Defining multistep cell fate decision pathways during pancreatic development at single-cell resolution

The generation of terminally differentiated cell lineages during organogenesis requires multiple, coordinated cell fate choice steps. However, this process has not been clearly delineated, especially in complex solid organs such as the pancreas. Here, we performed single-cell RNA-sequencing in pancreatic cells sorted from multiple genetically modified reporter mouse strains at embryonic stages E9.5–E17.5. We deciphered the developmental trajectories and regulatory strategies of the exocrine and endocrine pancreatic lineages as well as intermediate progenitor populations along the developmental pathways. Notably, we discovered previously undefined programs representing the earliest events in islet alpha- and beta cell lineage allocation as well as the developmental pathway of the “first wave” of alpha-cell generation. Furthermore, we demonstrated that repressing ERK pathway activity is essential for inducing both alpha- and beta-lineage differentiation. This study provides key insights into the regulatory mechanisms underlying cell fate choice and stepwise cell fate commitment and can be used as a resource to guide the induction of functional islet lineage cells from stem cells in vitro. The overall goal of this study was to comprehensively define a fate map of mouse pancreatic lineage differentiation using the Smart-seq2 single-cell RNA-seq method. We used a Pdx1-GFP transgenic mouse line to purify E9.5 – E11.5 pancreatic progenitor cells; Pdx1-Cre; ROSA-RFP to isolate all pancreatic lineages at each day from E10.5 to E15.5. Because the percentage of endocrine cells in the pancreas is relatively low, to enrich the pancreatic endocrine lineages, we used an Ngn3-GFP knock-in mouse strain to sort GFP lower (Ngn3-GFPlow) and higher (Ngn3-GFPhigh) expressing cells from E13.5 to E15.5. Because Ngn3-GFPlow and Ngn3-GFPhigh cells could not be effectively separated by FACS at an earlier developmental stage and later stages, we generally sorted Ngn3+ cells at E12.5, E16.5 and E17.5. To obtain the differentiated beta and alpha cells, we sorted RFP+ and GFP+ cells from E15.5 Ins1-RFP and Gcg-P2A-GFP embryos, respectively. We purified pancreatic ductal cells at E16.5 and E17.5 from Sox9-CreER; ROSA-RFP mouse. To investigate the characteristics of the earlier differentiated endocrine cells, also called “first wave” endocrine cells, we purified Ngn3-GFP+ cells at E10.5 and E12.5, and Ngn3-Cre; ROSA-RFP+ cells at E11.5. To study whether ERK pathway represses pancreatic endocrine differentiation, we performed single-cell RNA-seq in Ins1-RFP+ cells from pancreas explants treated with U0126 (a MEK1/2 inhibitor). We also analyzed the datasets of E14.5 pancreatic cells generated by the 10X Genomics platform to parallelly compare the datasets generated using Smart-seq2 method.