Robust genetic analysis of the X-linked anophthalmic (Ie) mouse

Abstract

Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families currently are left without a molecular diagnosis. Here we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked Ie (eye-ear reduction) X-ray induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~ 6Mb region on the X chromosome. Short and long read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the Ie linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to Ie. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male Ie embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) using cultured optic vesicle organoids were unable to determine any significant differences between Ie and wild type at the transcription or gene regulation levels. How-ever, an in silico genome-wide motif analysis in Ie accessible chromatin suggested there may be subtle mis-regulation of transcription factors associated with ventral eye development and in-creased Zic family transcription factor binding, including Zic3, which is located within the Ie re-fined critical interval. We conclude that the refined Ie critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that can be linked to altered cis regulation but does not contain a convincing causative mutation. Mis-regulation of transcription factor binding to chromatin causing altered gene expression during development, possibly through changes to Zic3, presents a plausible cause for the anophthalmia phenotype observed in Ie, but further work is required to determine the precise causative allele and its genetic mechanism.