Abstract
Purpose: :
We previously reported a two-fold increase in horizontal cell number from the A/J to the C56BL/6J (B6/J) strain (Reese et al., ARVO Abs. 2008, 49:3738). Using recombinant inbred strains we had mapped this variation to the distal end of Chr 13. Analysis of the interval underlying this QTL identified the transcription factor Isl1 as a promising candidate. Conditional knockout mice of Isl1 were shown to have a significant increase in horizontal cell number. The current study focuses on multiple lines of evidence supporting Isl1 as the causative gene.
Methods: :
Sequencing of the Isl1 cDNA was conducted following 5’ and 3’ UTR RACE. qPCR was used to determine the abundance of Isl1. Allelic-specific expression (ASE) of Isl1 was measured in retinas of reciprocal F1 crosses of B6/J and A/J. Two predictive programs of transcription factor binding, TESS and MATCH, were used to identify the functional impact of sequence differences between B6/J and A/J.
Results: :
We identified two SNPs in the promoter of Isl1 which may affect differences in the level of transcription between the two strains. Indeed, Isl1 expression is significantly higher in A/J relative to B6/J. Analysis of ASE showed significantly greater expression of Isl1 A alleles relative to B alleles, in both B6AF1 and AB6F1 samples, confirming the cis-regulation of this gene. Both predictive programs showed the binding of bHLH factors being affected by one of the promoter SNPs (rs545658554). This family of transcriptional regulators targets the sequence known as an E-box (CANNTG), present in the B allele and not the A allele. Interestingly, A is the conserved allele, seen in multiple other mammalian species and in several wild-derived mouse strains.
Conclusions: :
The creation of a novel E-box sequence within the promoter of B6/J provides a putative target for bHLH transcriptional regulators such as Neurod1 and E2a, each with documented roles in retinal neurogenesis. This differential control of Isl1 between B6/J and A/J may underlie the variation in horizontal cell number seen between the strains.
Keywords: candidate gene analysis • retinal development • transcription factors