Vsx2 is a paired-type homeodomain protein that is expressed in all retinal progenitor cells and in mature bipolar neurons and Mller glia. Mutations in the Vsx2 gene cause microphthalmia in humans and mice because it is required to regulate retinal progenitor cell proliferation. Due to this early developmental phenotype, it has been difficult to elucidate the role of Vsx2 in bipolar and Mller glial cell differentiation. To address this barrier in the field, we characterized the regulatory regions upstream of the Vsx2 gene. We identified a core regulatory circuit super-enhancer (CRC-SE) upstream of Vsx2 that is specific to bipolar neurons. Within the CRC-SE is a consensus binding site for Vsx2. When we deleted the Vsx2 CRC-SE in mice, there is a complete loss of bipolar cells with no effect on retinal progenitor cells or Mller glia. This is the first bipolar-less animal model and the first developmental-stage and cell-type-specific SE to be identified. The purpose of this study is to understand how the developmental-stage and cell-type-specific CRC-SE regulates Vsx2 expression for bipolar cell fate specification. First, I will dissect the CRC-SE and determine the genomic regions that are driving bipolar cell development. Four evolutionarily conserved regions within the CRC-SE including the Vsx2 consensus site will be knocked out in mice using CRISPR/Cas9. I will assess whether these regions are necessary for bipolar cell development through immunohistochemistry and RNA sequencing. Upon identification of a critical conserved region, I will assess its individual ability to drive bipolar cell development in a rescue experiment. Chromatin interactions involving the Vsx2 promoter during the birth of bipolar cells will be elucidated through chromosome conformation capture-on-chip (4C). Lastly, I will elucidate factors required for bipolar cell specification in the developing retina by examining the fate of retinal progenitor cells in the CRC-SE knockout mouse that would have normally differentiated into bipolar cells. We have extended our analysis beyond Vsx2 and found dozens of other cell and developmental-stage specific CRC-SEs in the developing retina. This suggests that our strategy may be broadly applied in the developing central nervous system to elucidate the cellular roles of key transcription factors in development and differentiation.
Recently we identified a regulatory element upstream of a key transcription factor gene that is necessary for retinal development, Vsx2. When this regulatory element is knocked out in mice there is no severe early developmental phenotype, but strikingly, there is a complete loss of a single retinal cell type, bipolar cells. Using this model I will elucidate the regulatory elements of Vsx2 that determine bipolar cell fate.
