical except for distinct first exons [16]. E2f3a is a strong activator, and, like other activating E2fs, its expression is induced when quiescent cells are stimulated to divide [16]. E2f3b, like repressive E2fs, is present in both quiescent and dividing cells, and in quiescent fibroblasts it associates primarily with Rb, suggesting that it mediates repression [16–18]. Indeed, silencing the Cdkn2d (p19Arf) locus in unstressed cells relies on E2f3b [19]. Other E2fs may also exist in isoforms since at least two mRNA species have been detected for E2f1 and E2f2 [16]. The roles of E2f isoforms in vivo are unknown. E2fs are also regulated by subcellular localization. Although this feature has been best characterized for repressive E2fs [20–22], it also affects activating E2fs [23–25]. The distribution of E2f isoforms has never been assessed. It has been known for many years that Rb loss perturbs neuronal differentiation [26–29]. However, prior work could not exclude the possibility that differentiation defects are simply an indirect consequence of abnormal division and death. If Rb does regulate differentiation directly it is unclear whether it does so in all or a subset of neurons. Moreover, the mechanism has never been solved. In other cell types where Rb may promote differentiation directly, such as muscle and bone, it seems to do so through E2f-independent means by potentiating tissue-specific transcription factors (Figure 1B) [30–33]. In the retina, others have noted abnormally shaped Rb KO rods and have suggested Rb may directly promote their morphogenesis by activating retina-specific factors [29]. However, differentiation defects in any Rb KO neuron could be an indirect effect of ectopic division and/or apoptosis (Figure 1B). Thus, it is critical to study differentiation of Rb KO cells in the absence of ectopic proliferation and death. Here, we establish that Rb suppresses RTC division and death by inhibiting E2f1, not E2f2 or E2f3. When these defects were rescued, most retinal neurons, including rods, survived, differentiated, and functioned normally. Thus, unexpectedly, retina-specific differentiation factors function independently of Rb. However, comprehensive assessment of the Rb/E2f1 double-null rescued retina revealed a differentiation defect in cholinergic starburst amacrine cells (SACs). Recent breakthroughs have revealed that these interneurons are critical for direction selectivity and developmentally important rhythmic bursts [34–36]. However, their differentiation is poorly understood. Contrary to the prevailing view that Rb promotes differentiation through E2f-independent tissue-specific transcription factors, we show that Rb facilitates SAC development through E2f3. Defects in Rb null SACs correlated with specific E2f3 expression in these cells, and E2f3 expression was absent in neurons that differentiated without Rb. E2f3 is also present in a specific subset of other CNS neurons, implying that this may be a general mechanism by which Rb facilitates neurogenesis. To define the mechanism in even more detail, we determined which E2f3 isoform Rb targets to control SAC differentiation. E2f3b mediates Rb function in quiescent fibroblasts [19], yet no prior studies to our knowledge have dissected E2f3a or E2f3b functions in vivo. Using an isoform-specific null mouse we show that Rb drives SAC differentiation through E2f3a. Thus, independent of E2f1-mediated effects on division and death, Rb does regulate neuronal differentiation, but only in specific neurons and, unexpectedly, through E2f3a, not tissue-specific differentiation factors.
We used the α-Cre transgene to delete floxed Rb exon 19 at embryonic day (E) 10 in peripheral retina [2]. RbloxP/loxP; α-Cre mice were bred with strains lacking E2f1 or E2f2 in the germ line, or a strain carrying a floxed E2f3 allele [5]. RbloxP/loxP; E2f1+/− and RbloxP/loxP; E2f1+/−; α-Cre mice were bred to produce RbloxP/loxP; E2f1−/−; α-Cre mice at a frequency of 1/8 and littermate controls at the same or higher (1/4) frequency. For simplicity we will refer to the RbloxP/loxP; E2f1−/−; α-Cre peripheral retina as the Rb/E2f1 double knockout (DKO) retina. Similar strategies were employed in the case of E2f2 or E2f3. Cre-mediated excision of Rb and E2f3 alleles in the retina was confirmed by PCR as described previously [2,5]. To measure ectopic cell division, mice were pulse-labelled with bromodeoxyuridine (BrdU) 2 h before sacrifice and the peripheral retina analyzed for BrdU incorporation by immunofluorescence. As reported before [2,3], Rb KO retinas exhibited both spatial and temporal ectopic DNA synthesis (Figures 1C and S1A). This is easily detected at E14, E16, and postnatal day (P) 0 in the inner retina where abnormal BrdU+ ganglion and amacrine RTCs are located, or on the outermost region of the P0 retina, where BrdU+ photoreceptor RTCs reside (Figures S1A and S2, arrows) [2]. Ectopic RTC division in Rb KO retinas is even more obvious at P8 or P18, when division is completed in wild-type (WT) retina (Figures 1C and S1A). Strikingly, the ectopically positioned S-phase cells at E14, E16, and P0 and all the abnormal division at P8 and P18 were completely suppressed in the Rb/E2f1 DKO retina (Figures 1C, 1E, 1F, S1A, and S2). In contrast, deletion of E2f2 or E2f3 had no effect at any stage of development. Analysis of mitotic cells with anti–phosphohistone 3 (PH3) –specific antibodies confirmed that loss of E2f1, but not E2f2 or E2f3, suppressed ectopic division (Figure S3). Deleting one E2f1 allele partially suppressed ectopic S-phase and mitosis in Rb KO RTCs (Figures 1C, 1E, 1F, S1A, S2, and S3), suggesting that E2f1 drives ectopic division in Rb KO RTCs in a dose-dependent fashion. These data contrast with previous findings in the lens and CNS of Rb KO embryos, where deletion of any activating E2f suppresses ectopic division to some extent [6–9]. Loss of Rb in the retina results in considerable RTC apoptosis, eliminating most bipolar and ganglion cells as well as many rods (Figure 2A–2D) [2,3]. The loss of Rb KO rods is evident from the thinner ONL, and the death of these cells as well as bipolar and ganglion neurons can be detected directly by double labelling for apoptotic and cell-type-specific markers [2] (M. P. and R. B. , unpublished data). Loss of peripheral Rb KO ganglion cells is also evident from thinning of the optic nerve (D. C. and R. B. , unpublished data). Deleting E2f1, but not E2f2 or E2f3, blocked this ectopic cell death in a dose-dependent fashion (Figures 1D, 1G, and S1B). To investigate the molecular mechanism that underlies the unique role of E2f1, we assessed the expression of known E2f targets as well as other genes that regulate the cell cycle and apoptosis. Numerous positive and negative cell cycle and apoptotic regulators were up-regulated in the Rb KO retina (Figure 1H). Among the E2f family, E2f1, E2f2, E2f3a, and E2f7 were induced following Rb loss, but E2f3b, E2f4, and E2f5 were unaffected. Consistent with the BrdU and terminal dUTP nick-end labelling (TUNEL) analyses, E2f1 deletion specifically reversed all these molecular defects, but E2f3 deletion had no effect (Figure 1H). Because E2f1 deletion blocks abnormal division and death in the Rb KO retina, the Rb/E2f1 DKO retina provided a unique opportunity to evaluate whether Rb controls differentiation independent of cell cycle effects. The Rb/E2f1 DKO retina had many Sag+ (S-antigen/rod arrestin) photoreceptors, Pou4f2+ (Brn3b) ganglion cells, and numerous Prkca+/Cabp5+ bipolar neurons (Figure 2A–2D). In contrast, there was no such rescue of cell types in Rb/E2f2 or Rb/E2f3 DKO retinas (Figure S4). Analysis with general neuronal markers Mtap2 (MAP2) and Snap25, as well as other markers expressed in bipolar cells (Chx10, Rcvrn, Vsx1, Tacr3, and Atp2b1) and rod photoreceptors (Rho and Rcvrn) confirmed rescue of the Rb/E2f1 DKO retina (Table S1). Moreover, neurons exhibited the same complex morphology as in WT retina. Bipolar cell bodies were located in the INL and had ascending and descending processes ending in the OPL and IPL, respectively (Figure 2A). In addition, the Rb/E2f1 DKO retina had a healthy appearing ONL consisting of morphologically normal rods with descending processes ending in the OPL and ascending processes that terminated in inner and outer segments (Figure 2A). It was suggested that Rb might regulate photoreceptor differentiation, possibly through rod-specific transcription factors (Figure 1B) [29]. However, if Rb does regulate photoreceptor differentiation, it does so by inhibiting E2f1, not by potentiating rod differentiation factors, such as Otx2, Crx, or Nrl. It is impossible to tell whether E2f1 perturbs differentiation directly, by affecting the expression of genes that modulate maturation, and/or indirectly through its effects on proliferation and survival (Figure 1B). As with ectopic division and apoptosis (Figure 1C and 1D), the rescue of Rb KO retinal bipolar, ganglion, and rod cells was dependent on E2f1 dose (Figure 2A–2D). Separate from its role in driving ectopic division of Rb KO RTCs, E2f1 also promotes normal RPC division since in its absence RPC proliferation drops ~2-fold (D. C. and R. B. , unpublished data). This modest reduction of RPC numbers in the absence of E2f1 accounts for the slight reduction in the number of ganglion cells at P0, in the number of bipolar cells at P18 or P30, and in the thickness of the ONL at P18 or P30 in the E2f1 KO and Rb/E2f1 DKO retina (Figure 2B–2D). The morphology of E2f1 KO neurons was WT (Figure 2A). Despite a slight drop in absolute cell numbers, the proportion of Rb/E2f1 DKO and E2f1 KO bipolar cells was the same as WT (data not shown). Slightly reduced cell numbers were not due to residual RTC death since we have not observed ectopic apoptosis at any embryonic or postnatal stage in the developing Rb/E2f1 DKO retina (Figures 1D, 1G, and S2). Moreover, deleting Ccnd1, which acts upstream of Rb proteins, also reduces RPC number, but does not suppress any defect in the Rb KO retina (D. C. and R. B. , unpublished data). Thus, slightly reduced RPC division and dramatic rescue of severe defects in Rb KO RTCs are distinct effects stemming from the deletion of E2f1. The discovery that E2f1 loss rescues even the morphology of Rb KO neurons is surprising because Rb is thought to regulate differentiation primarily through E2f-independent pathways [30–33]. However, normal morphology may not equate to completely normal differentiation. Thus, we compared the electroretinograms (ERGs) of adult WT (α-Cre), E2f1−/−, α-Cre; RbloxP/loxP, and α-Cre; RbloxP/loxP; E2f1−/− mice. ERGs functionally assess visual signalling in the mammalian retina from photoreceptors to amacrine cells (but usually not gangion cells), and are dominated by rod and cone bipolar cells. Typically, an ERG signal begins with a negative deflection initiated by the photoreceptors (the a-wave), which is terminated by a large positive deflection due to the activation of ON bipolar cells (the b-wave). Responses to dim light in dark-adapted (scotopic) conditions specifically assess the rod system, and were defective in the Rb KO retina (Figure 2E). The substantial reduction of both a- and b-waves is consistent with rod and bipolar cell apoptosis [2]. The sensitivity of the residual response appeared unchanged, suggesting it arose from the Cre-negative portions of the retina. Responses were about the same in the WT and E2f1 KO retina, and, most importantly, also the Rb/E2f1 DKO response median lay at the lower end of the normal range for most intensities (Figure 2F). Thus, E2f1 deletion almost completely rescued the rod system in the Rb KO retina. Light-adapted (photopic) recordings to specifically assess the cone system yielded comparable results. Cones represent only 3% of photoreceptors and, unlike rods, develop without Rb, but they require rods for survival, and in the Rb KO retina, they have abnormal morphology and their synaptic targets, bipolar cells, are much depleted [2]. The photopic response, a product of cone and mainly bipolar activity, was much reduced by Rb loss, but was rescued considerably in the Rb/E2f1 DKO retina (Figure S5). Again, the median amplitude lay at the lower end of the E2f1 KO range. The photopic response in E2f1 KO mice was slightly reduced relative to WT (Figure S5B), possibly because E2f1 is required for maximal expansion of embryonic RPCs, and the E2f1 KO retina has, as noted earlier, slightly fewer cells than the WT retina, although cell type proportions are unaffected (D. C. and R. B. , unpublished data). Thus, marginally subnormal photopic responses in the Rb/E2f1 DKO retina can be attributed to both a reduction of cone numbers in E2f1 KO mice alone, and a “genuine” slight reduction in cone function attributable to Rb loss relative to WT. This slight effect may relate to a true differentiation defect in a subset of amacrine cells discussed below. This discussion should not obscure the major outcome that E2f1 deletion recovers most of the ERG response. Thus, E2f1 deletion not only rescues morphology but also both rod and cone system function in the Rb KO retina. ERGs primarily assess photoreceptor and bipolar cell function, but may miss differentiation defects in other cells. To test for subtle differences we stained the Rb/E2f1 DKO retina with 43 markers (Table S1). Thirty-two proteins displayed identical patterns in WT, E2f1 KO, and Rb/E2f1 DKO retina (Table S1). The other 11 markers revealed a cell-cycle– and apoptosis-independent differentiation defect in SACs. We first studied Calb2 (calretinin), which marks a subset of amacrine and ganglion cell bodies as well as three tracks corresponding to their processes in the IPL (Figure 3A). Normal Calb2 staining was seen in the E2f1 KO IPL (data not shown). However, only one Calb2+ track was evident in the Rb KO IPL, and this defect was not rescued in the Rb/E2f1 DKO retina (Figure 3A). We quantified Calb2+ cell bodies in the Rb KO INL (corresponding to amacrine cell staining only) and observed a reduction from P8 onwards (Figures 3C and S6). Of the three Calb2+ tracks in the IPL, the two outer tracks are from SACs, named after their extensive dendritic-tree-like morphology [37]. SACs are cholinergic, represent ~5. 2% of amacrine neurons [38], and are critical for both direction selectivity [34,35] and spontaneous rhythmic activity that occurs during normal retinal development [36]. SACs in the INL synapse in the OFF layer of the IPL that responds to decreasing light, while displaced SACs in the GCL have processes that synapse in the nearby ON layer of the IPL that responds to increasing light (reviewed in [39]). Mature SAC processes stain specifically for Slc18a3 (vesicular acetyl choline transporter, VAChT) [37], and, significantly, this marker was absent in the peripheral Rb KO or Rb/E2f1 DKO P18 retina (Figures 3A and S7B). Chat, expressed from the same locus, is also SAC specific, but marks both cell bodies and processes of mature SACs [37]. Chat was seen in fewer cells in the mature Rb KO retina, and was present in the soma but absent from processes (Figure 3B). We obtained similar results for Sv2c, a synaptic vesicle protein found in SACs [40]; Kcnc1b and Kcnc2, potassium channels expressed on SAC soma and dendrites as well as a very small number of ganglion cells [41]; gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter present in about half of amacrine cells including SACs, as well as horizontal and some bipolar neurons [37]; and Calb1 (calbindin), which is expressed in many amacrine cells and labels SAC process faintly (Figure S7A and S7B; Table S1; and data not shown). Finally, we also examined the effect of Rb deletion on SAC differentiation using a Chx10-Cre transgene that is expressed in a mosaic pattern across the retina, generating patches of Cre-expressing cells [42]. Consistent with the mosaic deletion pattern, we observed markedly reduced Chat/Slc18a3 staining in the IPL of Chx10-Cre; RbloxP/loxP retina compared to WT (Figure S7C). Together, these results suggest a role for Rb in SAC differentiation. The above findings could indicate a defect in SAC specification, SAC survival, or the levels and/or transport of the markers described above. Camk2a marks both SACs and ganglion cells [37], but because ganglion cells are eliminated in the Rb KO retina, Camk2a is a specific SAC marker in this context. Importantly, Camk2a+ tracks and dendrites were present in both the WT and Rb KO retina (Figure 3B), and the number of Camk2a+ soma was similar in WT and Rb KO retina at P30 and beyond, although fewer cells stained in Rb KO retina at P18, suggesting a delay in its appearance (Figures 3C and S6B). Thus, Rb is not required for SAC survival or for process outgrowth, but rather it seems to regulate the expression and/or stability of Calb2, Calb1, Chat, Slc18a3, Sv2c, Kcnc1b, Kcnc2, and GABA in SACs, but leaves Camk2a expression virtually unaffected. The presence of Chat in some cell bodies but never in processes (Figure 3B) also suggests a transport defect. The developmental pattern of Slc18a3 expression also supported this notion. In mature WT SACs Slc18a3 is only in processes, but in early postnatal SACs, it is found in the cell body, and moves into emerging processes at approximately P4–P6. As noted above, Slc18a3 was absent at P18 in the Rb KO retina (Figure 3A); at P4 or P5 it was in cell bodies, yet was rarely present in Rb KO processes (Figures 4A and S6). Slc18a3 became virtually undetectable in Rb KO SACs by P8 (Figures 3C and S6C). These data suggest that Rb affects both the synthesis/stability and transport of SAC markers. Rb binds more than 100 proteins [43] and in some non-neuronal cells, such as skeletal muscle, adipocytes, and bone, Rb is thought to bind and potentiate tissue-specific transcription factors that promote differentiation [31–33]. Thus, we expected that Rb might interact with retina-specific factors to facilitate SAC differentiation. A direct role for E2f in mediating Rb-dependent differentiation defects (independent of cell cycle or death defects) has to our knowledge not been described, but because E2f can regulate some differentiation genes [44–48], we first tested whether E2f2 or E2f3 might perturb Rb KO SAC maturation. At multiple time points, E2f1 deletion suppressed ectopic mitosis (PH3+ cells), but did not reverse the SAC defect, and E2f2 deletion had no effect on either defect (Figure 4A). Remarkably, although E2f3 deletion did not reverse ectopic mitosis, it rescued Calb2, Slc18a3, Chat, GABA, Kcnc1b, Kcnc2, and Sv2c staining at multiple times (Figure 4A and data not shown). Rb/E2f3 DKO SAC tracks were slightly more disordered than in WT retina, most likely because of the absence of synaptic partner cells, which are killed by E2f1. Indeed, this minor defect was rescued in the Rb/E2f1/E2f3 triple knockout retina, where bipolar and ganglion cell death was rescued and SAC differentiation was restored (Figure 4A). E2f3 deletion alone did not affect SAC differentiation (Figure 4A); thus, it is unleashed E2f3 activity that is detrimental, and the critical role for Rb is to inhibit E2f3. We quantified the fraction of Camk2a+ SACs in different genotypes and found that 60% of WT P30 Camk2a+ cells expressed Chat and Slc18a3, which dropped to only 5. 6% in the Rb KO retina, and remained low at 3. 7% in the Rb/E2f1 DKO retina, but rose to 91% in the Rb/E2f3 DKO retina (Figure 4B). The latter fraction is higher than WT because ganglion cells, which normally make up ~40% of Camk2a+ cells, are killed by apoptosis. To quantify the effect of different E2fs on ectopic division specifically in SACs, we exploited Isl1 (Islet1). This marker is expressed in both SACs and ganglion cells, thus Isl1+ cells in the INL are exclusively SACs [49]. We found that 98. 2% ± 1. 8% of Isl1+ cells in the forming inner INL at P5 were also Slc18a3+, confirming that Isl1 is an excellent SAC marker (Figure 4C). Moreover, Isl1, unlike Slc18a3, is nuclear, which facilitates scoring of Isl1+/Mki67+ cells. It is also expressed earlier than Slc18a3, permitting analysis of SACs soon after their birth at ~E15; thus, we could study retina at P0, a time when ectopic division is high in the inner retina and prior to Rb-independent cell cycle exit associated with terminal differentiation [2]. At P0, no WT Isl1+ cells in the inner neuroblastic layer (NBL) (which is the future INL) were dividing, but 57 ± 14 Isl1+/Mki67+ cells were detected in the Rb KO inner NBL (Figure 4D). Indeed, about one-third of all Isl1+ cells in the entire inner NBL were dividing in the Rb KO retina, or ~50% in the periphery where Cre is expressed (Figure 4E and data not shown). This defect was suppressed in the Rb/E2f1 DKO retina, where we detected only 1 ± 1 dividing SAC, but not the Rb/E2f3 DKO retina, where there were 53 ± 8 dividing SACs (Figure 4D and 4E). We observed similar effects at P0 with Calb2, which marks newborn SACs and other amacrine cells (data not shown). Thus, in Rb KO SACs, E2f1 deletion suppresses ectopic division but not aberrant differentiation, whereas E2f3 deletion suppresses aberrant differentiation but not ectopic division. The unique effect of E2f3 in disrupting the differentiation of SACs but not other retinal neurons might be due to cell-type-specific expression or cell-type-specific activity of E2f3. Determining between these two possibilities is not easy, as E2f immunostaining in mouse tissues is problematic. We did not solve this issue for E2f1 or E2f2, but used a modified protocol [50] to successfully track E2f3 expression (Figure 5). At P0, E2f3 was detected in RPCs, consistent with a putative role in normal proliferation (Figure 5A). The signal was specific as it was absent in the E2f3 KO peripheral retina (Figure 5A). As the retina differentiated and RPC division diminished, the number of E2f3+ cells also dropped, and by P8, when division is virtually over, only a subset of post-mitotic cells in the inner retina expressed E2f3 (Figure 5A). By P18, E2f3 was also detected in two tracks in the IPL (Figure 5A and 5B), reminiscent of SAC markers such as Chat and Slc18a3 (c. f. Figures 3 and 4). This cytoplasmic E2f3 staining was also specific, as it was absent in the E2f3 KO peripheral retina of α-Cre; E2f3loxP/loxP mice (Figure 5A). Indeed, double labelling with E2f3 (red) and Chat plus Slc18a3 (green) confirmed that E2f3 is present in both SAC soma and dendrites (Figure 5B). Rb protein was also detected in the inner retina (Figure 5A), and showed a similar distribution as E2f3 in SACs (Figure 5B), and was also present in mature ganglion cells and Müller cells as reported [51]. Rb staining in SAC processes was specific as it was absent in the peripheral retina of αCre; RbloxP/loxP mice (Figure 5A). These data suggest that Rb and E2f3 colocalize in SACs and that E2f3 triggers defects in SAC differentiation because it is specifically expressed in these retinal neurons. We also found that E2f3 is present in a specific subset of mature neurons in various brain regions (data not shown). For example, in the P20 amygdala, E2f3 colocalized with the general neuronal markers Mtap2 and Mecp2 [52], but not with Calb2, which marks a subset of neurons, or with the glial marker Gfap (data not shown). Unlike in retinal SACs, E2f3 was not coexpressed in Chat+ or Slc18a3+ cholinergic neurons located in various regions of the brain and spinal cord (data not shown). In agreement, we could not detect defects in cholinergic Rb KO neurons in the developing forebrain, but other Rb KO neurons in this region showed differentiation defects that were rescued by deleting E2f3 [53]. Together, these results suggest that the common mechanism by which Rb promotes neural differentiation is through E2f3 inhibition. As noted above, E2f3 and Rb staining in SACs was both nuclear and cytoplasmic (Figure 5A and 5B). The antibody that worked in immunostaining recognizes a C-terminal region and thus, does not distinguish a/b isoforms. To our knowledge, the subcellular location of E2f3 isoforms has not been determined in any cell type. To verify the dual locations of E2f3 and to determine which isoforms were present in retina, we analyzed nuclear and cytoplasmic fractions by Western blot at different times during development. Analysis with the pan-E2f3 antibody (sc-878, Santa Cruz Biotechnology) detected a 55-kD E2f3a species and a 40-kD E2f3b polypeptide (Figure 6). To confirm that the upper species in our retinal lysates was E2f3a, we exploited novel mice that lack E2f3 exon 1a and thus express E2f3b exclusively (R. O. and G. L. , unpublished data). The genotyping strategy is discussed in detail later and is outlined in Figure 7A. Western analysis confirmed that the upper band was absent in E2f3a−/− mice (Figures 6 and S8). Consistent with the drop in E2f3-expressing cells during WT retinal maturation (Figure 5A), the total amount of E2f3a was less at P18 compared to P0 (Figure 6). E2f3b was present in similar amounts at both time points. At P0 and P18, E2f3a was present in both nuclear and cytoplasmic fractions, but in marked contrast, E2f3b was exclusively nuclear at both times (Figure 6). Two closely migrating E2f3a bands were detected, more clearly evident at P18, of which the faster migrating species was dominant in nuclear and the slower species was dominant in cytoplasm (Figure 6). The identity of both as E2f3a species was confirmed by their absence in the P18 E2f3a KO retina (Figure S8). Analysis of Pou4f2, a nuclear transcription factor expressed in ganglion cells, showed that nuclear proteins had not contaminated the cytoplasmic fraction, and analysis of Slc18a3, a cytoplasmic SAC marker, confirmed that the reverse had also not occurred (Figure 6). These data show, to our knowledge for the first time, that E2f3a and E2f3b exhibit distinct patterns of subcellular distribution, and raise the possibility that E2f3a localization may be regulated by as yet unknown post-translational modifications. We also examined the distribution of other cell cycle regulators during retinal development. Like E2f3a, Rb was present in both the WT cytoplasm and nucleus at P0, but at P18, when the levels of Rb had increased, it was primarily nuclear (Figure 6). A very faint cytoplasmic Rb signal was evident at P18, which is consistent with Rb staining of SAC processes (Figure 5B), and with the very small proportion of SACs in the retina [38]. E2f1 was also detected in both nuclear and cytoplasmic fractions, although unlike E2f3a it was predominantly nuclear both at P0 and P18 (Figure 6). The E2f dimerization partner, Tfdp1, which lacks a nuclear localization signal [54], was primarily cytoplasmic at both P0 and P18, and the Cdk inhibitors Cdkn1a and Cdkn1b showed a similar pattern of distribution (Figure 6). Thus, among the cell cycle regulators we examined, most showed bivalent distribution, and E2f3b was unusual in its solely nuclear compartmentalization. To test which E2f3 isoform is responsible for aberrant Rb KO SAC differentiation we exploited E2f3a−/− mice (Figure 7A). The genotyping strategy outlined in Figure 7A was used to distinguish the E2f3a, WT, and null alleles. Reverse transcriptase PCR (RT-PCR) confirmed the presence of both E2f3a and E2f3b RNA species in the developing WT retina, and the specific absence of E2f3a RNA in the E2f3a−/− retina (Figure 7B). E2f3a protein was absent in E2f3a−/− retinal lysate (Figures 6 and S8). Importantly, the levels of E2f3b message were similar in the Rb KO and Rb/E2f3a DKO retina, ruling out the possibility that any effects of E2f3a deletion we might observe were due to down-regulation of E2f3b (Figure 7C). Also, the levels of other E2fs were the same in the Rb KO, Rb/E2f3 DKO, and Rb/E2f3a DKO retina, ruling out any cross-regulatory effects (Figure 7C) [55]. E2f3a can trigger cell cycle induction, but because SAC defects are not linked to cell cycle perturbation (Figures 3A and 4), and in view of the predominant association between E2f3b and Rb in quiescent cells [16,19], we suspected that E2f3b may perturb differentiation in Rb KO SACs. Unexpectedly, however, E2f3a deletion suppressed the Rb KO SAC defect (Figure 7D). Thus, separate from its role in cell cycle control, Rb regulation of E2f3a is critical to ensure proper neuronal differentiation.
Work in the early 1990s showed that Rb loss triggers defects in neuronal cell cycle exit, survival, and differentiation [26–28]. Much of the death is an indirect consequence of probable hypoxia linked to placental defects [12–14]. However, targeted KO and chimeric studies reveal that Rb autonomously promotes cell cycle exit in newborn neurons, and is required for survival of a subset of neurons, particularly in the retina [2,3, 13,14,56–59]. However, whether Rb also regulates differentiation is obscured by potentially indirect effects of ectopic division and death. Moreover, a mechanism though which Rb may regulate neuronal maturation has not been elucidated. Here, deleting E2f1 specifically rescued ectopic division and death in the Rb KO retina. Importantly, major Rb/E2f1 DKO neurons differentiated normally, and ERGs revealed the rescue of rod- and cone-mediated function, implicating a regular signal flow from photoreceptors to bipolar and amacrine cells. Division and death genes were induced in Rb KO cells, and deleting E2f1, but not E2f2 or E2f3, reversed these molecular events. E2f1 may also regulate differentiation targets, but whether this contributes to defects in retinal cell maturation is impossible to separate from potentially indirect consequences of deregulated division and death. In any case, it is clear that in most retinal cells, including photoreceptors [29], transcription factors that promote differentiation function independently of Rb. We have also found that E2f1 deletion rescues cell-autonomous ectopic division, death, and differentiation defects in sporadic Rb KO clones generated using a Cre retrovirus vector (M. P. and R. B. , unpublished data). These data are consistent with the observation that E2f1 overexpression in newborn photoreceptors drives ectopic division and apoptosis [60], and add to the growing evidence indicating that E2f1 is the major, and perhaps only, member of the three activating E2fs required to induce apoptosis in Rb KO cells [10,15]. Thus, deregulated E2f1 activity in the retina, whether resulting from the inactivation of Rb or from overexpression, promotes unscheduled cell division and triggers apoptosis in susceptible RTCs. E2f1, rather than other E2fs, may be a potential target for novel therapeutics to prevent retinoblastoma in RB1+/− humans. Our ERG studies revealed rescue of the Rb KO rod–bipolar system, and almost complete restoration of the cone–bipolar system following E2f1 deletion. There was a slightly lower response in the Rb/E2f1 DKO retina relative to the E2f1 KO control retina. This difference might reflect a role for Rb in the development of cones, bipolar cells, or other cells that may contribute to the photopic ERG, including potentially SACs, which do have a serious defect in the Rb/E2f1 DKO retina. Comprehensive marker analysis revealed that, in striking contrast to other retinal neurons, E2f1 deletion did not suppress defects in Rb KO cholinergic SACs. Instead, we observed E2f1-independent defects in the synthesis and transport of a large cohort of SAC proteins. These data expand insight into the development of these important interneurons, but more critically, provide to our knowledge the first unambiguous evidence that Rb regulates neurogenesis beyond terminal mitosis. Rb binds more than 100 factors [43], and in several non-neuronal cells, such as skeletal muscle, adipocytes, and bone, it binds and potentiates tissue-specific transcription factors that promote differentiation [31–33]. The idea that Rb promotes muscle differentiation by potentiating Myod1 activity was contested [61], and other mechanisms proposed [62,63], but not involving E2f repression. Strikingly, however, we discovered that Rb promotes SAC differentiation through E2f3 (Figure 8). Rb regulation of SAC differentiation through E2f3 was independent of its role in controlling division or death: E2f3 deletion rescued Rb KO SAC defects but did not suppress aberrant proliferation or death, whereas E2f1 deletion reversed abnormal proliferation and death but did not rescue SAC differentiation. Double labelling confirmed that E2f1 but not E2f3 deletion reversed Rb KO SAC division. Moreover, deleting E2f1, but not E2f3, reversed deregulated expression of cell cycle and apoptotic genes in the Rb KO retina. E2f3 is expressed in a subset of CNS neurons (this work) and drives specific cell-cycle–independent defects in Rb KO forebrain neurons [53]. Thus, E2f3 inhibition is the first, and may be the only, mechanism by which Rb participates directly in neuronal differentiation. To further dissect the mechanism of action of Rb in SACs we determined the E2f3 isoform it targets to promote differentiation. E2f3b was the primary candidate, since Rb and E2f3b collaborate to repress targets in quiescent cells in vitro [19]. However, in the first work to our knowledge to examine the function of any E2f protein isoform in vivo, we made the surprising observation that Rb regulates SAC differentiation through E2f3a (Figure 8). Formally, we cannot exclude the possibility that deleting E2f3b might also rescue SAC differentiation, but definitive proof will require analysis of E2f3b null mice. Nevertheless, our data prove that Rb definitely regulates SAC differentiation through the activating E2f3 isoform. The subcellular location of E2f isoforms has not to our knowledge been addressed before. E2f3a and E2f3b share 110 C-terminal amino acids that encode the NLS, DNA-binding, marked box, transactivation, and Rb-binding domains [16], yet they exhibit different subcellular distribution in developing retinal cells. E2f3a is both nuclear and cytoplasmic, but E2f3b is always nuclear. The unique 121- and six-residue N-termini of E2f3a and E2f3b, respectively, likely mediate this difference. This region in E2f1, E2f2, and E2f3a binds Ccna2, establishing a negative regulatory loop that deactivates E2fs in mid-late S-phase [64,65]. However, even E2f3b, which lacks this domain, binds and is regulated by Ccna2 [18], so the domain difference may not explain the unique distributions we observed. Rb family and Tfdp proteins can also determine E2f localization [20–22], and we found that a portion of both Rb and Tfdp1 proteins are cytoplasmic in retinal cells. Indeed, immunostaining revealed that Rb and E2f3 colocalize to SAC processes. The nuclear localization of E2f3b contrasts with that of other repressive E2fs in differentiating muscle, where E2f5 switches from the nucleus to cytoplasm, while E2f4 remains in both compartments [23]. The distinct compartmentalization of E2f3a and E2f3b in the retina suggests temporally and functionally distinct activities. Rb distribution matches that of E2f3a, consistent with its critical role in supporting SAC differentiation through E2f3a. Rb is critical to ensure that many types of terminally differentiating cells leave the cell cycle (e. g. , neurons, gut and skin epithelia, muscle, and lens fibres) (reviewed in [66]). Early overexpression studies in vitro suggested Rb might temper expansion of cycling cells, but KO studies in vivo indicate that its major role is to block division in terminally differentiating cells. In its absence, many (but clearly not all) aspects of differentiation go ahead relatively unperturbed. In the retina, differentiating transition cells are born in the absence of Rb, migrate to the correct layer, and express appropriate markers ([2] and this work). In brain, Rb KO neurons migrate away from the ventricular zone and switch on Tubb3 (βIII-tubulin), but continue to incorporate BrdU [13], and in gut epithelia, differentiated enterocytes migrate up the villi and activate expression of serotonin, yet continue to incorporate BrdU [67]. In the case of SACs, the differentiation defects we observed (e. g. , loss of Slc18a3 and Chat) were not due to aberrant division, but it is possible there are other problems with these cells that are caused by ectopic division. Nevertheless, it is clear that many aspects of differentiation in multiple cell types are compatible with ectopic division. However, division of terminally differentiating cells is dangerous, since it may facilitate transformation, as is the case in retinoblastoma (reviewed in [66]). E2f3a could disrupt SAC differentiation through its well known role as a transcriptional activator, or, in view of the discovery that it is partially cytoplasmic, E2f3a may affect processes other than gene regulation. Both scenarios are feasible since E2fs regulate differentiation genes [44–48], and cell cycle regulators, such as Cdkn1b, have cytoplasmic activities that influence differentiation [68,69]. Many transcription factors shuttle between nucleus and cytoplasm during neurogenesis (e. g. , [70] and references therein). It may be difficult to identify E2f3a-specific target genes or cytoplasmic proteins in SACs since these neurons are a small proportion (<1%) of the total retina and only ~5. 2% of amacrine neurons [38]. Others have suggested that Rb promotes differentiation in non-neuronal cells through E2f-independent means [31–33]. However, these studies did not assess whether these cell types differentiate normally if Rb is deleted along with one or more E2f family members. One study reported that Rb mutants that do not bind E2f still induce differentiation [30]. However, the binding assays were performed in solution, and we have found that several of these mutants do bind E2f, albeit weakly, on chromatin (T. Yu and R. B. , unpublished data). It is possible that Rb-mediated potentiation of tissue-specific transcription factors may, at least in some cases, be a redundant activity, and that the only critical Rb function is to inhibit E2f. Our study is the first to our knowledge to assess comprehensively whether Rb KO cells can differentiate in the absence of different E2fs. In light of our findings, it will be important to reassess differentiation defects in other Rb KO tissues in the absence of individual and combined activating E2f family members.
Mice were treated according to institutional and national guidelines. α-Cre mice (P. Gruss), Chx10-Cre mice (C. Cepko), RbloxP/loxP mice (A. Berns), E2f1–/– mice, E2f2–/– mice, E2f3loxP/loxP mice, and E2f3a−/− mice were maintained on a mixed (NMRI × C57/Bl × FVB/N × 129sv) background. A detailed description of E2f3a−/− mice will be published elsewhere. Mice of different genotypes were compared within the same litter and across a minimum of three litters. We have not noted any phenotypic differences in separate litters. Genotyping was performed as before [2,5], and the primers used for genotyping E2f3a−/− mice were E2f3a KL (5′-CTCCAGACCCCCGATTATTT-3′), E2f3a KR1 (5′-TCCAGTGCACTACTCCCTCC-3′), and E2f3a KM (5′-GCTAGCAGTGCCCTTTTGTC-3′). Eyeballs were fixed in 4% paraformaldehyde for 1 h at 4 °C, embedded in OCT (TissueTek 4583, Sakura, http: //www. sakuraeu. com), frozen on dry ice, and cut into 12-μm sections on Superfrost plus slides (VWR, http: //www. vwr. com). For S-phase analysis, BrdU (100 μg/g of body weight) was injected intraperitoneally 2 h prior to sacrifice. BrdU+ cells were detected using a biotin-conjugated sheep polyclonal antibody (1: 500, Maine Biotechnology Services, http: //www. mainebiotechnology. com). All other antibodies are described in Table S1. For E2f3, Mki67, and Rb staining, antigen retrieval was performed by boiling sections in citric acid solution for 15 min according to Ino [50], except on frozen sections. TUNEL was performed as described [13]. Briefly, sections were incubated for 1 h at 37 °C with 75 μl of mixture solution consisting of 0. 5 μl of terminal deoxynucleotide transferase, 1 μl of biotin-16-dUTP, 7. 5 μl of CoCl2,15 μl of 5× terminal deoxynucleotide transferase buffer, and 51 μl of distilled water. After three washes in 4× SSC buffer, sections were incubated with Alexa 488– or Alexa 568−streptavidin (1: 1,000; Molecular Probes, http: //probes. invitrogen. com) for 1 h at room temperature. Primary antibodies or labelled cells were visualized using donkey anti-mouse Alexa 488 or Alexa 568, donkey anti-rabbit Alexa 488 or Alexa 568, donkey anti-goat Alexa 488 or Alexa 568, and streptavidin Alexa 488 or Alexa 568 (1: 1,000; Molecular Probes). Nuclei were counter-stained with 4,6-diamidino-2-phenyindole (DAPI; Sigma, http: //www. sigmaaldrich. com). Labelled cells were visualized using a Zeiss (http: //www. zeiss. com) Axioplan-2 microscope with Plan Neofluar objectives and images captured with a Zeiss AxionCam camera. For double-labelled samples, confocal images were obtained with a Zeiss LSM 5. 0 laser scanning microscope. The retina was separated into three bins by dividing the ventricular edge of the retina into equal parts and extending a line to the vitreal edge [2]. Bin 1 contains only cells that expressed Cre as progenitors; bin 3 is at central retina and contains cells derived from progenitors that did not express Cre. For cell counts or thickness measurement we used a region 0–100 μm peripheral to the boundary separating bins 1 and 2. Measurements were performed on an Axioplan-2 microscope using Axiovison software. Quantification of S-phase, M-phase, and apoptotic cells was performed on horizontal sections that included the optic nerve. Quantification of differentiated cell types was performed using horizontal sections at equal distances from the optic nerve. A minimum of three sections per eye and three eyes from different litters were counted. Total RNA was isolated from dissected peripheral retina using TRIzol reagent (Invitrogen, http: //www. invitrogen. com) followed by digestion with RNase-free DNase (DNA-free, Ambion, http: //www. ambion. com) to remove DNA contamination. First-strand cDNA was synthesized from 0. 2–0. 5 μg of total RNA using the SuperScript II first-strand synthesis system (Invitrogen). PCR primers are listed in Table S2. Real-time quantitative PCR was performed using an Applied Biosystems (http: //appliedbiosystems. com) PRISM 7900HT. Tests were run in duplicate on three separate biological samples with SYBR Green PCR Master Mix (Applied Biosystems) exactly as we described previously [71]. Briefly, master stocks were prepared such that each 10-μl reaction contained 5 μl of SYBR Green PCR Master Mix, 0. 1 μl of each forward and reverse primer (stock 50 μM), 0. 8 μl of blue H2O (0. 73% Blue Food Colour; McCormick, http: //www. mccormick. com), 2 μl of diluted cDNA template, and 2 μl of yellow H2O (0. 73% Yellow Food Colour). PCR consisted of 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 55 °C for 30 s. An additional cycle (95 °C, 15 s, 60 °C) generated a dissociation curve to confirm a single product. The cycle quantity required to reach a threshold in the linear range was determined and compared to a standard curve for each primer set generated by five 3-fold dilutions of genomic DNA or cDNA samples of known concentration. Values obtained for test RNAs were normalized to Hprt1 mRNA levels. Mouse retinas were homogenized by passing them through a 30-gauge BD 9 http: //www. bd. com) needle 5–10 times in 1× PBS solution. Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Product# 78833, Pierce Biotechnology, http: //www. piercenet. com). Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. After blocking overnight at 4 °C in 5% skim milk, membranes were incubated in the primary antibody for 2 h at room temperature. After three 10-min washes in TPBS (100 mM Na2HPO4,100 mM NaH2PO4,0. 5 N NaCl, 0. 1% Tween-20), membranes were incubated for 30 min at room temperature in the secondary horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, http: //www. jacksonimmuno. com). Blots were developed using the ECL-Plus chemiluminescent detection system (Amersham Pharmacia Biotech, http: //www. pharmacia. ca), according to the manufacturer' s instructions. The following primary antibodies were used: E2f-1 (SC-193), E2f-3 (SC-878), Cdkn1a (p21, SC-471), Cdkn1b (p27, SC-528), Pou4f2 (Brn3b, SC-6062), and Tfdp1 (Dp1, SC-610) from Santa Cruz Biotechnology (http: //www. scbt. com), pRB (554136) from BD Science-Pharmingen (http: //www. bdbiosciences. com), and Slc18a3 (VAChT, G448A) from Promega (http: //www. promega. com). ERGs were recorded from dark-adapted mice as described [72]. Briefly, mice were dark-adapted overnight and anaesthetized by subcutaneous injection of ketamine (66. 7 mg/kg body weight) and xylazine (11. 7 mg/kg body weight). The pupils were dilated and single-flash ERG recordings were obtained under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation was accomplished with a background illumination of 30 candela (cd) per square meter starting 10 min before recording. Single white-flash stimulation ranged from 10−4 to 25 cd·s/m2, divided into ten steps of 0. 5 and 1 log cd·s/m2. Ten responses were averaged with an inter-stimulus interval of either 5 s (for 10−4,10−3,10−2,3 × 10−2,10−1, and 3 × 10−1 cd·s/m2) or 17 s (for 1,3, 10, and 25 cd·s/m2). Band-pass filter cut-off frequencies were 0. 1 and 3,000 Hz. Different genotypes were evaluated using analysis of variance (ANOVA) followed by the Tukey honestly significant difference (HSD) test or Fisher test (XLSTAT program, http: //www. xlstat. com).
The GenBank (http: //www. ncbi. nlm. nih. gov/genbank) accession numbers for the major genes and gene products discussed in this paper are Camk2a (NM_009792), Chat (NM_009891), E2f1 (NM_007891), E2f2 (NM_177733), E2f3 (NM_010093), Rb (NM_009029), and Slc18a3 (NM_021712).
The retinoblastoma protein (Rb), an important tumor suppressor, blocks division and death by inhibiting the E2f transcription factor family. In contrast, Rb is thought to promote differentiation by potentiating tissue-specific transcription factors, although differentiation defects in Rb null cells could be an indirect consequence of E2f-driven division and death. Here, we resolve different mechanisms by which Rb controls division, death, and differentiation in the retina. Removing E2f1 rescues aberrant division of differentiating Rb-deficient retinal neurons, as well as death in cells prone to apoptosis, and restores both normal differentiation and function of major cell types, such as photoreceptors. However, Rb-deficient starburst amacrine neurons differentiate abnormally even when E2f1 is removed, providing an unequivocal example of a direct role for Rb in neuronal differentiation. Rather than potentiating a cell-specific factor, Rb promotes starburst cell differentiation by inhibiting another E2f, E2f3a. This cell-cycle–independent activity broadens the importance of the Rb–E2f pathway, and suggests we should reassess its role in the differentiation of other cell types.
developmental biology
cell biology
mus (mouse)
neuroscience
Rb-Mediated Neuronal Differentiation through Cell-Cycle–Independent Regulation of E2f3a