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Fisher otx12/20/2023 By determining which sequences were necessary for DHS-4 expression in the retina, we reasoned that the repertoire of transcription factors that activate Otx2 could be identified. Based on this pattern, we hypothesized that DHS-4 acts by transiently initiating Otx2 expression as progenitors exit the cell cycle. Compared with the other elements, DHS-4 expression was highest at postnatal day (P) 0 and became severely restricted by P7 ( Wilken et al., 2015). Three DHS sequence elements were able to drive reporter gene expression in OTX2+ retinal cells. We previously used DNase I hypersensitivity site (DHS) sequencing to identify candidates within the mouse retina ( Wilken et al., 2015). To reduce the number of potential Otx2 regulators, we searched for enhancers that coincide with Otx2 activation during retinal development. Enhancers provide spatial and temporal specificity to gene expression programs throughout development ( Benoist and Chambon, 1981 Gillies et al., 1983 Long et al., 2016). Which of these transcription factors, if any, controls Otx2 activation during retinal development is unknown.Ī major component of gene regulatory networks is noncoding cis-regulatory DNA sequences known as enhancers. Progenitors express a wide variety of transcription factors, including several from the basic helix-loop-helix (bHLH) class that control fate decisions throughout the central nervous system ( Hatakeyama and Kageyama, 2004 Hatakeyama et al., 2001 Wang and Harris, 2005). Given that this decision occurs in the last cell cycle, the gene regulatory events upstream of Otx2 occur in retinal progenitors. Thus, the decision to express Otx2 greatly influences the cell fate composition of the retina. Otx2 mutants lack photoreceptors and bipolar cells, but instead generate excess amacrine interneurons ( Ghinia Tegla et al., 2020 Nishida et al., 2003 Sato et al., 2007 Yamamoto et al., 2020). Loss-of-function studies show that Otx2 regulates fate choice in the retina. Otx2 is quickly lost in amacrines and horizontals, but is maintained by photoreceptors and bipolar cells into adulthood ( Emerson et al., 2013 Koike et al., 2007 Nishida et al., 2003). These Otx2+ cells appear to give rise to five neuronal types: horizontal, amacrine and bipolar interneurons, and rod and cone photoreceptors ( Baas et al., 2000 Brzezinski and Reh, 2015 Brzezinski et al., 2013 Emerson et al., 2013 Fossat et al., 2007 Nishida et al., 2003 Sato et al., 2007). Otx2 becomes upregulated in a large subset of progenitors as they exit the cell cycle ( Muranishi et al., 2011). Otx2 expression begins around embryonic day (E) 12, correlating with the earliest birthdates of photoreceptors ( Nishida et al., 2003). The homeobox transcription factor Otx2 is highly involved in retinal neurogenesis. Transcription factors play a major role in entrenching cell-fate choices within the retina. 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. Our findings suggest that Ascl1 and Neurog2 act either redundantly or in a compensatory fashion to activate the DHS-4D enhancer and Otx2 expression. CRISPR/Cas9 targeting of these factors showed that only the simultaneous loss of Ascl1 and Neurog2 prevented OTX2 expression. Single cell RNA-sequencing of nascent Otx2+ cells identified the bHLH factors Ascl1 and Neurog2 as candidate regulators. Systematic mutagenesis of the enhancer revealed that three basic helix-loop-helix (bHLH) transcription factor-binding sites were required for its activity. CRISPR/Cas9-mediated deletion of DHS-4D reduced OTX2 expression, but this effect was diminished in postnatal development. We identified a minimal enhancer element, DHS-4D, that drove expression in newly formed OTX2+ cells. To address this, we investigated the cis-regulatory network that controls Otx2 expression in mice. How these retinal progenitor cells initially activate Otx2 expression is unclear. During retinal development, a large subset of progenitors upregulates the transcription factor Otx2, which is required for photoreceptor and bipolar cell formation.
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