Characterization of an Eye Field-like State during Optic Vesicle Organoid Development [ATAC-seq]

Specification of the eye field (EF) within the neural plate marks the earliest detectable stage of eye development. Experimental evidence, primarily from non-mammalian model systems, indicates that the stable formation of this group of cells requires the activation of a set of key transcription factors (TFs). This critical event is challenging to probe in mammals and, quantitatively, little is known regarding the regulation of the transition of cells to this ocular fate. Using optic vesicle organoids to model the onset of the EF, we generate timecourse transcriptomic data allowing us to identify dynamic gene-expression programs that characterise this cellular-state transition. Integrating this with chromatin accessibility data suggests a direct role of canonical EFTFs in regulating these gene-expression changes, and highlights candidate cis-regulatory elements through which these TFs act. Finally, we begin to test a subset of these candidate enhancer elements, within the organoid system, by perturbing the underlying DNA sequence and measuring transcriptomic changes during EF activation. ATAC sequencing was performed on a timecourse through optic vesicle organoid differentiation grown from Rx-GFP mouse ESCs. A sample was collected every 24 hours from day 0 (defined as the day organoids are seeded) to day 3 and at day 5. On day 5, cells were sorted using FACS on the basis of GFP expression to produce 2 samples for this timpoint. GFP has been knocked into the Rax locus in this cell line, meaning GFP positive samples correspond to cells expressing Rax and committed to an eye field lineage.