Deletions of distant regulatory sequences upstream of zebrafish pitx2 result in a range of ocular phenotypes

Development of the anterior segment of the vertebrate eye is a highly coordinated process. Genetic mutations in factors guiding this process result in Anterior Segment Dysgenesis (ASD), a spectrum of disorders affecting the iris, cornea, trabecular meshwork and/or other iridocorneal angle structures and associated with glaucoma. One of the first factors linked to ASD in humans was PITX2, a homeodomain containing transcription factor with a role in Axenfeld-Rieger syndrome (ARS). In addition to pathogenic alleles within the coding region of PITX2, deletions affecting the distant upstream region, but not PITX2 itself, have also been reported in ARS. Consistent with this, the distant upstream region was shown to contain multiple conserved elements (CE) with pitx2-related enhancer activity identified through studies in zebrafish. The two smallest human deletions reported to date encompass conserved elements 5-11 (ΔCE5-11) or 5-7 (ΔCE5-7). We previously reported the generation of ΔCE5-11 in zebrafish and we have now replicated the smallest deletion, ΔCE5-7, in the same model and studied the associated phenotype, expression, and DNA methylation profiles; we also performed further phenotypic examinations of the pitx2ΔCE5-11 fish. We show that the expression changes and phenotypes observed in the two lines are variable but that the severity generally correlates with the size of the deletion and the number of affected CEs; pitx2 promoter and a nearby region were hypermethylated in the pitx2ΔCE5-7 embryonic eyes. In addition, a subset of pitx2ΔCE5-11 animals were found to have a severe retinal phenotype suggesting that additional factors may modify the effects of this allele. These data provide further insight into functional sequences in the PITX2/pitx2 genomic region that coordinate PITX2/pitx2 expression during eye development and provide the basis for future studies into PITX2/pitx2 upstream regulators and modifiers.