Figure 4.
Validation of TFs in RSCs using RNA interference. (a) Frequency of sphere formation from Creb1 mutant CE (wild type: WT, heterozygous hypomorphs: Het, and homozygous hypomorphs: Homo). Homo had a 45-50% decrease in stem cell frequency, and the total number of stem cells per eye was also decreased by 56-65% compared to WT and Het (WT=165 +/− 9.4, Het=207.4 +/−16.9, Homo=71.9 +/−16.7; F2,21=28.47, p<0.0001). (b) Creb1 RSC sphere diameter also decreased slightly in the Homo showing an effect on progenitor cells (WT=93.75 +/−1.72, Het: 86.19 +/−1.76, Homo: 77.92 +/−1.04; F2,74=26.35, p<0.0001). (c) Self-renewal of RSCs was tested by dissociating the primary spheres into single cells and plating them at 1000 cells/well. The Homo Creb1 RSCs had a diminished capacity to form new secondary spheres (WT: 13.2 +/−1.63, Het: 12.2 +/−1.10, Homo: 7.2 +/− 1.66, F2,12=5.516, p=0.02). (d) siRNA knockdown of Creb1 in adult RSCs in vitro reproduced the genetic homozygous hypomorph effect on RSC number with a 50% loss of sphere formation compared to siRNA controls (t5=5.217; p=0.0034). (e) Primary CE cells were kept undifferentiated for 5 days in the presence of Hdac10 siRNA or NT siRNA, dissociated and then sorted using either Cnr1 (cluster 2) or Grm7 (cluster 4) populations. Hdac10 significantly increased the number of sphere-forming cells in both the Cnr1 and the Grm7 populations as well as the negative populations compared to controls (F1,24=13.56, p=0.0012). Separate qPCR experiments demonstrated that the siRNAs caused 75%-90% decrease in the expression of the target genes (data not shown). Error bars=s.e.m. * indicates where a column is significantly different from its control: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.