Abstract / Description of output
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) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between Ie and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome wide reduction in binding of transcription factors associated with ventral eye development in Ie, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the Ie-refined critical interval.
We conclude that the refined Ie critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered cis regulation but does not contain a convincing causative mutation. Changes in the binding of key
transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of 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.
Keywords / Materials (for Non-textual outputs)
- X-ray induced allele
- linkage analysis
- genome wide analysis
- eye development