Abstract
Purpose:
BHLHB4 is a transcription factor essential to the maturation of rod bipolar cells (RBs). Adult Bhlhb4-/- mice lack RBs; whilst born in normal numbers, RBs die from postnatal day 8 (PN8) in Bhlhb4-/- retina, suggesting BHLHB4 is required for RB survival. Here we compare the transcriptomes of developing and adult Bhlhb4-/- retina to wildtype (WT) in order to identify novel genetic markers of RBs that may also be important for RB development and/or function.
Methods:
Bhlhb4-/- and WT retinal transcriptomes were compared using gene expression microarrays in adult mice (Affymetrix, n=4) and by RNAseq in PN7 mice (n=6): a time that immediately predates significant differences in RB numbers between Bhlhb4-/- and WT retina. Candidate genes were investigated for co-expression with known RB-specific markers in mature RBs using immunofluorescence (IF) and evaluated for differential expression in developing Bhlhb4-/- RBs compared to WT at PN7 with rtPCR and IF.
Results:
In adult mice, 97 genes are downregulated in Bhlhb4-/- retina compared to WT (false discovery rate (FDR) <10%). These genes are likely to include novel molecular markers of RBs, since RBs are absent in Bhlhb4-/- retina. Indeed, several known RB-specific genes are significantly downregulated in adult Bhlhb4-/- retina, including Prkca (fold change (FC) =0.43) and Car8 (FC=0.14). We have confirmed RB-specific expression of novel RB markers derived from this experiment including Lrrtm4 (FC=0.37), a known regulator of synaptic development which is expressed in RB dendrites.<br /> <br /> At PN7, 50 genes are misexpressed in Bhlhb4-/- retina compared to WT (FDR<10%): 23 of which are misregulated in both adult and PN7 Bhlhb4-/- retina (FDR<50%). These genes are likely to include novel specific markers of RBs and genes required for RB development and/or function. For example, Car8, which we identified as a RB-specific marker in the adult experiment, is also significantly misexpressed in PN7 Bhlhb4-/- retina compared to WT (FC=0.52, FDR<5%) and rtPCR (n=8, FC=0.67, p=0.013). Moreover, we have confirmed CAR8 protein expression is downregulated in developing RBs in Bhlhb4-/- retina compared to WT by IF of PN7 retinal sections.
Conclusions:
We have identified a number of novel molecular markers of RBs, likely to have important roles in RB development and/or function. In addition, we propose Car8 as a candidate target gene for regulation by BHLHB4.