Normal Differentiation in the Rb/E2f1 DKO Retina
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KO RTCs, E2f1 also promotes normal RPC division since in its absence RPC proliferation drops ~2-fold (D. C. and R. B., unpublished data).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.