Fetal Programming of the Endocrine Pancreas: Impact of a Maternal Low-Protein Diet on Gene Expression in the Perinatal Rat Pancreas.

In rats, the time of birth is characterized by a transient rise in beta cell replication, as well as beta cell neogenesis and the functional maturation of the endocrine pancreas. However, the knowledge of the gene expression during this period of beta cell expansion is incomplete. The aim was to characterize the perinatal rat pancreas transcriptome and to identify regulatory pathways differentially regulated at the whole organ level in the offspring of mothers fed a regular control diet (CO) and of mothers fed a low-protein diet (LP). We performed mRNA expression profiling via the microarray analysis of total rat pancreas samples at embryonic day (E) 20 and postnatal days (P) 0 and 2. In the CO group, pancreas metabolic pathways related to sterol and lipid metabolism were highly enriched, whereas the LP diet induced changes in transcripts involved in RNA transcription and gene regulation, as well as cell migration and apoptosis. Moreover, a number of individual transcripts were markedly upregulated at P0 in the CO pancreas: growth arrest specific 6 (Gas6), legumain (Lgmn), Ets variant gene 5 (Etv5), alpha-fetoprotein (Afp), dual-specificity phosphatase 6 (Dusp6), and angiopoietin-like 4 (Angptl4). The LP diet induced the downregulation of a large number of transcripts, including neurogenin 3 (Neurog3), Etv5, Gas6, Dusp6, signaling transducer and activator of transcription 3 (Stat3), growth hormone receptor (Ghr), prolactin receptor (Prlr), and Gas6 receptor (AXL receptor tyrosine kinase; Axl), whereas upregulated transcripts were related to inflammatory responses and cell motility. We identified differentially regulated genes and transcriptional networks in the perinatal pancreas. These data revealed marked adaptations of exocrine and endocrine in the pancreas to the low-protein diet, and the data can contribute to identifying novel regulators of beta cell mass expansion and functional maturation and may provide a valuable tool in the generation of fully functional beta cells from stem cells to be used in replacement therapy.

Deriving functional human enteroendocrine cells from pluripotent stem cells.

Enteroendocrine cells (EECs) are a minor cell population in the intestine yet they play a major role in digestion, satiety and nutrient homeostasis. Recently developed human intestinal organoid models include EECs, but their rarity makes it difficult to study their formation and function. Here, we used the EEC-inducing property of the transcription factor NEUROG3 in human pluripotent stem cell-derived human intestinal organoids and colonic organoids to promote EEC development in vitro An 8-h pulse of NEUROG3 expression induced expression of known target transcription factors and after 7 days organoids contained up to 25% EECs in the epithelium. EECs expressed a broad array of human hormones at the mRNA and/or protein level, including motilin, somatostatin, neurotensin, secretin, substance P, serotonin, vasoactive intestinal peptide, oxyntomodulin, GLP-1 and INSL5. EECs secreted several hormones including gastric inhibitory polypeptide (GIP), ghrelin, GLP-1 and oxyntomodulin. Injection of glucose into the lumen of organoids caused an increase in both GIP secretion and K-cell number. Lastly, we observed formation of all known small intestinal EEC subtypes following transplantation and growth of human intestinal organoids in mice.

Transcription factors that shape the mammalian pancreas.

Improving our understanding of mammalian pancreas development is crucial for the development of more effective cellular therapies for diabetes. Most of what we know about mammalian pancreas development stems from mouse genetics. We have learnt that a unique set of transcription factors controls endocrine and exocrine cell differentiation. Transgenic mouse models have been instrumental in studying the function of these transcription factors. Mouse and human pancreas development are very similar in many respects, but the devil is in the detail. To unravel human pancreas development in greater detail, in vitro cellular models (including directed differentiation of stem cells, human beta cell lines and human pancreatic organoids) are used; however, in vivo validation of these results is still needed. The current best 'model' for studying human pancreas development are individuals with monogenic forms of diabetes. In this review, we discuss mammalian pancreas development, highlight some discrepancies between mouse and human, and discuss selected transcription factors that, when mutated, cause permanent neonatal diabetes. Graphical abstract.

A novel NEUROG3 mutation in neonatal diabetes associated with a neuro-intestinal syndrome.

Neonatal diabetes mellitus (NDM) is a rare form of non-autoimmune diabetes usually diagnosed in the first 6 months of life. Various genetic defects have been shown to cause NDM with diverse clinical presentations and variable severity. Among transcriptional factor genes associated with isolated or syndromic NDM, a few cases of homozygous mutations in the NEUROG3 gene have been reported, all mutated patients presenting with congenital malabsorptive diarrhea with or without diabetes at a variable age of onset from early life to childhood. Through a targeted next-generation sequencing assay for monogenic diabetes genes, we aimed to search for pathogenic deleterious mutation in a Turkish patient with NDM, severe malabsorptive diarrhea, neurointestinal dysplasia and other atypical features. In this patient, we identified a novel homozygous nonsense mutation (p.Q4*) in NEUROG3. The same biallelic mutation was found in another affected family member. Of note, the study proband presents with abnormalities of the intrahepatic biliary tract, thyroid gland and central nervous system, which has never been reported before in NEUROG3 mutation carriers. Our findings extend the usually described clinical features associated with NEUROG3 deficiency in humans, and question the extent to which a complete lack of NEUROG3 expression may affect pancreas endocrine function in humans.

Identification of a small molecule that facilitates the differentiation of human iPSCs/ESCs and mouse embryonic pancreatic explants into pancreatic endocrine cells.

AIMS/HYPOTHESIS: Pancreatic beta-like cells generated from human induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs) offer an appealing donor tissue source. However, differentiation protocols that mainly use growth factors are costly. Therefore, in this study, we aimed to establish efficient differentiation protocols to change hiPSCs/hESCs to insulin (INS)+ cells using novel small-molecule inducers. METHODS: We screened small molecules that increased the induction rate of INS+ cells from hESC-derived pancreatic and duodenal homeobox 1 (PDX1)+ pancreatic progenitor cells. The differentiation protocol to generate INS+ cells from hiPSCs/hESCs was optimised using hit compounds, and INS+ cells induced with the compounds were characterised for their in vitro and in vivo functions. The inducing activity of the hit compounds was also examined using mouse embryonic pancreatic tissues in an explant culture system. Finally, RNA sequencing analyses were performed on the INS+ cells to elucidate the mechanisms of action by which the hit compounds induced pancreatic endocrine differentiation. RESULTS: One hit compound, sodium cromoglicate (SCG), was identified out of approximately 1250 small molecules screened. When SCG was combined with a previously described protocol, the induction rate of INS+ cells increased from a mean ± SD of 5.9 ± 1.5% (n = 3) to 16.5 ± 2.1% (n = 3). SCG induced neurogenin 3-positive cells at a mean ± SD of 32.6 ± 4.6% (n = 3) compared with 14.2 ± 3.6% (n = 3) for control treatment without SCG, resulting in an increased generation of endocrine cells including insulin-producing cells. Similar induction by SCG was confirmed using mouse embryonic pancreatic explants. We also confirmed that the mechanisms of action by which SCG induced pancreatic endocrine differentiation included the inhibition of bone morphogenetic protein 4 signalling. CONCLUSIONS/INTERPRETATION: SCG improves the generation of pancreatic endocrine cells from multiple hiPSC/hESC lines and mouse embryonic pancreatic explants by facilitating the differentiation of endocrine precursors. This discovery will contribute to elucidating the mechanisms of pancreatic endocrine development and facilitate cost-effective generation of INS+ cells from hiPSCs/hESCs. DATA AVAILABILITY: The RNA sequencing data generated during the current study are available in the Gene Expression Omnibus ( www.ncbi.nlm.nih.gov/geo ) with series accession number GSE89973.

Interaction of sex chromosome complement, gonadal hormones and neuronal steroid synthesis on the sexual differentiation of mammalian neurons.

Female mouse hippocampal and hypothalamic neurons growing in vitro show a faster development of neurites than male mouse neurons. This sex difference in neuritogenesis is determined by higher expression levels of the neuritogenic factor neurogenin 3 in female neurons. Experiments with the four core genotype mouse model, in which XX and XY animals with male gonads and XX and XY animals with female gonads are generated, indicate that higher levels of neurogenin 3 in developing neurons are determined by the presence of the XX chromosome complement. Female XX neurons express higher levels of estrogen receptors than male XY neurons. In female XX neurons, neuronal derived estradiol increases neurogenin 3 expression and neuritogenesis. In contrast, neuronal-derived estradiol is not able to upregulate neurogenin 3 in male XY neurons, resulting in decreased neuritogenesis compared to female neurons. However, exogenous testosterone increases neurogenin 3 expression and neuritogenesis in male XY neurons. These findings suggest that sex differences in neuronal development are determined by the interaction of sex chromosomes, neuronal derived estradiol and gonadal hormones.

Enteroendocrine, Musashi 1 and neurogenin 3 cells in the large intestine of Thai and Norwegian patients with irritable bowel syndrome.

OBJECTIVES: The prevalence, gender distribution and clinical presentation of IBS differ between Asian and Western countries. This study aimed at studying and comparing enteroendocrine, Musashi 1 (Msi 1) and neurogenin 3 (neurog 3) cells in Thai and Norwegian IBS patients. MATERIAL AND METHODS: Thirty Thai and 61 Norwegian IBS patients as well as 20 Thai and 24 Norwegian controls were included. Biopsy samples were taken from each of the sigmoid colon and the rectum during a standard colonoscopy. The samples were immunostained for serotonin, peptide YY, oxyntomodulin, pancreatic polypeptide, somatostatin, Msi 1 and neurog 3. The densities of immunoreactive cells were determined with computerized image analysis. RESULTS: The densities of several enteroendocrine cell types were altered in both the colon and rectum of both Thai and Norwegian IBS patients. Some of these changes were similar in Thai and Norwegian IBS patients, while others differed. CONCLUSIONS: The findings of abnormal densities of the enteroendocrine cells in Thai patients support the notion that enteroendocrine cells are involved in the pathophysiology of IBS. The present observations highlight that IBS differs in Asian and Western countries, and show that the changes in large-intestine enteroendocrine cells in Thai and Norwegian IBS patients might be caused by different mechanisms.

Abnormalities in ileal stem, neurogenin 3, and enteroendocrine cells in patients with irritable bowel syndrome.

BACKGROUND: This study examined whether the densities of stem- and enteroendocrine cell progenitors are abnormal in the ileum of patients with irritable bowel syndrome (IBS), and whether any abnormalities in ileal enteroendocrine cells are correlated with abnormalities in stem cells and enteroendocrine cell progenitors. METHODS: One hundred and one IBS patients covering all IBS subtypes were recruited, and 39 non-IBS subjects were included as a control group. The patients and controls underwent standard colonoscopies, during which biopsy specimens were obtained from the ileum. The biopsy specimens were stained with hematoxylin-eosin and immunostained for Musashi-1 (Msi-1), neurogenin 3 (NEUROG3), chromogranin A (CgA), serotonin, peptide YY (PYY), oxyntomodulin (enteroglucagon), pancreatic polypeptide, and somatostatin. The immunoreactive cells were quantified by computerized image analysis. RESULTS: The densities of Msi-1, NEUROG3, CgA, and serotonin cells were reduced in all IBS patients and in patients with diarrhea-predominant IBS (IBS-D), mixed-diarrhea-and-constipation IBS (IBS-M), and constipation-predominant (IBS-C) relative to the control subjects. While the PYY cell density was increased in IBS-C relative to controls, it did not differ between control subjects and IBS-D and IBS-M patients. The densities of Msi-1 and NEUROG3 cells were strongly correlated with that of CgA cells. CONCLUSIONS: The abnormalities in the ileal enteroendocrine cells appear to be caused by two mechanisms: (1) decreases in the clonogenic activity of the stem cells and in the endocrine-cell progenitors differentiating into enteroendocrine cells, and (2) switching on the expression of PYY and switching off the expression of certain other hormones in other types of the enteroendocrine cells.

A combination of cytokines EGF and CNTF protects the functional beta cell mass in mice with short-term hyperglycaemia.

AIMS/HYPOTHESIS: When the beta cell mass or function declines beyond a critical point, hyperglycaemia arises. Little is known about the potential pathways involved in beta cell rescue. As two cytokines, epidermal growth factor (EGF) and ciliary neurotrophic factor (CNTF), restored a functional beta cell mass in mice with long-term hyperglycaemia by reprogramming acinar cells that transiently expressed neurogenin 3 (NGN3), the current study assesses the effect of these cytokines on the functional beta cell mass after an acute chemical toxic insult. METHODS: Glycaemia and insulin levels, pro-endocrine gene expression and beta cell origin, as well as the role of signal transducer and activator of transcription 3 (STAT3) signalling, were assessed in EGF+CNTF-treated mice following acute hyperglycaemia. RESULTS: The mice were hyperglycaemic 1 day following i.v. injection of the beta cell toxin alloxan, when the two cytokines were applied. One week later, 68.6 ± 4.6% of the mice had responded to the cytokine treatment and increased their insulin(+) cell number to 30% that of normoglycaemic control mice, resulting in restoration of euglycaemia. Although insulin(-) NGN3(+) cells appeared following acute EGF+CNTF treatment, genetic lineage tracing showed that the majority of the insulin(+) cells originated from pre-existing beta cells. Beta cell rescue by EGF+CNTF depends on glycaemia rather than on STAT3-induced NGN3 expression in acinar cells. CONCLUSIONS/INTERPRETATION: In adult mice, EGF+CNTF allows the rescue of beta cells in distress when treatment is given shortly after the diabetogenic insult. The rescued beta cells restore a functional beta cell mass able to control normal blood glucose levels. These findings may provide new insights into compensatory pathways activated early after beta cell loss.

Neurogenin 3 is regulated by neurotrophic tyrosine kinase receptor type 2 (TRKB) signaling in the adult human exocrine pancreas.

BACKGROUND: Reports of exocrine-to-endocrine reprogramming through expression or stabilization of the transcription factor neurogenin 3 (NGN3) have generated renewed interest in harnessing pancreatic plasticity for therapeutic applications. NGN3 is expressed by a population of endocrine progenitor cells that give rise exclusively to hormone-secreting cells within pancreatic islets and is necessary and sufficient for endocrine differentiation during development. In the adult human pancreas, NGN3 is expressed by dedifferentiating exocrine cells with a phenotype resembling endocrine progenitor cells and the capacity for endocrine differentiation in vitro. Neurotrophic tyrosine kinase receptor type 2 (TRKB), which regulates neuronal cell survival, differentiation and plasticity, was identified as highly overexpressed in the NGN3 positive cell transcriptome compared to NGN3 negative exocrine cells. This study was designed to determine if NGN3 is regulated by TRKB signaling in the adult human exocrine pancreas. METHODS: Transcriptome analysis, quantitative reverse transcriptase polymerase chain reaction (RTPCR) and immunochemistry were used to identify TRKB isoform expression in primary cultures of human islet-depleted exocrine tissue and human cadaveric pancreas biopsies. The effects of pharmacological modulation of TRKB signaling on the expression of NGN3 were assessed by Student's t-test and ANOVA. RESULTS: Approximately 30 % of cultured exocrine cells and 95 % of NGN3+ cells express TRKB on their cell surface. Transcriptome-based exon splicing analyses, isoform-specific quantitative RTPCR and immunochemical staining demonstrate that TRKB-T1, which lacks a tyrosine kinase domain, is the predominant isoform expressed in cultured exocrine tissue and is expressed in histologically normal cadaveric pancreas biopsies. Pharmacological inhibition of TRKB significantly decreased the percentage of NGN3+ cells, while a TRKB agonist significantly increased this percentage. Inhibition of protein kinase B (AKT) blocked the effect of the TRKB agonist, while inhibition of tyrosine kinase had no effect. Modulation of TRKB and AKT signaling did not significantly affect the level of NGN3 mRNA. CONCLUSIONS: In the adult human exocrine pancreas, TRKB-T1 positively regulates NGN3 independent of effects on NGN3 transcription. Targeting mechanisms controlling the NGN3+ cell population size and endocrine cell fate commitment represent a potential new approach to understand pancreas pathobiology and means whereby cell populations could be expanded for therapeutic purposes.

Revisiting the immunocytochemical detection of Neurogenin 3 expression in mouse and man.

During embryonic development, endocrine cells of the pancreas are specified from multipotent progenitors. The transcription factor Neurogenin 3 (NEUROG3) is critical for this development and it has been shown that all endocrine cells of the pancreas arise from endocrine progenitors expressing NEUROG3. A thorough understanding of the role of NEUROG3 during development, directed differentiation of pluripotent stem cells and in models of cellular reprogramming, will guide future efforts directed at finding novel sources of ß-cells for cell replacement therapies. In this article, we review the expression and function of NEUROG3 in both mouse and human and present the further characterization of a monoclonal antibody directed against NEUROG3. This antibody has been previously been used for detection of both mouse and human NEUROG3. However, our results suggest that the epitope recognized by this antibody is specific to mouse NEUROG3. Thus, we have also generated a monoclonal antibody specifically recognizing human NEUROG3 and present the characterization of this antibody here. Together, these antibodies will provide useful tools for future studies of NEUROG3 expression, and the data presented in this article suggest that recently described expression patterns of NEUROG3 in human foetal and adult pancreas should be re-examined.

Mutant neurogenin-3 in a Turkish boy with congenital malabsorptive diarrhea.

Congenital diarrheal disorders are caused by disruption in nutrient digestion, absorption, or transport, enterocyte development and functioning, or enteroendocrine functioning. Many additional rare forms of congenital diarrhea are expected to be linked to genes associated with appropriate intestinal fluid and electrolyte balance. Neurogenin-3 mutation, a very rare form of congenital diarrhea, disrupts enteroendocrine cell differentiation and is characterized by malabsorption and the absence of pancreatic islet cells. Diabetes mellitus is typically associated with malabsorptive diarrhea at early onset or at later presentation in neurogenin-3 mutation. Here, we describe the case of an infant with homozygous neurogenin-3 mutation who had severe malabsorptive diarrhea and episodes of hyperchloremic metabolic acidosis after birth. Remarkably, cholestyramine was effective at reducing stool volume and frequency and improved the consistency of the stools; diabetes was not present in this patient.

Ectopic Ptf1a expression in murine ESCs potentiates endocrine differentiation and models pancreas development in vitro.

Besides its role in exocrine differentiation, pancreas-specific transcription factor 1a (PTF1a) is required for pancreas specification from the foregut endoderm and ultimately for endocrine cell formation. Examining the early role of PTF1a in pancreas development has been challenging due to limiting amounts of embryonic tissue material for study. Embryonic stem cells (ESCs) which can be differentiated in vitro, and without limit to the amount of experimental material, can serve as a model system to study these early developmental events. To this end, we derived and characterized a mouse ESC line with tetracycline-inducible expression of PTF1a (tet-Ptf1a mESCs). We found that transient ectopic expression of PTF1a initiated the pancreatic program in differentiating ESCs causing cells to activate PDX1 expression in bud-like structures resembling pancreatic primordia in vivo. These bud-like structures also expressed progenitor markers characteristic of a developing pancreatic epithelium. The epithelium differentiated to generate a wave of NGN3+ endocrine progenitors, and further formed cells of all three pancreatic lineages. Notably, the insulin+ cells in the cultures were monohormonal, and expressed PDX1 and NKX6.1. PTF1a-induced cultures differentiated into significantly more endocrine and exocrine cells and the ratio of endocrine-to-exocrine cell differentiation could be regulated by retinoic acid (RA) and nicotinamide (Nic) signaling. Moreover, induced cultures treated with RA and Nic exhibited a modest glucose response. Thus, this tet-Ptf1a ESC-based in vitro system is a valuable new tool for interrogating the role of PTF1a in pancreas development and in directing differentiation of ESCs to endocrine cells.

Reprogramming of enteroendocrine K cells to pancreatic ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic ß-cells in a process called cell reprogramming. Enteroendocrine cells and ß-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in ß-cells. Thus, K cells could be reprogrammed to ß-cells under certain conditions. However, there is no clear evidence on whether these cells convert to ß-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1(+)-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1(+)-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1(+)-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to ß-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture.

Deletion of retinoic acid receptor ß (RARß) impairs pancreatic endocrine differentiation.

All-trans retinoic acid (RA) signals via binding to retinoic acid receptors (RARs α, ß, and γ). RA directly influences expression of Pdx1, a transcription factor essential for pancreatic development and beta-cell (ß-cell) maturation. In this study we follow the differentiation of cultured wild-type (WT) vs. RARß knockout (KO) embryonic stem (ES) cells into pancreatic islet cells. We found that RARß KO ES cells show greatly reduced expression of some important endocrine markers of differentiated islet cells, such as glucagon, islet amyloid polypeptide (Iapp), and insulin 1 (Ins1) relative to WT. We conclude that RARß activity is essential for proper differentiation of ES cells to pancreatic endocrine cells.

Neuroendocrine carcinoma arising in a hepatitis C virus-infected liver: mechanism of the tumor development may be similar to that of development of pancreatic neuroendocrine cells.

We experienced a case of neuroendocrine carcinoma (NC). The tumor developed in the cirrhotic liver of a 62-year-old Japanese man who had been infected with hepatitis C virus. The tumor cells showed high N/C ratio, formed many rosettes, and expressed CD56, synaptophysin, HepPar1 and pancreatic and duodenal homeobox 1. MIB1 expression was 65%. Because both liver and pancreas are derived from a common endodermal layer during fetal development, we speculated that the tumor may have formed via the interaction of neurogenin 3, insulinoma-associated 1 gene and NeuroD/beta2, which are involved in the stage at which some pancreatic cells commit to becoming endocrine cells. Molecular analysis revealed that the NC had higher relative expression levels of mRNA of the three molecules than did the nontumorous liver. The results indicate that the NC in this patient may have formed via the same mechanism that acts in the development of pancreatic neuroendocrine cells.

Co-ordinated regulation of neurogenin-3 expression in the maternal and fetal pancreas during pregnancy.

OBJECTIVE: Several studies have shown increased beta cell mass during pregnancy in both rodents and humans. Proliferation of existing beta cells seems to be the predominant mechanism in rodents, whereas the mechanism in humans is unclear. We hypothesized that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved. SAMPLES: Pancreatic tissue from mice and rat and serum from pregnant women. METHOD: Morphometric analysis of pancreas of pregnant and nonpregnant mice was carried out by immunocytochemical staining for the neogenic marker neurogenin-3. Messenger RNA levels of neurogenin-3 and the transcription factor musculoaponeurotic fibrosarcoma oncogene family protein B in fetal rat pancreas cells, cultured with serum from pregnant women, were measured by quantitative polymerase chain reaction. MAIN OUTCOME MEASURES: The number of neurogenin-3-positive cells present in pregnant mice was increased compared with nonpregnant mice. Neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA was detected in fetal rat pancreas exposed to serum from pregnant women. RESULTS: In pregnant mice we found a 3.6-fold increase in beta cell volume at day 18 compared with nonpregnant mice and a 3.5-fold increase in neurogenin-3 volume at day 14, mainly located in the acinar compartment where it was eightfold higher than in nonpregnant mice. In fetal rat pancreatic cells exposed to serum from pregnant women we found a marked increase in both neurogenin-3 and musculoaponeurotic fibrosarcoma oncogene family protein B mRNA levels in fibroblast-like cells. CONCLUSION: These results suggest that neogenesis contributes to the increased beta cell mass in pregnancy and that circulating factors are involved in beta cell formation in both the maternal and fetal pancreas during pregnancy.

Protein arginine methyltransferase 1 regulates mouse enteroendocrine cell development and homeostasis.

BACKGROUND: The adult intestinal epithelium is a complex, self-renewing tissue composed of specialized cell types with diverse functions. Intestinal stem cells (ISCs) located at the bottom of crypts, where they divide to either self-renew, or move to the transit amplifying zone to divide and differentiate into absorptive and secretory cells as they move along the crypt-villus axis. Enteroendocrine cells (EECs), one type of secretory cells, are the most abundant hormone-producing cells in mammals and involved in the control of energy homeostasis. However, regulation of EEC development and homeostasis is still unclear or controversial. We have previously shown that protein arginine methyltransferase (PRMT) 1, a histone methyltransferase and transcription co-activator, is important for adult intestinal epithelial homeostasis. RESULTS: To investigate how PRMT1 affects adult intestinal epithelial homeostasis, we performed RNA-Seq on small intestinal crypts of tamoxifen-induced intestinal epithelium-specific PRMT1 knockout and PRMT1fl/fl adult mice. We found that PRMT1fl/fl and PRMT1-deficient small intestinal crypts exhibited markedly different mRNA profiles. Surprisingly, GO terms and KEGG pathway analyses showed that the topmost significantly enriched pathways among the genes upregulated in PRMT1 knockout crypts were associated with EECs. In particular, genes encoding enteroendocrine-specific hormones and transcription factors were upregulated in PRMT1-deficient small intestine. Moreover, a marked increase in the number of EECs was found in the PRMT1 knockout small intestine. Concomitantly, Neurogenin 3-positive enteroendocrine progenitor cells was also increased in the small intestinal crypts of the knockout mice, accompanied by the upregulation of the expression levels of downstream targets of Neurogenin 3, including Neuod1, Pax4, Insm1, in PRMT1-deficient crypts. CONCLUSIONS: Our finding for the first time revealed that the epigenetic enzyme PRMT1 controls mouse enteroendocrine cell development, most likely via inhibition of Neurogenin 3-mediated commitment to EEC lineage. It further suggests a potential role of PRMT1 as a critical transcriptional cofactor in EECs specification and homeostasis to affect metabolism and metabolic diseases.

Extremely rare cause of congenital diarrhea: enteric anendocrinosis.

Congenital diarrheal disorders consist of a variety of chronic enteropathies. There are approximately 30 different diseases that can be classified into four groups according to the mechanisms involved in pathogenesis: (i) absorption and transport of nutrients and electrolytes; (ii) enterocyte differentiation and polarization; (iii) enteroendocrine cell differentiation; and (iv) modulation of the intestinal immune response. Affected patients often present with life-threatening diarrhea, in the first few weeks of life. A new disorder, enteric anendocrinosis, which is characterized by severe malabsorptive diarrhea and a lack of intestinal enteroendocrine cells has recently been described in six patients with recessively inherited mutations in the Neurogenin-3 gene. In this report we describe a seventh case with a review of the literature.

Integrating single-cell imaging and RNA sequencing datasets links differentiation and morphogenetic dynamics of human pancreatic endocrine progenitors.

Basic helix-loop-helix genes, particularly proneural genes, are well-described triggers of cell differentiation, yet information on their dynamics is limited, notably in human development. Here, we focus on Neurogenin 3 (NEUROG3), which is crucial for pancreatic endocrine lineage initiation. By monitoring both NEUROG3 gene expression and protein in single cells using a knockin dual reporter in 2D and 3D models of human pancreas development, we show an approximately 2-fold slower expression of human NEUROG3 than that of the mouse. We observe heterogeneous peak levels of NEUROG3 expression and reveal through long-term live imaging that both low and high NEUROG3 peak levels can trigger differentiation into hormone-expressing cells. Based on fluorescence intensity, we statistically integrate single-cell transcriptome with dynamic behaviors of live cells and propose a data-mapping methodology applicable to other contexts. Using this methodology, we identify a role for KLK12 in motility at the onset of NEUROG3 expression.