June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Identification of an enhancer that regulates Otx2 expression during early retinal differentiation
Author Affiliations & Notes
  • Punita Bhansali
    Ophthalmology, Albert Einstein College of Medicine , New York, New York, United States
  • Ales Cvekl
    Ophthalmology, Albert Einstein College of Medicine , New York, New York, United States
  • Wei Liu
    Ophthalmology, Albert Einstein College of Medicine , New York, New York, United States
  • Footnotes
    Commercial Relationships   Punita Bhansali, None; Ales Cvekl, None; Wei Liu, None
  • Footnotes
    Support  NH Grant EY022645, EY012200, RPB unrestricted grant
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 123. doi:
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      Punita Bhansali, Ales Cvekl, Wei Liu; Identification of an enhancer that regulates Otx2 expression during early retinal differentiation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):123.

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

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Abstract

Purpose : Photoreceptors are critical for proper functioning of the eye and their dysfunction is the cause of various retinal disorders. Transcription factor Otx2 is at the top of a gene regulatory network for photoreceptor differentiation. To dissect the regulatory elements that guide expression of Otx2, we focus our attention on a highly conserved enhancer upstream of this gene. We hypothesize that this enhancer directs proper expression of Otx2 during early retinal development and aim to dissect the core regulatory elements of this enhancer and identify regulators of this enhancer.

Methods : The enhancer sequence was identified using the VISTA Enhancer Browser. We generated and characterized a transgenic reporter mouse line to assess whether this enhancer, which we will refer to as ERDE (early retinal development enhancer), is active in Otx2-positive cells fated to become photoreceptors. Moreover, we generated two additional lines to assess which regions of the enhancer are critical for its regulation of Otx2. Thus, LacZ was expressed under the control of the full-length 2kb enhancer (2K-ERDE), or distinct fragments of the enhancer that exhibit evolutionary conservation across species (0.4kb region and 1.2kb region, referred to as 0.4K-ERDE and 1.2K-ERDE, respectively). To ascertain whether reporter expression can replicate Otx2 expression in retinal cells during early eye development, we used Xgal staining in wholemount embryos and sections through the retina, as well as immunohistochemistry in sections through the retina.

Results : These experiments have revealed that LacZ expression in all three reporter mouse lines can recapitulate early expression of Otx2 in the retina during embryonic development. LacZ expression starts in the neuroretina at around E11.5-E12.5, and is co-localized with Otx2 expression at its early stages. These results suggest that ERDE is active in cells fated to become photoreceptors when Otx2 is first turned on. The peak of co-localization between Otx2 and LacZ in the neural retina in all three reporter lines occurs between E12.5-E14.5. Our data from 1.2K-ERDE and 0.4K-ERDE reporter lines suggest redundant mechanisms of these two fragments for regulation of Otx2.

Conclusions : We identified an enhancer that regulates Otx2 expression in the retina during early photoreceptor specification and differentiation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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