Single-cell chromatin accessibility identifies enhancer networks driving gene expression during spinal cord development in mouse.

Spinal cord development is precisely orchestrated by spatiotemporal gene regulatory programs. However, the underlying epigenetic mechanisms remain largely elusive. Here, we profiled single-cell chromatin accessibility landscapes in mouse neural tubes spanning embryonic days 9.5-13.5. We identified neuronal-cell-cluster-specific cis-regulatory elements in neural progenitors and neurons. Furthermore, we applied a novel computational method, eNet, to build enhancer networks by integrating single-cell chromatin accessibility and gene expression data and identify the hub enhancers within enhancer networks. It was experimentally validated in vivo for Atoh1 that knockout of the hub enhancers, but not the non-hub enhancers, markedly decreased Atoh1 expression and reduced dp1/dI1 cells. Together, our work provides insights into the epigenetic regulation of spinal cord development and a proof-of-concept demonstration of enhancer networks as a general mechanism in transcriptional regulation.

Three distinct Atoh1 enhancers cooperate for sound receptor hair cell development.

Cochlear hair cells (HCs) in the inner ear are responsible for sound detection. For HC fate specification, the master transcription factor Atoh1 is both necessary and sufficient. Atoh1 expression is dynamic and tightly regulated during development, but the cis-regulatory elements mediating this regulation remain unresolved. Unexpectedly, we found that deleting the only recognized Atoh1 enhancer, defined here as Eh1, failed to impair HC development. By using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we discovered two additional Atoh1 enhancers: Eh2 and Eh3. Notably, Eh2 deletion was sufficient for impairing HC development, and concurrent deletion of Eh1 and Eh2 or all three enhancers resulted in nearly complete absence of HCs. Lastly, we showed that Atoh1 binds to all three enhancers, consistent with its autoregulatory function. Our findings reveal that the cooperative action of three distinct enhancers underpins effective Atoh1 regulation during HC development, indicating potential therapeutic approaches for HC regeneration.

Single-cell transcriptomic landscapes of the otic neuronal lineage at multiple early embryonic ages.

Inner ear vestibular and spiral ganglion neurons (VGNs and SGNs) are known to play pivotal roles in balance control and sound detection. However, the molecular mechanisms underlying otic neurogenesis at early embryonic ages have remained unclear. Here, we use single-cell RNA sequencing to reveal the transcriptomes of mouse otic tissues at three embryonic ages, embryonic day 9.5 (E9.5), E11.5, and E13.5, covering proliferating and undifferentiated otic neuroblasts and differentiating VGNs and SGNs. We validate the high quality of our studies by using multiple assays, including genetic fate mapping analysis, and we uncover several genes upregulated in neuroblasts or differentiating VGNs and SGNs, such as Shox2, Myt1, Casz1, and Sall3. Notably, our findings suggest a general cascaded differentiation trajectory during early otic neurogenesis. The comprehensive understanding of early otic neurogenesis provided by our study holds critical implications for both basic and translational research.

Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells.

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represents a barrier to restore auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single-cell RNA sequencing revealed the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.