Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals putative regulatory toggles operating in native and iPS-derived retina
AbstractBackgroundIt has recently become possible to recapitulate retinal development from induced pluripotent stem cells, opening new investigative and therapeutic opportunities. Single cell RNA sequencing allows comparison of transcriptome unfolding during in vivo and in vitro development at single cell resolution, which can be integrated with information about accessible regulatory elements identified by ATAC-Seq.ResultsWe report the generation and analysis of single-cell RNA-Seq data (> 38,000 cells) from native and iPSC-derived murine retina at four matched developmental stages spanning the emergence of the major retinal cell types. We combine information from temporal sampling, visualization of 3D UMAP manifolds, and RNA velocity to show that iPSC-derived 3D retinal aggregates broadly recapitulate the native developmental trajectories with evidence supporting re-specification from amacrine cells to horizontal and photoreceptor precursor cells, as well as a direct differentiation of Tbr1+ retinal ganglion cells from neuro-epithelium cells. We show relaxation of spatial and temporal transcriptome control, premature emergence and dominance of photoreceptor precursor cells, and susceptibility of dynamically regulated pathways and transcription factors to culture conditions in iPSC-derived retina. We generate bulk ATAC-Seq data for native and iPSC-derived murine retina identifying ∼125,000 peaks. We combine single-cell RNA-Seq with ATAC-Seq information and obtain evidence that approximately halve the transcription factors that are dynamically regulated during retinal development may act as repressors rather than activators. We propose that sets of activators and repressors with cell-type specific expression control “regulatory toggles” that lock cells in distinct transcriptome states underlying differentiation, with subtle but noteworthy differences between native and iPSC-derived retina.ConclusionsCombined analysis of single-cell RNA-Seq and ATAC-Seq information has refined the comparison of native and iPS-derived retinal development.