Initiation of Otx2 expression in the developing mouse retina requires a unique enhancer and either Ascl1 or Neurog2 activity

Development. 2021 Jun 15;148(12):dev199399. doi: 10.1242/dev.199399. Epub 2021 Jun 18.


During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation. How these retinal progenitor cells initially activate Otx2 expression is unclear. To address this, we investigated the cis-regulatory network that controls Otx2 expression in mice. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. CRISPR/Cas9-mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor-binding sites were required for its activity. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Our findings suggest that Ascl1 and Neurog2 act either redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. We observed redundancy or compensation at both the transcriptional and enhancer utilization levels, suggesting that the mechanisms governing Otx2 regulation in the retina are flexible and robust.

Keywords: Cis-regulation; Ascl1; Enhancers; Neurog2; Otx2; Retinal development.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Transport System y+ / genetics*
  • Animals
  • Base Sequence
  • Basic Helix-Loop-Helix Transcription Factors / genetics*
  • E-Box Elements
  • Enhancer Elements, Genetic*
  • Fluorescent Antibody Technique
  • Gene Expression Regulation, Developmental*
  • Mice
  • Mice, Knockout
  • Mice, Transgenic
  • Nerve Tissue Proteins / genetics*
  • Nucleotide Motifs
  • Organogenesis / genetics*
  • Otx Transcription Factors / genetics*
  • Otx Transcription Factors / metabolism
  • Retina / embryology
  • Retina / metabolism*


  • Amino Acid Transport System y+
  • Basic Helix-Loop-Helix Transcription Factors
  • Nerve Tissue Proteins
  • Neurog2 protein, mouse
  • Otx Transcription Factors
  • Otx2 protein, mouse
  • Slc7a10 protein, mouse