Figure 4

Ptf1a misexpression dramatically affects the ratio of GABAergic versus glutamatergic neurons. (A-J) In situ hybridization analysis of glutamic acid decarboxylase (Gad) and glutamate transporter 1 (Vglut1) in stage 39/40 retinas, following Ptf1a-GR or Ptf1a Mo injection in two-cell stage embryos. (A-F) Ptf1a overexpression results in a drastic increase of Gad staining (A-C) at the expense of VGlut1 staining (D-F). Shown in C and F are strong phenotypes compared to milder ones in B and E. (G-J) Conversely, Ptf1a knocked-down retinas display virtual absence of Gad expression (G, H), while that of VGlut1 is highly expanded (I, J). Arrow and arrowhead in A and G point to Gad expression domain in amacrine and horizontal layers, respectively. Black arrow, white arrow and black arrowhead in D and I point to VGlut1 expression domain in ganglion, bipolar and photoreceptor cells, respectively. Ctrl: control; L: lens; NT: neural tube. Scale Bar represents 50 μm. (K) Real-time RT-PCR analysis of Gad and Hox11L2 expression in animal cap assays (equivalent stage 16), following X-ngnr-1-GR (25 pg) or Ptf1a-GR (50 pg) overexpression. N-tubulin was used as a control for X-ngnr-1 and Ptf1a neuralizing activities. Expression levels were normalized to ornithine decarboxylase (ODC). The expression levels were measured using a standard curve for each analyzed gene. All measurements were done in duplicates and the values in the figures represent the mean of a representative experiment. (L-Q) In situ hybridization analysis of N-tubulin (N-tub) and Gad expression in whole embryos (stage 28 in L-N and 24 in O-Q), following X-ngnr-1 or Ptf1a mRNA injection in one of two blastomere of two-cell stage embryos. (R, S) In situ hybridization analysis of Gad expression in the neural tube (stage 28), following Ptf1a-GR or Ptf1a Mo injection in one of two blastomere of two-cell stage embryos. The Ptf1a-GR injected embryos were induced with dexamethasone at stage 11. NT: neural tube; NC: notocord; inj: injected side. Scale Bar represents 100 μm.