A population of ipRGCs, which survive combined loss of Brn3a and Brn3b, might be transcriptionally regulated by Isl1.

<p>A–D', Retinal sections from genotypes identical to Fig. 2 A–D were triple stained for Isl1, Opn4 and Brn3a^AP. A–D, Pie charts showing relative frequencies of single and double stained cells. No Opn4+ only, Brn3a^AP+ Opn4+ or triple labeled cells were observed. A',D', Examples of stainings quantitated in A and D show each fluorescent channel merged to DAPI, (panels 1–3) and all channels merged in 4. Note colocalization of Isl1 with either Brn3a^AP (green arrow) or Opn4 (red arrow). E–H', Quantitation (E–H) and examples (E', H') of retinal sections triple stained for Isl1, Opn4 and ChAT. Note that Isl1+ (RGCs) and Isl1+ Opn4+ (ipRGCs – red arrows) cells are reduced in numbers in Brn3b KO or Brn3a–Brn3b DKO retinas, but Isl1+ ChAT+ (Starburst amacrines – white arrows) frequencies remain relatively unaffected. I–J', Retinal sections triple stained for Isl1, Opn4 and Brn3. One example from <i>Six3: Cre; Isl1 ^CKO/+</i> control (I') and three examples from <i>Six3</i>: Cre; <i>Isl1 ^CKO/CKO</i> (J') mice are shown. Green, red and white arrows point at double labeled cells. I, J pie charts of single, double or triple labeled cells. Note that, in Isl1 KO retinas, total Brn3+ RGCs are reduced to about half and Opn4+ cells are essentially missing. A total of 9 sections for <i>Six3: Cre; Isl1 ^CKO/+</i> control and 11 sections from <i>Six3</i>: Cre; <i>Isl1 ^CKO/CKO</i> mice were analyzed. Mean Opn4/DAPI ratios were 10.63% for <i>Six3: Cre; Isl1 ^CKO/+</i> and 0.63% for <i>Six3</i>: Cre; <i>Isl1 ^CKO/CKO</i> (t test p value = 2.4 e-7). In Six3: Cre; <i>Isl1^CKO/CKO</i> retinas rare Isl1+ are observed as result of a low level of mosaicism of the Six3: Cre transgene. Some of these are also Opn4 positive, as seen in panel J', top and bottom row. Scale bar in J' is 25 μm.