Prdm8 and Bhlhb4 in the retina and CNS

Abstract

Purpose: Understanding the genetic regulation of retinal development is key to advancing therapies for disease. Bhlhb4 is a transcription factor (TF) essential to the maturation of the major class of retinal interneurons, rod bipolar cells (RBs). Adult Bhlhb4^-/- mice have b wave deficits on dark adapted ERGs, a defect of RB signalling that models the human disorder Congenital Stationary Night Blindness. RBs are born in normal numbers in Bhlhb4^-/- retina but die from PN8, suggesting Bhlhb4 is required for RB survival. In the CNS, Bhlhb4 is expressed the developing forebrain and in adult cerebellum and hippocampus. We describe a second mouse model lacking the TF, Prdm8, that phenocopies the Bhlhb4^-/- model. Both TFs regulate the final stages of RB development in the retina and have parallel functions in the regulation of interneuron development elsewhere in the CNS.

Methods: Prdm8 and Bhlhb4 expression in wildtype (wt) retina and CNS was analysed by immunofluorescence using cell specific markers. Retinal transcriptomes of Prdm8^-/-, Bhlhb4^-/- and wt mice at PN6 were compared using Affymetrix Murine Genome 430 2.0 arrays with a false discovery rate (FDR) of up to 10%. Differential gene expression between mutant and littermatched wt retina was quantified by rtPCR. Morphological differences between mutant and wt brains were analysed by MRI; and neurobehavioural phenotypes with SHIRPA screening tests and the Morris water maze.

Results: Adult Prdm8^-/- and Bhlhb4^-/- retinas lack RBs and demonstrate b wave deficits on dark adapted ERGs, consistent with compromised RB circuitry. In both models, RBs are born in normal numbers but die apoptotically from PN8. Prdm8 and Bhlhb4 are co-expressed in RBs at PN5 to PN12 and from PN14 to adulthood. At PN6, Bhlhb4 is downregulated in Prdm8^-/- retina (p=0.003) whilst Prdm8 expression is unchanged in Bhlhb4^-/- retina (p=0.5). Non-overlapping genes are differentially expressed in Prdm8^-/- and Bhlhb4^-/- retina at PN6. In the latter, the most significantly downregulated genes, c-Rel and Neu3 (FDR=0%), are implicated in apoptosis. In the CNS, Prdm8 and Bhlhb4 are coexpressed in adult cerebellum and hippocampus. Prdm8^-/- mice demonstrate neurobehavioural deficits and MRI changes consistent with impaired hippocampal and cerebellar development that overlap with deficits in Bhlhb4^-/- mice.

Conclusions: We propose a model in which Prdm8 and Bhlhb4 coordinately regulate the development of specific interneuron subtypes in retina and brain.

Keywords: 435 bipolar cells • 497 development • 739 transcription factors