Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation

How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag-seq, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2, and Isl1, and uncover their roles in shaping the epigenetic landscape and controlling gene expression in a sequential and combinatorial fashion along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs). There are 6 sample for scATAC-seq with cells isolated by FACS from E14.5 and E17.7 mouse retinas: Atoh7-Null-E14.5 (zsGreen+ cells from Atoh7-zsGreen/lacZ retinas), Atoh7-Null-E17.5 (zsGreen+ cells from Atoh7-zsGreen/lacZ retina), Atoh7-WT-E14.5 (zsGreen+ cells from Atoh7-zsGreen/+ retinas), Atoh7-WT-E17.5 (zsGreen+ cells from Atoh7-zsGreen/+ retinas), Pou4f2-WT-E14.5 (tdTomato cells from Pou4f2-tdTomato/+ retinas), Pou4f2-WT-E17.5 (tdTomato cells from Pou4f2-tdTomato/+ retinas). scRNA-seq were also performed with the same cell populations. There were also one sample of E14.5 Atoh7/Pou4f2-double negative sample (zsGreen/tdTomaton double negative cells from Atoh7-zsGreen/+;Pou4f2-tdTomaton/+ retinas) for scATAC-seq. There are 4 targets for CUT&Tag on wild type E14.5 mouse retina cells with 2 replicates: Atoh7, Pou4f2, ISL1, OTX2. For Atoh7, anti-HA was used with Atoh7-HA/HA retinas. For Pou4f2, anti-HA was used with Pou4f2-HA/HA retinas. For Otx2, multiple preparations were pooled into two independent replicates. Normal rabbit IgG was used as target blank control. Note that some libraries (Pou4f2_HA Rep1, Isl1 Rep2, and are sequenced twice, and Rabbit IgG Rep1) were sequenced twice. These read were combined as one replicate in the analysis.