Moreover Sox2-CreER is also expressed in a small number of amacrine cells (Fig
July 5, 2021
Moreover Sox2-CreER is also expressed in a small number of amacrine cells (Fig.?S1A,E). neuronal/RPC phenotype. Our results suggest that the Asarinin differences in miRNA expression between MG and RPCs contribute to their difference in neurogenic potential, and that manipulations in miRNAs provide a new tool with which to reprogram MG for retinal regeneration. (Jorstad et al., 2017; Ueki et al., 2015). Comparable studies of other candidate reprogramming factors further exhibited that miRNAs miR-124, miR-9 and miR-9* (alone or in combination with Ascl1) (Wohl and Reh, 2016b) were effective in stimulating the conversion of mouse MG to RPCs and/or neurons. However, a comprehensive survey of miRNAs that differ between progenitors and glia, similar to that carried out for mRNAs, has not been reported. We therefore used fluorescence-activated cell sorting (FACS) to purify RPCs from postnatal day 2 mice and MG from P8, P11 and adult mice. The RNA was extracted from purified RPCs and MG, and miRNA expression was analyzed by means of the molecular barcode technology called NanoStrings (Dennis et al., 2015; Geiss et al., 2008). We recognized the miRNAs that were more highly expressed in RPCs, when compared with MG, as well as miRNAs that were more highly expressed in MG than in RPCs. For the miRNAs that were enriched in the FACS-purified RPCs when compared with the MG, we experimentally overexpressed these in MG cultures to determine whether neurogenic competency could be restored. Similarly, for miRNAs that were enriched in the MG relative to the RPCs, we antagonized these in the MG to determine whether this would restore neurogenic competency to the MG. We found that manipulations in two miRNAs, miR-25 (mimic) and let-7 (antagomiR), stimulated neural reprogramming of MG with a neuronal conversion of up to 40% of young MG The combination of miR-25 overexpression and let-7 inhibition was even more effective than either treatment alone, with 60% of the Ascl1-expressing MG developing neuronal phenotypes. This reprogramming capacity was decreased in adult MG cultures (range 1-4?months) to 20%. Single cell RNA-seq of reprogrammed MG confirmed that many of the cells acquired a gene Asarinin expression profile much like RPCs and retinal neurons. Together, our data show that miRNAs are important in regulating the development of MG, and at least one of these, let-7, has a conserved role in the neurogenic competence of both mouse and fish MG. RESULTS The miRNA profile of retinal progenitor cells and Mller glia in the mouse retina We have previously reported miRNA expression in MG, using FACS to purify the cells from mature retina (Wohl and Reh, 2016a). To determine which miRNAs are uniquely expressed in RPCs, we used a similar strategy and FACS-purified RPCs from postnatal day 2 (P2) Sox2-CreER: tdTomatoflSTOP/flSTOP mice. We induced expression of the reporter by tamoxifen injections at P0 and P1, resulting in tdTomato expression in many cells of the neuroblastic layer (NBL). The majority of these cells also expressed progenitor markers Sox9 and Sox2. The portion of the tdTomato+ cells was 50% of the total, somewhat higher than expected (Fig.?S1A,A,F). In addition to the RPCs, it is likely that some of the tdTomato+ cells were also the neuronal progeny of the RPCs. Moreover Sox2-CreER is also expressed in a small number of amacrine cells (Fig.?S1A,E). These two Sox2+ populations thus reduce the purity of the final sample. To label MG, we used a different strategy that allowed for greater purity of the cells. We FACS purified the MG at the ages P8, P11 and >P21 from Rlbp1-CreER:tdTomatoflSTOP/flSTOP mice, as previously explained (Wohl et al., 2017; Wohl Asarinin and Reh, 2016a). After tamoxifen application, the majority of MG [Sox9, Sox2 and glutamine synthetase (GS)] were labeled; the MG represented 1.5-2.1% of all cells (Fig.?S1B-D,F), consistent with previous estimates of MG in the mouse retina (Grosche et al., 2016; Jeon et al., 1998). To quantify the miRNAs expressed in RPCs and MG, total RNA was extracted from FACS-purified Sox2:tdTomato+ and Rlbp1:tdTomato+ cells. The miRNAs were measured by answer hybridization using the NanoString nCounter assay (Geiss et al., 2008). The expression profiles of RPCs and MG are shown in Fig.?1A as a heatmap of all ages. We found three main clusters, a green cluster with miRNAs moderately expressed, a blue cluster with miRNAs with low expression and a pink cluster with Mouse monoclonal to CD21.transduction complex containing CD19, CD81and other molecules as regulator of complement activation the most highly expressed miRNAs. A list of the miRNAs in each of the three clusters can be found in Table?S1. Interestingly, the overall.