Fig. 4.
Dicer-CKO Müller glia express Brevican, which leads to disorganization. a Percent decline of the top 10 highly expressed MG miRNAs in the Dicer-CKOMG, listed in order of their highest expression levels, retinas of 40 wt and 26 CKO retinas were pooled. b RT-qPCR comparison of the top five highly expressed miRNAs for Nanostring validation (40 wt and 26 CKO retinas). c Scatter plot of genes differently regulated in the MG from Dicer-CKOMG compared with MG from wt, shown as log2 of fold change and of counts per million (CPM). Genes showing the greatest increase are highlighted in red. d Bcan gene model (top) with the mapped reads from RNA-Seq in the wt and the Dicer-CKOMG. e RT-qPCR confirmation of the top four genes upregulated in RNA-Seq in the Dicer-CKOMG (26 retinas, pooled) when compared to wt MG (32 retinas, pooled). f Comparison of ΔΔC t means CKO vs. wt from RT-pPCR of four highly expressed genes in the Dicer-CKOMG from FAC-sorted MG (26 CKO and 32 wt retinas pooled, normalized against beta-actin) vs. log2 CPM from RNA-Seq, regression coefficient R 2 = 0.6. g–s Immunofluorescent labeling for tdTomato, Brevican, and DAPI. g–j 1 month after deletion: in wt mice, Brevican was expressed in neurons (asterisks) in the GCL and INL but not in MG (neither somata nor endfeet, unfilled arrows, g, h). Most MG in Dicer-CKOMG mice expressed Brevican, i, j, in their endfeet in the GCL and in their somata in the INL (filled arrows). Unfilled arrows point to rare MG without Brevican. k Percentage of MG expressing Brevican in wt (n = 7) and MG from Dicer-CKOMG mice (n = 7, mice of at least two different litters were analyzed). l–s 6 months after deletion: while the wt remains unchanged (l–n), tdTomato+Brevican+ MG in the Dicer-CKOMG form aggregates in the IPL/GCL (o–q) and INL/ONL (o, r, s). Statistics: mean ± S.D., Shapiro–Wilk test for normality and Mann–Whitney U-test, significant differences: **p < 0.01. Scale bars in g, i, o: 50 μm, in n, q, s: 25 μm. MG, wt, retinal layers as defined in Fig. 1 legend