Increasing evidence suggests that loss of β cell characteristics may cause insulin secretory deficiency in diabetes, but the underlying mechanisms remain unclear. Here, we show that Rfx6, whose ...mutation leads to neonatal diabetes in humans, is essential to maintain key features of functionally mature β cells in mice. Rfx6 loss in adult β cells leads to glucose intolerance, impaired β cell glucose sensing, and defective insulin secretion. This is associated with reduced expression of core components of the insulin secretion pathway, including glucokinase, the Abcc8/SUR1 subunit of KATP channels and voltage-gated Ca2+ channels, which are direct targets of Rfx6. Moreover, Rfx6 contributes to the silencing of the vast majority of “disallowed” genes, a group usually specifically repressed in adult β cells, and thus to the maintenance of β cell maturity. These findings raise the possibility that changes in Rfx6 expression or activity may contribute to β cell failure in humans.
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•Inactivation of Rfx6 in adult β cells leads to impaired insulin secretion•Glucose-induced Ca2+ influx is reduced in β cells lacking Rfx6•Rfx6 activates components of the insulin secretion pathway•Rfx6 maintains β cell maturity by repressing “disallowed” genes
Rfx6 transcription factor is critical for the development of pancreatic β cells, and Rfx6 mutations cause neonatal diabetes in humans. Using adult β cell deletion of Rfx6, Piccand et al. show that Rfx6 controls glucose homeostasis and maintains the differentiated state by activation of the insulin secretory pathway and repression of disallowed genes, a group of genes selectively repressed in mature β cells.
The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic ...programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.
During embryogenesis, the pancreas arises from dorsal and ventral pancreatic protrusions from the primitive gut endoderm upon induction by different stimuli from neighboring mesodermal tissues. ...Recent studies have shown that Retinoic Acid (RA) signaling is essential for the development of the pancreas in non-mammalian vertebrates. To investigate whether RA regulates mouse pancreas development, we have studied the phenotype of mice with a targeted deletion in the
retinaldehyde dehydrogenase 2 (
Raldh2) gene, encoding the enzyme required to synthesize RA in the embryo. We show that
Raldh2 is expressed in the dorsal pancreatic mesenchyme at the early stage of pancreas specification. RA-responding cells have been detected in pancreatic endodermal and mesenchymal cells.
Raldh2-deficient mice do not develop a dorsal pancreatic bud. Mutant embryos lack Pdx1 expression, an essential regulator of early pancreas development, in the dorsal but not the ventral endoderm. In contrast to
Pdx1-deficient mice, the early glucagon-expressing cells do not develop in
Raldh2 knockout embryos.
Shh expression is, as in the wild-type embryo, excluded from the dorsal endodermal region at the site where the dorsal bud is expected to form, indicating that the dorsal bud defect is not related to a mis-expression of Shh. Mesenchymal expression of the LIM homeodomain protein Isl1, required for the formation of the dorsal mesenchyme, is altered in
Raldh2−/− embryos. The homeobox gene
Hlxb9, which is essential for the initiation of the pancreatic program in the dorsal foregut endoderm, is still expressed in
Raldh2−/− dorsal epithelium but the number of HB9-expressing cells is severely reduced. Maternal supplementation of RA rescues early dorsal pancreas development and restores endodermal Pdx1 and mesenchymal Isl1 expression as well as endocrine cell differentiation. These findings suggest that RA signaling is important for the proper differentiation of the dorsal mesenchyme and development of the dorsal endoderm. We conclude that RA synthesized in the mesenchyme is specifically required for the normal development of the dorsal pancreatic endoderm at a stage preceding Pdx1 function.
Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous ...mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)–derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets.
We generated a novel hiPSC line (NEUROG3-HA-P2A-Venus) where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)–associated variants at the PEP stage.
CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3−/− PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions.
Mapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation.
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•NEUROG3 CUT&RUN analysis revealed 1263 target genes in human pancreatic endocrine progenitors (PEPs).•NEUROG3 binding sites overlap with active chromatin regions in PEPs.•1/5 of the genes downregulated in NEUROG3−/− hESC-derived PEPs are bound by NEUROG3.•NEUROG3 targets islet-specific TFs and regulators of insulin secretion.•Several T2DM risk alleles lie within NEUROG3-bound regions.
Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher ...the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell–Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated.
We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes.
Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine.
These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell–Riley syndrome.
•The lack of Rfx6 impairs the differentiation of peptide-producing enteroendocrine cells.•The number of 5-HT-expressing-cells is increased in Rfx6-deficient intestine.•Intestinal inactivation of Rfx6 leads to lipid malabsorption and decreased food efficiency.
GPR64/ADGRG2 is an orphan Adhesion G protein-coupled receptor (ADGR) known to be mainly expressed in the parathyroid gland and epididymis. This investigation aimed to delineate the cellular ...expression of GPR64 throughout the body with focus on the gastrointestinal (GI) tract.
Transgenic Gpr64mCherry reporter mice were histologically examined throughout the body and reporter protein expression in intestinal tuft cells was confirmed by specific cell ablation. The GPCR repertoire of intestinal Gpr64mCherry-positive tuft cells was analyzed by quantitative RT-PCR analysis and in situ hybridization. The Gpr64mCherry was crossed into the general tuft cell reporter Trpm5GFP to generate small intestinal organoids for time-lapse imaging. Intestinal tuft cells were isolated from small intestine, FACS-purified and transcriptionally compared using RNA-seq analysis.
Expression of the Gpr64mCherry reporter was identified in multiple organs and specifically in olfactory microvillous cells, enteric nerves, and importantly in respiratory and GI tuft cells. In the small intestine, cell ablation targeting Gpr64-expressing epithelial cells eliminated tuft cells. Transcriptional analysis of small intestinal Gpr64mCherry -positive tuft cells confirmed expression of Gpr64 and the chemo-sensors Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3. Time-lapse studies of organoids from Trpm5GFP:Gpr64mCherry mice revealed sequential expression of initially Trpm5GFP and subsequently also Gpr64mCherry in maturing intestinal tuft cells. RNA-seq analysis of small intestinal tuft cells based on these two markers demonstrated a dynamic change in expression of transcription factors and GPCRs from young to mature tuft cells.
GPR64 is expressed in chemosensory epithelial cells across a broad range of tissues; however, in the GI tract, GPR64 is remarkably selectively expressed in mature versus young immunoregulatory tuft cells.
•GPR64-RFP is expressed mainly in chemosensory epithelial cells, including tuft cells.•Maturing intestinal tuft cells initially express Trpm5 and subsequently Gpr64.•Mature intestinal Gpr64+ tuft cells express Sucnr1, Gprc5c, Drd3, and Gpr41/Ffar3.•RNAseq analysis reveals dynamic transcriptional change of mature vs. young tuft cells.
The Drosophila transcription factor Prospero functions as a tumor suppressor, and it has been suggested that the human counterpart of Prospero, PROX1, acts similarly in human cancers. However, we ...show here that PROX1 promotes dysplasia in colonic adenomas and colorectal cancer progression. PROX1 expression marks the transition from benign colon adenoma to carcinoma in situ, and its loss inhibits growth of human colorectal tumor xenografts and intestinal adenomas in Apcmin/+ mice, while its transgenic overexpression promotes colorectal tumorigenesis. Furthermore, in intestinal tumors PROX1 is a direct and dose-dependent target of the β-catenin/TCF signaling pathway, responsible for the neoplastic transformation. Our data underscore the complexity of cancer pathogenesis and implicate PROX1 in malignant tumor progression through the regulation of cell polarity and adhesion.
Endocrine cells of the pancreas and the gastrointestinal tract derive from multipotent endodermal stem cells. We have shown previously that the basic helix– loop–helix (bHLH) transcription factor ...neurogenin3 (ngn3) is required for the specification of the endocrine lineage in uncommitted progenitors in the developing pancreas. We investigate herein the expression and the function of ngn3 in the control of endocrine cell development in the intestinal and gastric epithelium. Our results indicate that as in the pancreas, gastrointestinal endocrine cells derive from ngn3‐expressing progenitors. Mice homozygous for a null mutation in ngn3 fail to generate any intestinal endocrine cells, and endocrine progenitor cells are lacking. The other main intestinal epithelial cell types differentiate properly. In contrast, in the glandular stomach, the differentiation of the gastrin‐ (G cells) and somatostatin (D cells)‐secreting cells is impaired whereas serotonin‐ (enterochromaffin EC cells), histamine‐ (enterochromaffin‐like ECL cells) and ghrelin (X/A cells)‐expressing cells are still present. Thus, ngn3 is strictly required for endocrine cell fate specification in multipotent intestinal progenitor cells, whereas gastric endocrine development is both ngn3 dependent and independent.
The basic helix-loop-helix transcription factor Neurogenin 3 (NGN3) controls endocrine cell fate specification in uncommitted pancreatic progenitor cells. Ngn3-deficient mice do not develop any islet ...cells and are diabetic. All the major islet cell types, including insulin-producing beta-cells, derive from Ngn3-positive endocrine progenitor cells. Therefore, the characterization of this population of immature cells is of particular interest for the development of novel strategies for cell replacement therapies in type 1 diabetes. To explore further the biology of islet progenitor cells we have generated a mouse in which Ngn3-expressing cells are labeled with the enhanced yellow fluorescent protein (EYFP) using a knock-add-on strategy. In this approach, the EYFP cDNA is introduced into the 3'-untranslated region of the proendocrine transcription factor, Neurogenin 3, without deleting any endogenous coding or regulatory sequences. In Ngn3(EYFP/+) and Ngn3(EYFP/EYFP) mice, the EYFP protein is targeted to Ngn3-expressing progenitors in the developing pancreas, and islets develop normally. Islet progenitors can be purified from whole embryonic pancreas by fluorescence-activated cell sorting from Ngn3(EYFP/+) mice and their development can be monitored in real time in pancreas explant cultures. These experiments showed that endocrine progenitors can form de novo and expand, in vitro, in the absence of signals from the surrounding mesenchyme, suggesting that endocrine commitment is a default pathway. The Ngn3(EYFP) mice represent a valuable tool to study islet cell development and neogenesis in normal and diabetic animals as well as for the determination of the conditions to generate beta-cells in vitro.
The lineage of olfactory neurons has been relatively well characterized at the cellular level, but the genes that regulate the proliferation and differentiation of their progenitors are currently ...unknown. In this study, we report the isolation of a novel murine gene, Math4C/neurogenin1, which is distantly related to the Drosophila proneural gene atonal. We show that Math4C/neurogenin1 and the basic helix-loop-helix gene Mash1 are expressed in the olfactory epithelium by different dividing progenitor populations, while another basic helix-loop-helix gene, NeuroD, is expressed at the onset of neuronal differentiation. These expression patterns suggest that each gene marks a distinct stage of olfactory neuron progenitor development, in the following sequence: Mash1>Math4C/neurogenin1>NeuroD. We have previously reported that inactivation of Mash1 function leads to a severe reduction in the number of olfactory neurons. We show here that most cells in the olfactory epithelium of Mash1 mutant embryos fail to express Math4C/neurogenin1 or NeuroD. Strikingly, a subset of progenitor cells in a ventrocaudal domain of Mash1 mutant olfactory epithelium still express Math4C/neurogenin1 and NeuroD and differentiate into neurons. Cells in this domain also express Math4A/neurogenin2, another member of the Math4/neurogenin gene family, and not Mash1. Our results demonstrate that Mash1 is required at an early stage in the olfactory neuron lineage to initiate a differentiation program involving Math4C/neurogenin1 and NeuroD. Another gene activates a similar program in a separate population of olfactory neuron progenitors.
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