June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
An in vivo investigation of the upstream regulatory elements of PITX2/pitx2 through generation of large deletions of conserved enhancer elements in zebrafish
Author Affiliations & Notes
  • Eric Weh
    Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Elena Semina
    Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Eric Weh, None; Elena Semina, None
  • Footnotes
    Support  R01EY015518
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3003. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Eric Weh, Elena Semina; An in vivo investigation of the upstream regulatory elements of PITX2/pitx2 through generation of large deletions of conserved enhancer elements in zebrafish. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3003.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
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.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×