Selective RGC loss in Brn3b – Brn3c double KO retinas documented by losses of general RGC markers.

<p>Retinal sections cut in a naso-temporal plane, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076347#pone-0076347-g001" target="_blank">Figure 1D</a>, 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. 2E–H. 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 Brn3c^AP 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, 3–7 sections from 2–3 mice were analyzed for each marker and genotype; for AP, 17–22 sections have been quantitated for each genotype. Marker density averages, expressed as Marker/DAPI cells in GCL, are: AP: E' = 10.18%, F' = 11.63%, G' = 6.57% and H' = 6.16%; Brn3a: E' = 34.34%, F' = 40.65%, G' = 17.6% and H' = 8.26%; NFL: E' = 48.47%, F' = 46.41%, G' = 34.03%, and H' = 9.86%; Opn4: E' = 4.21%, F' = 4.36%, G' = 3.3%, and H' = 1.77% (significance levels ^*p<0.05, ^**p<0.005, ^**p<0.001). Note that retinas lacking Brn3b alone, or in conjunction with Brn3c have preserved AP positivity only in a restricted lamina in the centre of the IPL. Brn3a and NFL, show cumulative losses in Brn3b–Brn3c DKO retinas when compared to Brn3b single KO. Scale bar in D is 25 μm.