Abstract
Purpose :
The molecular mechanisms underlying developmental regulation of the transcription factor PITX2/pitx2 remain unknown. In this study we create zebrafish models of large genomic deletions to determine the regulatory regions upstream of PITX2/pitx2 critical for ocular development.
Methods :
CRISPR/Cas9 was used to generate deletions removing different conserved elements (CEs) upstream of pitx2, including CE4, CE5, CE7, CE10, predicted to be associated with its ocular expression/function. Mutant zebrafish were analyzed for abnormal phenotypes using light microscopy and alcian blue staining. In situ hybridization was used to detect distribution of both pitx2 isoforms during development and qPCR to assess their expression level. In silico transcription factor binding predictions were performed using MatInspector, CONSITE, JASPAR and TRAP.
Results :
Zebrafish lines carrying 4.5-kb and 60-kb genomic deletions encompassing CE4 and CE5-7, respectively, have been established. Embryos homozygous for a deletion encompassing CE4 or CE5-7 were grossly normal during the first 5 days of development. By 7dpf, embryos displayed significantly reduced volume of the anterior chamber: CE4 (24.86 + 3.7, p<0.029, n=10 eyes) or CEs 5-7 (18.76 + 3.3, p<2.5E-05, n=14 eyes) compared to WT (29.75 + 5.9, n=16 eyes). Alcian blue revealed no abnormal development of craniofacial cartilages. In situ hybridization to detect pitx2 transcript did not show any obvious differences in transcript distribution; qPCR analyses are ongoing. Bioinformatic analysis of the CE4 and 5-7 showed enrichment for binding sites for transcription factors from the SOX (2, 3, 9, 10) family and retinoic acid binding proteins as well as many other ocular factors. Luciferase assays to verify their interactions with the regulatory elements is underway.
Conclusions :
Our data show that removing conserved sequences upstream of pitx2 previously shown to drive reporter expression in a pattern consistent with pitx2 ocular activity results in abnormal anterior chamber development. This further confirms the important role of these elements in the regulation and maintenance of normal pitx2 dosage during eye development. Identification of transcription factors that interact with these elements will help to dissect the upstream signaling cascade leading to proper expression of pitx2 during ocular development.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.