Abstract
Purpose :
Mutations in Paired-like homeodomain transcription factor 2 (PITX2 [MIM 601542]) have been implicated in Axenfeld-Rieger syndrome (ARS [MIM 180500]), yet the pathways by which disrupted PITX2 causes the disease phenotype have yet to be fully elucidated. To address the question of downstream targets of PITX2, we generated and characterized zebrafish pitx2 knockout lines and utilized tissues from said mutant lines and wild-types for comparative transcriptome analyses.
Methods :
TALEN-mediated genome editing produced four mutant alleles within the conserved homeobox domain of both pitx2 isoforms; all alleles were predicted to be complete or partial loss-of-function. The c.190_197delATGTCGAC, p.(Met64*) frameshift line was selected for further studies. Phenotypic analysis of homozygous pitx2M64* mutant embryos and adults included gross morphology/light microscopy, Alcian blue staining, in situ hybridization/immunohistochemistry, histology, and transmission electron microscopy (TEM). Microarray analysis of RNA from 23 hours post fertilization (hpf) wild-type vs. homozygous pitx2M64* mutant whole eyes was performed and potential targets verified by quantitative PCR.
Results :
The pitx2M64* homozygous mutant phenotype involves a severely underdeveloped anterior chamber (100% of embryos) with a gap in the ventral ocular tissues (94-100%) and craniofacial abnormalities (60-70%). TEM of 72-hpf and 14 days post fertilization (dpf) pitx2M64* homozygous embryos indicated delayed differentiation of periocular mesenchyme-derived structures, including corneal endothelium, iris stroma, and iridocorneal angles, as well as defects in the neighboring tissues including corneal epithelium and ciliary zone non-pigmented epithelium. Comparison of whole eye mutant versus whole eye wild-type transcriptome identified 2226 downregulated and 2044 upregulated transcripts. RNA transcripts of seven wnt signaling pathway components and four collagens were confirmed to be reduced at 24-hpf, and nine of these transcripts were also down-regulated through 48- and 72-hpf.
Conclusions :
Disruption to the pitx2 homeodomain in zebrafish produced a mutant phenotype consistent with the features found in patients with ARS. The observed changes may be caused by disruption of localized Wnt signaling or reduced levels of some collagens during early stages of eye development.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.