PMC:1914394 / 29528-33117 JSON TXT

Annnotations TAB JSON ListView MergeView

craft-sa-dev

craft-ca-core-ex-dev

craft-ca-core-dev

2_test

{"project":"2_test","denotations":[{"id":"17608565-11447760-84868995","span":{"begin":79,"end":81},"obj":"11447760"},{"id":"17608565-8381715-84868996","span":{"begin":267,"end":269},"obj":"8381715"},{"id":"17608565-11545733-84868996","span":{"begin":267,"end":269},"obj":"11545733"},{"id":"17608565-8946919-84868996","span":{"begin":267,"end":269},"obj":"8946919"},{"id":"17608565-11159908-84868997","span":{"begin":595,"end":597},"obj":"11159908"},{"id":"17608565-12110166-84868997","span":{"begin":595,"end":597},"obj":"12110166"},{"id":"17608565-12975318-84868997","span":{"begin":595,"end":597},"obj":"12975318"},{"id":"17608565-15479636-84868997","span":{"begin":595,"end":597},"obj":"15479636"},{"id":"17608565-16081278-84868997","span":{"begin":595,"end":597},"obj":"16081278"},{"id":"17608565-9315903-84868998","span":{"begin":2204,"end":2206},"obj":"9315903"},{"id":"17608565-15193257-84868999","span":{"begin":2737,"end":2738},"obj":"15193257"},{"id":"T27346","span":{"begin":79,"end":81},"obj":"11447760"},{"id":"T67368","span":{"begin":267,"end":269},"obj":"8381715"},{"id":"T65967","span":{"begin":267,"end":269},"obj":"11545733"},{"id":"T79277","span":{"begin":267,"end":269},"obj":"8946919"},{"id":"T48689","span":{"begin":595,"end":597},"obj":"11159908"},{"id":"T1759","span":{"begin":595,"end":597},"obj":"12110166"},{"id":"T21385","span":{"begin":595,"end":597},"obj":"12975318"},{"id":"T89101","span":{"begin":595,"end":597},"obj":"15479636"},{"id":"T28753","span":{"begin":595,"end":597},"obj":"16081278"},{"id":"T59642","span":{"begin":2204,"end":2206},"obj":"9315903"},{"id":"T26396","span":{"begin":2737,"end":2738},"obj":"15193257"}],"text":"Rb Regulates SAC Differentiation through E2f3\nRb 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.\nWe 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.\nTo 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."}

PMC:1914394 / 29528-33117 JSONTXT

Annnotations TAB JSON ListView MergeView

craft-sa-dev

craft-ca-core-ex-dev

craft-ca-core-dev

2_test

PMC:1914394 / 29528-33117 JSON TXT