Figure 6

Haploinsufficiency of p107 in the developing mouse retina. Immunostaining of P30 Rb ^+/-; p107 ^-/- retinas in the region of the retinal dysplasia. (A, B) Antibodies against calbindin were used to identify horizontal cells; (C, D) those against Chx10, bipolar cells; (F, G) those against glutamine synthetase, Müller glia; and (H, I) those against bassoon, synapses in the OPL and IPL. (E) EM analysis of the P30 Rb ^+/-; p107 ^-/- retinas showing the OPL with normal organization of synaptic connections in contrast to the Rb-deficient retinas. (J-T) Immunostaining of P14 Chx10-Cre;Rb ^Lox/-; p107 ^+/- retinas in the region of Cre-mediated Rb inactivation using antibodies to OPL synapses (PSD-95, J-M), horizontal cells (calbindin, O, P), amacrine/progenitor cells (Pax6, Q, R), and reactive Müller glia (GFAP, S, T). (N) EM analysis of the P14 Chx10-Cre;Rb ^Lox/-; p107 ^+/- retinas showing the OPL with disrupted synaptic connections and an apical horizontal process (arrows). (U-Y) To determine if there are any ectopically dividing cells in Rb ^+/-; p107 ^-/-, Rb ^-/-; p107 ^+/- or Rb ^-/-; p107 ^-/- retinas at E14.5, P0, P6, P12 and P30, we labeled retinas with [³H]thy and BrdU for 1 h and then dissociated and immunostained the cells with antibodies against 12 markers of different retinal cell types. BrdU and [³H]thy always colocalized. (W) The only markers that colocalized with [³H]thy were amacrine/progenitor cell markers Pax6 and syntaxin and bipolar/progenitor cell marker Chx10. There were also some reactive Müller glia, as indicated by GFAP, that incorporated [³H]thy. Ectopic proliferation was more prevalent in the double-knockout retinas than in the other genotypes. Each bar is the average of triplicate samples scored in duplicate. (X, Y) Real-time PCR analysis demonstrated that throughout development there were more progenitor cell markers such as Cdk2 and Sfrp1 expressed in the double-knockout and Rb ^-/-; p107 ^+/- retinas. Abbreviations: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: B, D, G, I, K, M, P, Q, T, U and V, 10 μm.