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.

Extracellular signals in development, physiology, homeostasis and disease often act by regulating transcription. Herein we describe a general method and specific resources for determining where and when such signaling occurs in live animals and for systematically comparing the timing and extent of different signals in different cellular contexts. We used recombinase-mediated cassette exchange (RMCE) to test the effect of successively deleting conserved genomic regions of the ubiquitously active Rosa26 promoter and substituting the deleted regions for regulatory sequences that respond to diverse extracellular signals. We thereby created an allelic series of embryonic stem cells and mice, each containing a signal-responsive sentinel with different fluorescent reporters that respond with sensitivity and specificity to retinoic acids, bone morphogenic proteins, activin A, Wnts or Notch, and that can be adapted to any pathway that acts via DNA elements.

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.

A complete molecular understanding of β-cell mass expansion will be useful for the improvement of therapies to treat diabetic patients. During normal periods of metabolic challenges, such as pregnancy, β-cells proliferate, or self-renew, to meet the new physiological demands. The transcription factor Forkhead box D3 (Foxd3) is required for maintenance and self-renewal of several diverse progenitor cell lineages, and Foxd3 is expressed in the pancreatic primordium beginning at 10.5 d postcoitum, becoming localized predominantly to β-cells after birth. Here, we show that mice carrying a pancreas-specific deletion of Foxd3 have impaired glucose tolerance, decreased β-cell mass, decreased β-cell proliferation, and decreased β-cell size during pregnancy. In addition, several genes known to regulate proliferation, Foxm1, Skp2, Ezh2, Akt2, and Cdkn1a, are misregulated in islets isolated from these Foxd3 mutant mice. Together, these data place Foxd3 upstream of several pathways critical for β-cell mass expansion in vivo.

Myelin transcription factor 1 (Myt1) is one of the three vertebrate C2HC-type zinc finger transcription factors that include Myt1 (Nzf1), Myt1L (Png1), and Myt3 (Nzf3, St18). All three paralogs are widely expressed in developing neuronal cells. Yet their function for mammalian development has not been investigated directly. Here we report that only Myt1 is expressed in the embryonic pancreas, in both endocrine progenitors and differentiated islet cells. Myt1(-/-) animals die postnatally, likely due to confounding effects in multiple tissues. The endocrine tissues in the embryonic Myt1(-/-) pancreas contained abnormal islet cells that expressed multiple hormones; although hormone levels were normal. We also created pancreas-specific Myt1 knockout mice. These mutant animals had no obvious physical defects from their wild-type littermates. Male mutant animals had reduced glucose-clearing abilities and abnormal multi-hormone-expressing cells present in their endocrine islets. In addition, they also had reduced Glut2 expression, and attenuated glucose-induced insulin secretion in the adult islets. Surprisingly, the expression of the Myt1 paralogs, Myt1l and Myt3, was induced in the embryonic Myt1(-/-) pancreas. The consequences of Myt1 inactivation in the developing pancreas could be masked by activation of its paralogs, Myt1l and Myt3. These findings suggest Myt1 is involved in proper endocrine differentiation and function.

Neural networks are balanced by inhibitory and excitatory neuronal activity. The formation of these networks is initially generated through neuronal subtype specification controlled by transcription factors. The basic helix-loop-helix (bHLH) transcription factor Ptf1a is essential for the generation of GABAergic inhibitory neurons in the dorsal spinal cord, cerebellum, and retina. The transcription factor Rbpj is a transducer of the Notch signaling pathway that functions to maintain neural progenitor cells. Here we demonstrate Ptf1a and Rbpj interact in a complex that is required in vivo for specification of the GABAergic neurons, a function that cannot be substituted by the classical form of the bHLH heterodimer with E-protein or Notch signaling through Rbpj. We show that a mutant form of Ptf1a without the ability to bind Rbpj, while retaining its ability to interact with E-protein, is incapable of inducing GABAergic (Pax2)- and suppressing glutamatergic (Tlx3)-expressing cells in the chick and mouse neural tube. Moreover, we use an Rbpj conditional mutation to demonstrate that Rbpj function is essential for GABAergic specification, and that this function is independent of the Notch signaling pathway. Together, these findings demonstrate the requirement for a Ptf1a-Rbpj complex in controlling the balanced formation of inhibitory and excitatory neurons in the developing spinal cord, and point to a novel Notch-independent function for Rbpj in nervous system development.

Islet β cells from newborn mammals exhibit high basal insulin secretion and poor glucose-stimulated insulin secretion (GSIS). Here we show that β cells of newborns secrete more insulin than adults in response to similar intracellular Ca2+ concentrations, suggesting differences in the Ca2+ sensitivity of insulin secretion. Synaptotagmin 4 (Syt4), a non-Ca2+ binding paralog of the β cell Ca2+ sensor Syt7, increased by ∼8-fold during β cell maturation. Syt4 ablation increased basal insulin secretion and compromised GSIS. Precocious Syt4 expression repressed basal insulin secretion but also impaired islet morphogenesis and GSIS. Syt4 was localized on insulin granules and Syt4 levels inversely related to the number of readily releasable vesicles. Thus, transcriptional regulation of Syt4 affects insulin secretion; Syt4 expression is regulated in part by Myt transcription factors, which repress Syt4 transcription. Finally, human SYT4 regulated GSIS in EndoC-βH1 cells, a human β cell line. These findings reveal the role that altered Ca2+ sensing plays in regulating β cell maturation.

Transcription factors positively and/or negatively impact gene expression by recruiting coregulatory factors, which interact through protein-protein binding. Here we demonstrate that mouse pancreas size and islet β-cell function are controlled by the ATP-dependent Swi/Snf chromatin remodeling coregulatory complex that physically associates with Pdx1, a diabetes-linked transcription factor essential to pancreatic morphogenesis and adult islet cell function and maintenance. Early embryonic deletion of just the Swi/Snf Brg1 ATPase subunit reduced multipotent pancreatic progenitor cell proliferation and resulted in pancreas hypoplasia. In contrast, removal of both Swi/Snf ATPase subunits, Brg1 and Brm, was necessary to compromise adult islet β-cell activity, which included whole-animal glucose intolerance, hyperglycemia, and impaired insulin secretion. Notably, lineage-tracing analysis revealed Swi/Snf-deficient β-cells lost the ability to produce the mRNAs for Ins and other key metabolic genes without effecting the expression of many essential islet-enriched transcription factors. Swi/Snf was necessary for Pdx1 to bind to the Ins gene enhancer, demonstrating the importance of this association in mediating chromatin accessibility. These results illustrate how fundamental the Pdx1:Swi/Snf coregulator complex is in the pancreas, and we discuss how disrupting their association could influence type 1 and type 2 diabetes susceptibility.

The mechanisms that restrict regeneration and maintain cell identity following injury are poorly characterized in higher vertebrates. Following β-cell loss, 1-2% of the glucagon-producing α-cells spontaneously engage in insulin production in mice. Here we explore the mechanisms inhibiting α-cell plasticity. We show that adaptive α-cell identity changes are constrained by intra-islet insulin- and Smoothened-mediated signalling, among others. The combination of β-cell loss or insulin-signalling inhibition, with Smoothened inactivation in α- or δ-cells, stimulates insulin production in more α-cells. These findings suggest that the removal of constitutive 'brake signals' is crucial to neutralize the refractoriness to adaptive cell-fate changes. It appears that the maintenance of cell identity is an active process mediated by repressive signals, which are released by neighbouring cells and curb an intrinsic trend of differentiated cells to change.

A gain-of-function mutation in the TALK-1 K+ channel (p.L114P) associated with maturity-onset diabetes of the young (MODY) was recently reported in two distinct families. TALK-1 is a key regulator of β-cell electrical activity and glucose-stimulated insulin secretion (GSIS). KCNK16, the gene that encodes TALK-1, is the most abundant and β-cell-restricted K+ channel transcript; polymorphisms in the KCNK16 locus are also associated with an increased risk of type-2 diabetes. To investigate the impact of TALK-1-L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the Kcnk16 L114P mutation was generated. Heterozygous and homozygous Kcnk16 L114P mice exhibit increased neonatal lethality in the C57BL/6J and the mixed C57BL/6J:CD-1(ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous Kcnk16 L114P neonates due to lack of GSIS and can be reduced with insulin treatment. TALK-1-L114P drastically increased whole-cell β-cell K+ currents resulting in blunted glucose-stimulated Ca2+ entry and loss of glucose-induced Ca2+ oscillations. Thus, adult Kcnk16 L114P mice have reduced GSIS and plasma insulin levels, which significantly impaired glucose homeostasis. Taken together, this study determined that the MODY-associated TALK-1-L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by altering islet hormone secretion during development. These data strongly suggest that TALK-1 is an islet-restricted target for the treatment of diabetes.

Type 2 diabetes (T2D) is associated with compromised identity of insulin-producing pancreatic islet β cells, characterized by inappropriate production of other islet cell-enriched hormones. Here, we examined how hormone misexpression was influenced by the MAFA and MAFB transcription factors, closely related proteins that maintain islet cell function. Mice specifically lacking MafA in β cells demonstrated broad, population-wide changes in hormone gene expression with an overall gene signature closely resembling islet gastrin+ (Gast+) cells generated under conditions of chronic hyperglycemia and obesity. A human β cell line deficient in MAFB, but not one lacking MAFA, also produced a GAST+ gene expression pattern. In addition, GAST was detected in human T2D β cells with low levels of MAFB. Moreover, evidence is provided that human MAFB can directly repress GAST gene transcription. These results support a potentially novel, species-specific role for MafA and MAFB in maintaining adult mouse and human β cell identity, respectively. Here, we discuss the possibility that induction of Gast/GAST and other non-β cell hormones, by reduction in the levels of these transcription factors, represents a dysfunctional β cell signature.

Pancreatic alpha-cells, like beta-cells, express ATP-sensitive K(+) (K(ATP)) channels. To determine the physiological role of K(ATP) channels in alpha-cells, we examined glucagon secretion in mice lacking the type 1 sulfonylurea receptor (Sur1). Plasma glucagon levels, which were increased in wild-type mice after an overnight fast, did not change in Sur1 null mice. Pancreas perfusion studies showed that Sur1 null pancreata lacked glucagon secretory responses to hypoglycemia and to synergistic stimulation by arginine. Pancreatic alpha-cells isolated from wild-type animals exhibited oscillations of intracellular free Ca(2+) concentration ([Ca(2+)](i)) in the absence of glucose that became quiescent when the glucose concentration was increased. In contrast, Sur1 null alpha-cells showed continuous oscillations in [Ca(2+)](i) regardless of the glucose concentration. These findings indicate that K(ATP) channels in alpha-cells play a key role in regulating glucagon secretion, thereby adding to the paradox of how mice that lack K(ATP) channels maintain euglycemia.

Using a transgenic mouse model to express MafA, Pdx1, and Neurog3 (3TF) in a pancreatic acinar cell- and doxycycline-dependent manner, we discovered that the outcome of transcription factor-mediated acinar to β-like cellular reprogramming is dependent on both the magnitude of 3TF expression and on reprogramming-induced inflammation. Overly robust 3TF expression causes acinar cell necrosis, resulting in marked inflammation and acinar-to-ductal metaplasia. Generation of new β-like cells requires limiting reprogramming-induced inflammation, either by reducing 3TF expression or by eliminating macrophages. The new β-like cells were able to reverse streptozotocin-induced diabetes 6 days after inducing 3TF expression but failed to sustain their function after removal of the reprogramming factors.

The transcription factor encoded by the Pdx1 gene is a critical transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet β-cell development, and adult islet β-cell function. Transgenic- and cell line-based experiments have identified 5'-flanking conserved sequences that control pancreatic and β-cell type-specific transcription, which are found within areas I (bp -2694 to -2561), II (bp -2139 to -1958), III (bp -1879 to -1799), and IV (bp -6200 to -5670). Because of the presence in area IV of binding sites for transcription factors associated with pancreas development and islet cell function, we analyzed how an endogenous deletion mutant affected Pdx1 expression embryonically and postnatally. The most striking result was observed in male Pdx1ΔIV mutant mice after 3 weeks of birth (i.e., the onset of weaning), with only a small effect on pancreas organogenesis and no deficiencies in their female counterparts. Compromised Pdx1 mRNA and protein levels in weaned male mutant β-cells were tightly linked with hyperglycemia, decreased β-cell proliferation, reduced β-cell area, and altered expression of Pdx1-bound genes that are important in β-cell replication, endoplasmic reticulum function, and mitochondrial activity. We discuss the impact of these novel findings to Pdx1 gene regulation and islet β-cell maturation postnatally.

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules (MTs) affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that MT stability in the β-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable MTs. Consistently, MT hyper-stabilization prevents, and MT depolymerization promotes the capacity of single β-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that MT depolymerization activates otherwise dormant β-cells via initiation of secretion clusters (hot spots). MT depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without MTs, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca2+ influx was not affected by MT depolymerization yet required for secretion under these conditions, indicating that MT-dependent regulation of secretion hot spots acts in parallel with Ca2+ signaling. Our findings uncover a novel MT function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional β-cell heterogeneity.

Repressive KRAB domain-containing zinc-finger proteins (KRAB-ZFPs) are abundant in mammalian genomes and contribute both to the silencing of transposable elements (TEs) and to the regulation of developmental stage- and cell type-specific gene expression. Here we describe studies of zinc finger protein 92 (Zfp92), an X-linked KRAB-ZFP that is highly expressed in pancreatic islets of adult mice, by analyzing global Zfp92 knockout (KO) mice. Physiological, transcriptomic and genome-wide chromatin binding studies indicate that the principal function of ZFP92 in mice is to bind to and suppress the activity of B1/Alu type of SINE elements and modulate the activity of surrounding genomic entities. Deletion of Zfp92 leads to changes in expression of select LINE and LTR retroelements and genes located in the vicinity of ZFP92-bound chromatin. The absence of Zfp92 leads to altered expression of specific genes in islets, adipose and muscle that result in modest sex-specific alterations in blood glucose homeostasis, body mass and fat accumulation. In islets, Zfp92 influences blood glucose concentration in postnatal mice via transcriptional effects on Mafb, whereas in adipose and muscle, it regulates Acacb, a rate-limiting enzyme in fatty acid metabolism. In the absence of Zfp92, a novel TE-Capn11 fusion transcript is overexpressed in islets and several other tissues due to de-repression of an IAPez TE adjacent to ZFP92-bound SINE elements in intron 3 of the Capn11 gene. Together, these studies show that ZFP92 functions both to repress specific TEs and to regulate the transcription of specific genes in discrete tissues.

Sox17 gene expression is essential for both endothelial and endodermal cell differentiation. To better understand the genetic basis for the expression of multiple Sox17 mRNA forms, we identified and performed CRISPR/Cas9 mutagenesis of two evolutionarily conserved promoter regions (CRs). The deletion of the upstream and endothelial cell-specific CR1 caused only a modest increase in lympho-vasculogenesis likely via reduced Notch signaling downstream of SOX17. In contrast, the deletion of the downstream CR2 region, which functions in both endothelial and endodermal cells, impairs both vascular and endodermal development causing death by embryonic day 12.5. Analyses of 3D chromatin looping, transcription factor binding, histone modification, and chromatin accessibility data at the Sox17 locus and surrounding region further support differential regulation of the two promoters during the development.

ASCL1, a pioneer transcription factor, is essential for neural cell differentiation and function. Previous studies have shown that Ascl1 expression is increased in pancreatic β-cells lacking functional KATP channels or after feeding of a high fat diet (HFD) suggesting that it may contribute to the metabolic stress response of β-cells.

The Notch signaling pathway regulates embryonic development of the pancreas, inhibiting progenitor differentiation into exocrine acinar and endocrine islet cells. The adult pancreas appears to lack progenitor cells, and its mature cell types are maintained by the proliferation of pre-existing differentiated cells. Nonetheless, Notch remains active in adult duct and terminal duct/centroacinar cells (CACs), in which its function is unknown. We previously developed mice in which cells expressing the Notch target gene Hes1 can be labeled and manipulated, by expression of Cre recombinase, and demonstrated that Hes1(+) CACs do not behave as acinar or islet progenitors in the uninjured pancreas, or as islet progenitors after pancreatic duct ligation. In the current study, we assessed the function of Notch signaling in the adult pancreas by deleting the transcription factor partner of Notch, Rbpj, specifically in Hes1(+) cells. We find that loss of Rbpj depletes the pancreas of Hes1-expressing CACs, abrogating their ongoing contribution to growth and homeostasis of more proximal duct structures. Upon Rbpj deletion, CACs undergo a rapid transformation into acinar cells, suggesting that constitutive Notch activity suppresses the acinar differentiation potential of CACs. Together, our data provide direct evidence of an endogenous genetic program to control interconversion of cell fates in the adult pancreas.

The National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) supports multiple basic science consortia that generate high-content datasets, reagent resources, and methodologies, in the fields of kidney, urology, hematology, digestive, and endocrine diseases, as well as metabolic diseases such as diabetes and obesity. These currently include the Beta Cell Biology Consortium, the Nuclear Receptor Signaling Atlas, the Diabetic Complications Consortium, and the Mouse Metabolic Phenotyping Centers. Recognizing the synergy that would accrue from aggregating information generated and curated by these initiatives in a contiguous informatics network, we created the NIDDK Consortium Interconnectivity Network (dkCOIN; www.dkcoin.org). The goal of this pilot project, organized by the NIDDK, was to establish a single point of access to a toolkit of interconnected resources (datasets, reagents, and protocols) generated from individual consortia that could be readily accessed by biologists of diverse backgrounds and research interests. During the pilot phase of this activity dkCOIN collected nearly 2000 consortium-curated resources, including datasets (functional genomics) and reagents (mouse strains, antibodies, and adenoviral constructs) and built nearly 3000 resource-to-resource connections, thereby demonstrating the feasibility of further extending this database in the future. Thus, dkCOIN promises to be a useful informatics solution for rapidly identifying useful resources generated by participating research consortia.