Fig. S3

mRNA was extracted from total retinal lysates from three independent tamoxifeninduced CKO and corn oil-injected control retinae. We performed qrtPCR analysis and found statistically significant reductions in Yap, Taz, Cyclin D1,  and Cyclin D3 (A) . It is important to note that the varying levels of mRNA reduction between mice likely reflect a combination of Cre mosaicism, variation in tamoxifen induction, and inefficiency in recombining four independent floxed loci in a given cell. Furthermore, Yap  and Taz  are also expressed in retinal endothelial cells, which are expected to contribute to mRNA transcripts within total retinal lysate preparations (Neto et al., 2018; Sakabe et al., 2017). For qrtPCR, mRNA levels are relative to the corn oil control (set to 1) and depicted as fold change ±SEM (n = 3 independent pooled samples per group; Student’s t test). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. To further illustrate that the loss of Cyclin D1  in MGs is coincident with loss of Yap  and Taz , we generated Chx10-Cre+/tg; Yapflox/flox; Tazflox/flox ; ROSA26R-mTmG+/tg mice. Chx10-Cre  (Rowan and Cepko, 2004) is expressed broadly in retinal progenitor cells during development and provides large regions of Cre-mediated recombination that can be easily visualized by GFP expression from the R26R-mTmG  Cre reporter. In GFP+ regions of the retina, we observed coincident loss of YAP and CYCLIN D1 immunofluorescence in the INL (white arrowheads in B) . In adjacent tdTomato+ regions, which did not experience Cre-mediated recombination, CYCLIN D1 and YAP coexpression persisted (yellow arrowheads in B) . Importantly, immunofluorescence also showed that SOX9+/YAPMGs are still present in the CKO retinae indicating that loss of CYCLIN D1 expression in MGs is not due to loss of MGs, but rather loss of Yap  and Taz  expression (yellow arrowheads in C) . The same results were observed for CYCLIN D3 expression (data not shown). N = 3. Map of the Cyclin D1  gene showing the location of two putative TEAD binding sites (red arrowheads in D) . Immunofluorescence showed that the ImM10 MG-like cell line expresses YAP, TEAD1, CYCLIN D1 and SOX9 (E) . N = 3 per sample. ImM10 cell lysates were immunoprecipitated with antibodies against YAP or an IgG negative control. Western blot with anti-TEAD1 and YAP antibodies showed that YAP and TEAD1 coimmunoprecipitate (F) . N = 3 per sample. YAP chromatin immunoprecipitation was performed on ImM10 cells followed by qPCR analysis of the 2 putative TEAD motifs. YAP was enriched on motif #1 that lies 3.7 kb upstream of the Cyclin D1  start site, but not on motif #2 in intron 3 (G) . N = 3 per sample.