NKX2.2 is required for timely NEUROG3 extinction and segregation of competing lineage programs in human pancreatic endocrine progenitors

Pancreatic endocrine progenitors commit to the NEUROG3+ lineage before resolving into mono-hormonal cell types. The homeodomain factor NKX2.2 is required for islet formation in mice and humans, but its role at the endocrine progenitor stage is undefined. We generated NKX2.2-null human embryonic stem cells and differentiated them towards endocrine lineages. Mutant progenitors induced NEUROG3 and formed endocrine committed cells at near wild-type frequency. Single cell transcriptomics showed persistent NEUROG3 in mutant cells running , {beta} and {delta} programs, co-activation of the mutually exclusive ARX and PAX4 networks, and no terminal lineage factor induction. Reanalysis of published islet NKX2.2 ChIP-seq showed that chromatin state at bound elements, not occupancy alone, predicted the direction of transcriptional change, including at NEUROG3, NKX6.1, ARX and PAX4. End-stage cultures yielded few insulin+ cells, with excess somatostatin, ghrelin, polyhormonal and hormone-negative cells. NKX2.2 is therefore dispensable for endocrine specification but required for resolution of endocrine fates.