Figure 2

Ptf1a is required for proper amacrine and horizontal cell genesis. (A-T) In situ hybridization (A-B, E-F, I-J, K-L, O-P, S-T) or immunofluorescence (C-D, G-H, M-N, Q-R) analysis of cell-type specific marker expression in stage 39/40 retinas, following Ptf1a-GR or Ptf1a Mo injection in two cell stage embryos. (A-J) Prox1 (horizontal marker, arrows in A and B) and syntaxin (optic nerve and inner plexiform layer marker, arrows in C and D) expressions are expanded in the central retina following Ptf1a overexpression. In contrast, both Brn3 (ganglion cell marker, arrows in E and F) and rhodopsin (photoreceptor marker, arrows in G, H) display highly diminished expression. Expression of Vsx1 (bipolar marker, arrows in I and J) does not show any clear decrease although significant disorganization is occasionnaly observed in severe phenotypes (not shown). (K-T) Compared to control ones, Ptf1a Mo injected retinas display virtual absence of Prox1 expression (arrows in K, L) and reduced syntaxin staining in the IPL (arrows in M and N). Note the increased size of the optic nerve in Ptf1a knocked down retinas (arrowheads in M and N) that is consistent with the increased expression of the ganglion cell marker Brn3 (arrows in O and P). Both rhodopsin (arrows in Q and R) and Vsx1 (arrows in S and T) appear normally expressed albeit with a disorganized expression pattern. Ctrl: control; L: lens. Scale Bar represents 50 μm. (U-Y) Test of Ptf1a Morpholino efficiency. In vivo GFP fluorescence was analysed following co-injection of either Ptf1a Mo or Ctrl Mo with a chimeric GFP construct fused downstream Ptf1a Mo complementary region. (U) Schematic representation of the construct and sequences of Morpholino oligonucleotides. (V-Y) GFP expression can be observed in Ctrl Mo injected neurulas, while it is efficiently inhibited in Ptf1a Mo injected ones.