June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Functional characterization of Otx2 enhancers used in mouse retinal development
Author Affiliations & Notes
  • IAN PURVIS
    Ophthalmology, University of Colorado, Denver, Colorado, United States
  • Michael Kaufman
    RNA Bioscience Initiative, University of Colorado, Denver, Colorado, United States
  • Omar Ochoa
    Ophthalmology, University of Colorado, Denver, Colorado, United States
  • Ko Park
    Ophthalmology, University of Colorado, Denver, Colorado, United States
  • Charles Henry
    Clemson University, Clemson, South Carolina, United States
  • Ami Haas
    Pathology, University of Colorado, Denver, Colorado, United States
  • Joseph A Brzezinski
    Ophthalmology, University of Colorado, Denver, Colorado, United States
  • Footnotes
    Commercial Relationships   IAN PURVIS None; Michael Kaufman None; Omar Ochoa None; Ko Park None; Charles Henry None; Ami Haas None; Joseph Brzezinski None
  • Footnotes
    Support  R01- EY024272
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1353 – F0284. doi:
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    • Get Citation

      IAN PURVIS, Michael Kaufman, Omar Ochoa, Ko Park, Charles Henry, Ami Haas, Joseph A Brzezinski; Functional characterization of Otx2 enhancers used in mouse retinal development. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1353 – F0284.

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

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Abstract

Purpose : Otx2 is a transcription factor necessary for the development of photoreceptor and bipolar cells in the retina. How Otx2 expression is regulated to produce these cell types is unknown. We identified three enhancers (DHS2, 4, and 15) that potentially explain the spatial and temporal regulation of Otx2 expression and perturbed them to determine how they control retinal development.

Methods : To determine enhancer activity, we constructed plasmids containing enhancer sequences driving a GFP or Cre recombinase reporter. These plasmids were electroporated into retinal explants cultured in vitro or into live newborn mouse retinas. To remove enhancer function, we electroporated CRISPR/Cas9 plasmid systems to delete the targeted sequences. To prevent enhancer activity, we epigenetically silenced the sequences by electroporating a dead Cas9 CRISPRi system. Immunohistochemistry was conducted to determine enhancer activity, OTX2 expression, and changes in cell fate in the electroporated retinas.

Results : Reporter assays and lineage tracing showed that the DHS2 and DHS15 enhancers were expressed in the developing retina, gave rise to photoreceptor and bipolar cells, and remained active in adult photoreceptors. In contrast, DHS4 had an earlier expression pattern that was limited to development. CRISPR-mediated knock-out of DHS4 caused a strong reduction in OTX2 expression embryonically, whereas targeting DHS2 and DHS15 resulted in a pronounced reduction of OTX2 expression postnatally. Targeting DHS4 with CRISPRi caused a strong reduction in OTX2 both embryonically and postnatally and resulted in an increase in amacrine cells.

Conclusions : DHS4 acts early in Otx2 regulation, while DHS2 and DHS15 enhancers act later. Perturbing these enhancers showed that each is important for Otx2 expression. The incomplete phenotypes seen upon deletion suggest that these enhancers act in a partially redundant or compensatory fashion during development, either with each other or with unidentified enhancers. This is supported by the stronger CRISPRi phenotype, which suggests that the Otx2 enhancers are looped in a complex where they can act interchangeably. Ongoing experiments are testing how these enhancers work together to control Otx2 expression and cell fate choice in the developing retina.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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