Figure 7

Ptf1a overexpression triggers the conversion of glutamatergic bipolar interneurons into GABAergic cells. (A-D) Double in situ hybridization analysis of Vsx1/VGlut1 or Vsx1/Gad co-expression (stage 39/40), following Ptf1a-GR injection in both blastomeres of two cell stage embryos. (A) In control retinas, VGlut1 stains ganglion (black arrow) and photoreceptor cells (black arrowhead) and colocalizes with Vsx1 in bipolar cells (white arrow). (B) Double VGlut1/Vsx1 staining is highly reduced in Ptf1a overexpressing retinas (white arrow: region of double staining persistence, red arrow: Vsx1-positive region with no VGlut1 staining). (C) Gad (white arrow) and Vsx1 (black arrow) have exclusive expression patterns in control retinas. (D) Upon Ptf1a overexpression, regions of double Gad/Vsx1 staining become apparent (arrow). (E-K) Analysis of GABAergic bipolar cell proportion in stage 40/41 retinas, following Ptf1a-VP16 lipofection. (E-J) Typical sections of retinas transfected with gap-GFP alone (E-G) or gap-GFP plus Ptf1a-VP16 (H-J), immunostained with an anti-GABA antibody. Arrows point to a transfected GABA-negative bipolar cell in a control retina (E-G) and to a GABA-positive one in Ptf1a-VP16 overexpressing retina (H-J). (K) Quantification of GABA-positive bipolar cells among transfected cells. (L-N) Immunofluorescence analysis of Otx2 expression (stage 39), following Ptf1a-GR injection in one of two blastomere of two cell stage embryos. (L, M) Typical sections of control (L) and Ptf1a overexpressing (M) retinas immunostained for Otx2 (arrow). (N) Quantification of Otx2-positive cells per retinal section in each condition. (O-W) Analysis of Otx2-positive bipolar cell proportion in stage 40/41 retinas, following Ptf1a-VP16 lipofection. (O, V) Typical sections of retinas transfected with GFP alone (O-R) or GFP plus Ptf1a-VP16 (S-V), immunostained with an anti-Otx2 antibody. (P-R) and (T-V) are higher magnifications of the delineated regions in O and S, respectively. Arrows point to transfected Otx2-positive bipolar cells in a control retina (P-R) and Otx2-negative ones in Ptf1a-VP16 overexpressing retina (P-R). (W) Quantification of Otx2-positive bipolar cells among transfected cells. Values are given as mean +/- s.e.m. p < 0.001 (***)(Student's t test in N, binomial test in K and W). Ctrl: control; L: lens; INL: inner nuclear layer; GCL: ganglion cell layer. Scale Bar represents 50 μm in A-D, L, M and 30 in E-J, O-V.