For P3 bipolar neuron birthdates, we quantified Vsx2+ BrdU+ nuclei and divided by the total number of INL DAPI+ nuclei (Fig
August 5, 2021
For P3 bipolar neuron birthdates, we quantified Vsx2+ BrdU+ nuclei and divided by the total number of INL DAPI+ nuclei (Fig. 3/genotype. There is a 100% autonomous loss of Neurog2 in both conditional mutants, with a trend towards an additional, simultaneous loss of Neurog2+ cells outside of each Cre lineage (non-autonomous effect). Scale bars in A,E = 50 pm. NIHMS1502182-supplement-2.tif (8.2M) GUID:?A43F381C-A95B-4F8B-8D51-61D352A0DF36 3: Supplemental Fig 3. Extent of Crx and Neurog2 coexpression at two embryonic ages. A) Representative El3.5 colabeling. Boxed areas shown at higher magnification, merged and for each channel alone. B) Representative E16.5 colabeling, with boxed areas shown at higher magnification, merged and for each channel alone. In all panels, arrows point to coexpressing RPCs. C) Quantification at both ages, average number of cells per 200x images, s.d. = standard deviation, n 3/age; EIF4EBP1 apical is up, scale bar = 50 m. DY131 NIHMS1502182-supplement-3.tif (10M) GUID:?A4D2ADBF-B6F4-4487-847F-E346E810D0AC 4: Supplemental Fig 4. Additional E17.5 and P3 retinal birthdating data. A-F) Double antibody labeling for incorporated BrdU and retinal marker of interest. A-C) Arrows point to examples of BrdU+Vsx2+ double positive bipolar neurons. Ds-F) Arrows point to BrdU+ only rod photoreceptor (cones = BrdU+Arr3+ double positive cells). G) Quantification of El 7.5 BrdU bipolar data. H) Quantification of rod birthdates used same strategy as P21 rods in Figure 2. Quantification of P3 BrdU rod data, (n = 3/age + genotype; scale bar in D = 50pm; NS = not significant; error bars = SEM) NIHMS1502182-supplement-4.tif (8.5M) GUID:?EBB9A179-2053-45D6-9399-D5F219448B44 5: Supplemental Fig 5. Genomic view of RNA-sequencing reads. RNA-sequencing reads aligned against the mm 10 genome and viewed by the IGV browser comparing and Chx 10-Cre;individuals. A) Reads aligned to the gene. B) Reads aligned to the gene. A,B) Blue dotted boxes represent qRT-PCR amplicon (Fig. 7G; Primers in Suppl. Table 1; n = 5/genotype) NIHMS1502182-supplement-5.tif (17M) GUID:?A0E65837-DC06-4226-95BC-DFE0958D9985 6: Supplemental Table 1. List of qPCR primers used for validation of RNA-seq outcomes NIHMS1502182-supplement-6.docx (11K) GUID:?0B3F157C-34E3-4C9A-BDE3-6CCC113F5F24 Abstract During embryonic retinal development, the bHLH factor regulates the temporal progression of neurogenesis, but no role has been assigned for this gene in the postnatal retina. Using conditional DY131 mutants, we found that is necessary for the development of an early, embryonic cohort of rod photoreceptors, but also required by both a subset of cone bipolar subtypes, and rod bipolars. Using transcriptomics, we identified a subset of downregulated genes in P2 mutants, which act during rod differentiation, outer segment morphogenesis or visual processing. We also uncovered defects DY131 in neuronal cell culling, which suggests that the rod and bipolar cell phenotypes may arise via more complex mechanisms rather than a simple cell fate shift. However, given an overall phenotypic resemblance between and mutants, we explored the relationship between these two factors. We found that is downregulated between E12-birth in mutants, which probably reflects a dependence on in embryonic progenitor cells. Overall, we conclude that the gene is expressed and active prior to birth, but also exerts an influence on postnatal retinal neuron differentiation. and are expressed by RPCs that produce the first RGCs (Brown et al., 1998; Brown et al., 2001b; Gradwohl et al., 1996; Sommer et al., 1996; Wang et al., 2001; Yan et al., 2001). Previously was shown to activate transcription directly, plus control the spatiotemporal progression of the initial wave of retinal neurogenesis (Hufnagel et al., 2010; Skowronska-Krawczyk et al., 2009). However, does DY131 not instruct early cell fates per se, given that in E18.5 germline mutants there was only a 2% increase in RGCs, and no impact on the proportions of RPCs, cone photoreceptor, amacrine or horizontal neurons (Hufnagel et al., 2010). However, the requirements for this gene in the postnatal retina have not been explored, since.