FIGURE 2.

Cplx mRNA levels in wild-type (WT) and cone and rod specific BMAL1 knockouts. (a) Cplx3 is the only member of the Cplx family which is differentially expressed in WT and cone-Bmal1^−/− cones. Cones also show high Cplx4 expression in both WT and cone-Bmal1^−/− cones. (b) Cplx3 is the only member of the Cplx family which is differentially expressed in WT and rod-Bmal1^−/−. A small difference was found between WT and mutant rods for Cplx2 but the levels were overall very low. Rods show a very high expression of Cplx4, consistent with previous research indicating that it is the primary Cplx found in rods. Error bars are SEM. Dots show individual data points. n = 1 animal (two retinas)/point, three animals/group. Tissue was collected in the middle of the light phase (ZT05-07). p-Values: unpaired t-test, two-tailed. Note the difference in y-axis scale for Cplx4 in rods and cones. (c) RNAscope in situ hybridization (ISH) followed by immunocytochemistry (ICC) revealed that Cplx3 mRNA (green dots) is found primarily in the outer layer of the outer nuclear layer (ONL), where the somas of cones primarily stratify, in WT retinas. Cplx3 mRNA is primarily surrounding cone somas (white), designated by white arrows. In cone-Bmal1^−/− retinas, there is less Cplx3 mRNA in the outer ONL. DAPI (cyan) stains the cell nuclei. Scale bar: 25 μm (applies to all). (d) Vertical intensity plot of Cplx3 mRNA in WT (green) and cone-Bmal1^−/− (purple). Cplx3 mRNA was higher in the upper ONL where the somas of cones stratify. (e) Expression of Cplx mRNA in cone photoreceptors, rod bipolar cells, and rod photoreceptors based on published single-cell RNA-seq data (Shekhar et al., 2016). Cplx1 and Cplx2 are not highly expressed in any of these cell types. Cplx3 mRNA is primarily in cones and rod bipolar cells. Cplx4 is primarily expressed in rods and cones. Cones (n = 14); rod bipolar cells (n = 7,784); and rods (n = 32). Error bars show SEM