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

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 with 25% families currently 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 read whole-genome sequencing (WGS) of affected and unaffected male littermates identified no plausible causative variants, and long-read WGS failed to identify any structural rearrangements. In addition, embryonic head cDNA analysis using a tiled expression array for the mouse X chromosome did not identify any major transcriptomic anomaly. To investigate earlier embryonic events, we 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. A genome-wide analysis of the bound regions in the accessible chromatin suggests there may be a subtle mis-regulation of Zic3, which is a strong candidate gene within the linkage interval. We conclude that multiple mutations in the region chrX: 56,145,000-58,385,000 are linked to altered Zic3 cis-regulation and together are the likely cause of Ie anophthalmia.