Introduction: All forms of diabetes result from loss or impairment of pancreatic beta cell function. Recent work demonstrating the feasibility of replacing beta cells with insulin-producing cells generated from renewable sources such as embryonic stem cells, has sparked great excitement. However, the molecular and cellular processes leading to beta cell differentiation are not well understood. During embryonic development, the activation of Neurog3 in select pancreatic duct cells specifies the endocrine progenitors, which will then differentiate into hormone producing cells, such as beta, alpha, and delta cells. Despite recent progress, molecular features of endocrine progenitors remain elusive, and how subsequent hormone cell fates are determined is largely unknown.

Method and Results: In this study, we used lineage-traced Neurog3 cells from mouse embryos to perform single cell transcriptome analysis to elucidate gene expression changes during differentiation. By establishing pseudotime trajectories and using differential gene expression analysis, we identified unique combinations of transcription factors for each of the beta, alpha, and delta lineages. We found that these transcription factors turn on and off at precise moments to guide endocrine cell differentiation. In addition, we performed ATAC-seq to investigate chromatin conformation changes in endocrine progenitor cells. We discovered genomic regions that undergo substantial transformation during endocrine differentiation as well as enriched motifs in differentiation-stage specific open regions. Further, we applied genomic footprinting analysis to identify transcription factor activity in open chromatin regions and found evidence of specific transcription factor footprints linked to their associated motifs. Our analysis suggests a revised model for endocrine pancreas development by providing evidence for direct development of this lineage from duct cells and the absence of a bipotent progenitor.

Conclusions: Our results demonstrate the feasibility of using a combined scRNA-seq and ATAC-seq analysis to gain insights into gene regulatory networks that define cell lineages. We anticipate our findings from these and similar efforts will reveal gene regulatory networks governing pancreatic endocrine development, and facilitate the efforts toward stem cell based therapies and tissue replacement.