Genetic Interactions between Brn3 Transcription Factors in Retinal Ganglion Cell Type Specification
Figure 5
Cumulative RGC loss in Brn3a – Brn3b double KO retinas is confirmed by drastic reduction in other general RGC markers.
Retinal sections cut in a naso-temporal plane, as shown in Figure 1D, where stained with antibodies against AP and a variety of markers. Imaged and quantitated regions fall within the Pax6α: Cre full recombination domain. Genotypes indicated at the top are the same as the ones seen in Fig. 2A–D. A–D, examples of retinas from the four genotypes stained with antibodies against AP and various RGC markers, as quantitated in E–H. E–H, Pie charts represent quantitations of Brn3aAP RGCs stained for AP in conjunction with either antibodies against the Brn3a (row 1), Brn3b (row 2) or Brn3c (row3) proteins or the RGC markers Neurofilament Light chain (NFL, row 4) and Melanopsin (Opn4, row 5). To normalize the observed frequencies of AP only (green), marker only (red), or double positive cells (yellow), the number of DAPI only cells in the GCL is included (blue). Numbers next to the pie sectors are percent of total. E'–H', box whisker plots for relative densities of four RGC markers in the GCL, for the same experiments and genotypes presented in E–H. For Brn3a, NFL and Opn4, 4–7 sections from 2–3 mice were analyzed for each marker and genotype; for AP, 16–21 sections have been quantitated for each genotype. Marker density averages, expressed as Marker/DAPI cells in GCL, are: AP: E' = 39.08%, F' = 21.21%, G' = 11.54% and H' = 0.20%; Brn3a: E' = 41.31%, F' = 6.2%, G' = 11.62% and H' = 0.00%; NFL: E' = 39.64%, F' = 33.04%, G' = 15.4%, and H' = 5.55%; Opn4: E' = 5.84%, F' = 7.83%, G' = 4.43%, and H' = 5.04% (significance levels *p<0.05, **p<0.005, **p<0.001). Note that retinas lacking both Brn3a and Brn3b are completely devoid of Brn3aAP RGCs, Brn3a, Brn3b and Brn3c, but preserve a few NFL and Opn4 positive RGCs, representing each about 5% of DAPI positive cells in the GCL (a–d, rows 4 and 5). Scale bar in D is 25 μm.