Insulinoma associated 1 (Insm1) plays an important role in regulating the development of cells in the central and peripheral nervous systems, olfactory epithelium and endocrine pancreas. To better define the role of Insm1 in pancreatic endocrine cell development we generated mice with an Insm1(GFPCre) reporter allele and used them to study Insm1-expressing and null populations. Endocrine progenitor cells lacking Insm1 were less differentiated and exhibited broad defects in hormone production, cell proliferation and cell migration. Embryos lacking Insm1 contained greater amounts of a non-coding Neurog3 mRNA splice variant and had fewer Neurog3/Insm1 co-expressing progenitor cells, suggesting that Insm1 positively regulates Neurog3. Moreover, endocrine progenitor cells that express either high or low levels of Pdx1, and thus may be biased towards the formation of specific cell lineages, exhibited cell type-specific differences in the genes regulated by Insm1. Analysis of the function of Ripply3, an Insm1-regulated gene enriched in the Pdx1-high cell population, revealed that it negatively regulates the proliferation of early endocrine cells. Taken together, these findings indicate that in developing pancreatic endocrine cells Insm1 promotes the transition from a ductal progenitor to a committed endocrine cell by repressing a progenitor cell program and activating genes essential for RNA splicing, cell migration, controlled cellular proliferation, vasculogenesis, extracellular matrix and hormone secretion.

Pancreatic multipotent progenitor cells (MPCs) produce acinar, endocrine and duct cells during organogenesis, but their existence and location in the mature organ remain contentious. We used inducible lineage-tracing from the MPC-instructive gene Ptf1a to define systematically in mice the switch of Ptf1a(+) MPCs to unipotent proacinar competence during the secondary transition, their rapid decline during organogenesis, and absence from the mature organ. Between E11.5 and E15.5, we describe tip epithelium heterogeneity, suggesting that putative Ptf1a(+)Sox9(+)Hnf1β(+) MPCs are intermingled with Ptf1a(HI)Sox9(LO) proacinar progenitors. In the adult, pancreatic duct ligation (PDL) caused facultative reactivation of multipotency factors (Sox9 and Hnf1β) in Ptf1a(+) acini, which undergo rapid reprogramming to duct cells and longer-term reprogramming to endocrine cells, including insulin(+) β-cells that are mature by the criteria of producing Pdx1(HI), Nkx6.1(+) and MafA(+). These Ptf1a lineage-derived endocrine/β-cells are likely formed via Ck19(+)/Hnf1β(+)/Sox9(+) ductal and Ngn3(+) endocrine progenitor intermediates. Acinar to endocrine/β-cell transdifferentiation was enhanced by combining PDL with pharmacological elimination of pre-existing β-cells. Thus, we show that acinar cells, without exogenously introduced factors, can regain aspects of embryonic multipotentiality under injury, and convert into mature β-cells.

The timing and gene regulatory logic of organ-fate commitment from within the posterior foregut of the mammalian endoderm is largely unexplored. Transient misexpression of a presumed pancreatic-commitment transcription factor, Ptf1a, in embryonic mouse endoderm (Ptf1a(EDD)) dramatically expanded the pancreatic gene regulatory network within the foregut. Ptf1a(EDD) temporarily suppressed Sox2 broadly over the anterior endoderm. Pancreas-proximal organ territories underwent full tissue conversion. Early-stage Ptf1a(EDD) rapidly expanded the endogenous endodermal Pdx1-positive domain and recruited other pancreas-fate-instructive genes, thereby spatially enlarging the potential for pancreatic multipotency. Early Ptf1a(EDD) converted essentially the entire glandular stomach, rostral duodenum and extrahepatic biliary system to pancreas, with formation of many endocrine cell clusters of the type found in normal islets of Langerhans. Sliding the Ptf1a(EDD) expression window through embryogenesis revealed differential temporal competencies for stomach-pancreas respecification. The response to later-stage Ptf1a(EDD) changed radically towards unipotent, acinar-restricted conversion. We provide strong evidence, beyond previous Ptf1a inactivation or misexpression experiments in frog embryos, for spatiotemporally context-dependent activity of Ptf1a as a potent gain-of-function trigger of pro-pancreatic commitment.

The transcription factor Pdx1 is required for multiple aspects of pancreatic organogenesis. It remains unclear to what extent Pdx1 expression and function depend upon trans-activation through 5' conserved cis-regulatory regions and, in particular, whether the mammal-specific Area II (-2139 to -1958 bp) affects minor or major aspects of organogenesis. We show that Area II is a primary effector of endocrine-selective transcription in epithelial multipotent cells, nascent endocrine progenitors, and differentiating and mature β cells in vivo Pdx1ΔAREAII/- mice exhibit a massive reduction in endocrine progenitor cells and progeny hormone-producing cells, indicating that Area II activity is fundamental to mounting an effective endocrine lineage-specification program within the multipotent cell population. Creating an Area II-deleted state within already specified Neurog3-expressing endocrine progenitor cells increased the proportion of glucagon+ α relative to insulin+ β cells, associated with the transcriptional and epigenetic derepression of the α-cell-determining Arx gene in endocrine progenitors. There were also glucagon and insulin co-expressing cells, and β cells that were incapable of maturation. Creating the Pdx1ΔAREAII state after cells entered an insulin-expressing stage led to immature and dysfunctional islet β cells carrying abnormal chromatin marking in vital β-cell-associated genes. Therefore, trans-regulatory integration through Area II mediates a surprisingly extensive range of progenitor and β-cell-specific Pdx1 functions.