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Abstract The efficiency of inducing human embryonic stem cells into NEUROG3+ pancreatic endocrine cells is a bottleneck in stem cell therapy for diabetes. To understand the cell properties and fate decisions during differentiation, we analyzed the modified induction method using single-cell transcriptome and found that DAPT combined with four factors (4FS): nicotinamide, dexamethasone, forskolin and Alk5 inhibitor II (DAPT + 4FS) increased the expression of NEUROG3 to approximately 34.3%. The increased NEUROG3+ cells were mainly concentrated in Insulin + Glucagon + (INS + GCG+) and SLAC18A1 + Chromogranin A+(SLAC18A1 + CHGA +) populations, indicating that the increased NEUROG3+ cells promoted the differentiation of pancreatic endocrine cells and enterochromaffin-like cells. Single-cell transcriptome analysis provided valuable clues for further screening of pancreatic endocrine cells and differentiation of pancreatic islet cells. The gene set enrichment analysis (GSEA) suggest that we can try to promote the expression of INS + GCG+ population by up-regulating G protein-coupled receptor (GPCR) and mitogen-activated protein kinase signals and down-regulating Wnt, NIK/NF-KappaB and cytokine-mediated signal pathways. We can also try to regulate GPCR signaling through PLCE1, so as to increase the proportion of NEUROG3+ cells in INS+GCG+ populations. To exclude non-pancreatic endocrine cells, ALCAMhigh CD9low could be used as a marker for endocrine populations, and ALCAMhigh CD9lowCDH1low could remove the SLC18A1 + CHGA+ population.

Objective: 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. Methods: 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. Results: 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. Conclusion: 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.

In the context of type 1 diabetes research and the development of insulin-producing β-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene Neurog3 in ductal cells in vivo. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary β-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new β-like cells and that strategies aiming at controlling the expression of Neurog3, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes.

Purpose: Our previous study identified a significant association between a single nucleotide polymorphism (SNP) located in the neurogenin3 (NEUROG3) gene and post-stroke depression (PSD) in Chinese populations. The present work explores whether polymorphism rs144643855 affects regional brain activity and clinical phenotypes in major depressive disorder (MDD)., Method: A total of 182 participants were included: 116 MDD patients and 66 normal controls. All participants underwent resting-state functional magnetic resonance imaging (rs-fMRI) scanning at baseline. Spontaneous brain activity was assessed using amplitude of low-frequency fluctuation (ALFF). The Hamilton Depression Scale-24 (HAMD-24) and Snaith-Hamilton Pleasure Scale (SHAPS) were used to assess participants at baseline. Two-way analysis of covariance (ANCOVA) was used to explore the interaction between diagnostic groups and NEUROG3 rs144643855 on regional brain activity. We performed correlation analysis to further test the association between these interactive brain regions and clinical manifestations of MDD., Results: Genotype and disease significantly interacted in the left inferior frontal gyrus (IFG-L), right superior frontal gyrus (SFG-R), and left paracentral lobule (PCL-L) (P < 0.05). ALFF values of the IFG-L were found to be significantly associated with anhedonia in MDD patients., Conclusion: These findings suggest a potential relationship between rs144643855 variations and altered frontal brain activity in MDD. NEUROG3 may play an important role in the neuropathophysiology of MDD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Many pancreatic transcription factors that are essential for islet cell differentiation have been well characterized; however, because they are often expressed in several different cell populations, their functional hierarchy remains unclear. To parse out the spatiotemporal regulation of islet cell differentiation, we used a Neurog3-Cre allele to ablate Nkx2.2, one of the earliest and most broadly expressed islet transcription factors, specifically in the Neurog3+ endocrine progenitor lineage (Nkx2.2△endo). Remarkably, many essential components of the β cell transcriptional network that were down-regulated in the Nkx2.2KO mice, were maintained in the Nkx2.2△endo mice - yet the Nkx2.2△endo mice displayed defective β cell differentiation and recapitulated the Nkx2.2KO phenotype. This suggests that Nkx2.2 is not only required in the early pancreatic progenitors, but has additional essential activities within the endocrine progenitor population. Consistently, we demonstrate Nkx2.2 functions as an integral component of a modular regulatory program to correctly specify pancreatic islet cell fates.

Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption - due to a complete absence of enteroendocrine cells - followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic and, hence, may have had residual function in pancreas. We report 2 patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy but instead developed permanent IDDM during middle childhood ages. The variants showed no evidence of function in traditional promoter-based assays of NEUROG3 function and also failed to exhibit function in a variety of potentially novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that, unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression and, hence, suggest the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding β cell mass sufficient to maintain euglycemia until early childhood.

Neurogenin3 (NEUROG3) is a basic helix-loop-helix transcription factor required for development of the endocrine pancreas in mice. In contrast, humans with NEUROG3 mutations are born with endocrine pancreas function, calling into question whether NEUROG3 is required for human endocrine pancreas development. To test this directly, we generated human embryonic stem cell (hESC) lines where both alleles of NEUROG3 were disrupted using CRISPR/Cas9-mediated gene targeting. NEUROG3-/- hESC lines efficiently formed pancreatic progenitors but lacked detectible NEUROG3 protein and did not form endocrine cells in vitro. Moreover, NEUROG3-/- hESC lines were unable to form mature pancreatic endocrine cells after engraftment of PDX1+/NKX6.1+ pancreatic progenitors into mice. In contrast, a 75-90% knockdown of NEUROG3 caused a reduction, but not a loss, of pancreatic endocrine cell development. We conclude that NEUROG3 is essential for endocrine pancreas development in humans and that as little as 10% NEUROG3 is sufficient for formation of pancreatic endocrine cells. [ABSTRACT FROM AUTHOR]

Neurog3 is expressed transiently in pancreatic endocrine progenitors where it is responsible for activating a transcription factor cascade which eventually defines the mature endocrine cells. However, the mechanism by which Neurog3 regulates different aspects of the endocrine differentiation program is less clear. In this report we used in ovo electroporation to investigate how manipulation of Neurog3 protein activity affected migration, differentiation and fate determination. We found that changes in the onset of Neurog3 expression only had minor effect on differentiation. However increasing the transcriptional activity of Neurog3 by fusing it to VP16 or co-electroporating with Ep300 caused the electroporated cells to migrate rather than differentiate. In contrast, reducing the transcriptional activity of Neurog3 by deleting parts of the activation domain, by fusing Neurog3 to the engrailed repressor domain, or co-electroporating with Hdac1 greatly increased the proportion of glucagon expressing cells. Developmental Dynamics 239:1950-1966, 2010. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2009.12.009 Byline: Sui Wang, Jingbo Yan, Daniel A. Anderson, Yanwen Xu, Maneesh C. Kanal, Zheng Cao, Christopher V.E. Wright, Guoqiang Gu Keywords: Diabetes; Islet; Neurog3; Ngn3; Lateral inhibition; Notch; Endocrine progenitor; Dosage; Haploinsufficiency; Pancreas Abstract: The basic helix-loop-helix transcription factor Neurog3 (Neurogenin3 or Ngn3) actively drives endodermal progenitor cells towards endocrine islet cell differentiation during embryogenesis. Here, we manipulate Neurog3 expression levels in endocrine progenitor cells without altering its expression pattern using heterozygosity and a hypomorph. Lowered Neurog3 gene dosage in the developing pancreatic epithelium reduces the overall production of endocrine islet cells without significantly affecting the proportions of various islet cell types that do form. A reduced Neurog3 production level in the endocrine-directed pancreatic progenitor population activates the expression of Neurog3 in an increased number of epithelial progenitors. Yet a significant number of these Neurog3.sup.+ cells detected in heterozygous and hypomorphic pancreata, possibly those that express low levels of Neurog3, move on to adopt pancreatic ductal or acinar fates. These data directly demonstrate that achieving high levels of Neurog3 expression is a critical step for endocrine commitment from multipotent pancreatic progenitors. These findings also suggest that a high level of Neurog3 expression could mediate lateral inhibition or other unknown feedback mechanisms to regulate the number of cells that initiate Neurog3 transcription and protein production. The control of Neurog3.sup.+ cell number and the Neurog3 threshold-dependent endocrine differentiation mechanism combine to select a specific proportion of pancreatic progenitor cells to adopt the islet cell fate. Author Affiliation: Program in Developmental Biology and Department of Cell and Developmental Biology, Center for Stem Cell Biology, Vanderbilt University Medical Center, 465 21st Avenue South, Rm 4128, Vanderbilt Medical Center, Nashville, TN 37232, USA Article History: Received 1 July 2009; Revised 5 December 2009; Accepted 7 December 2009

Context: Biallelic pathogenic variants in the NEUROG3 gene cause malabsorptive diarrhea, insulin-dependent diabetes mellitus (IDDM), and rarely hypogonadotropic hypogonadism. With only 17 reported cases, the clinical and mutational spectra of this disease are far from complete. Objective: To identify the underlying genetic etiology in 3 unrelated Thai patients who presented with early-onset malabsorptive diarrhea, endocrine abnormalities, and renal defects and to determine the pathogenicity of the newly identified pathogenic variants using luciferase reporter assays and western blot. Methods: Three unrelated patients with congenital diarrhea were recruited. Detailed clinical and endocrinological features were obtained. Exome sequencing was performed to identify mutations and in vitro functional experiments including luciferase reporter assay were studied to validate their pathogenicity. Results: In addition to malabsorptive diarrhea due to enteric anendocrinosis, IDDM, short stature, and delayed puberty, our patients also exhibited pituitary gland hypoplasia with multiple pituitary hormone deficiencies (Patient 1, 2, 3) and proximal renal tubulopathy (Patient 2, 3) that have not previously reported. Exome sequencing revealed that Patient 1 was homozygous for c.371C>G (p.Thr124Arg) while the other 2 patients were homozygous for c.284G>C (p.Arg95Pro) in NEUROG3. Both variants have never been previously reported. Luciferase reporter assay demonstrated that these 2 variants impaired transcriptional activity of NEUROG3. Conclusions: This study reported pituitary gland hypoplasia with multiple pituitary hormone deficiencies and proximal renal tubulopathy and 2 newly identified NEUROG3 loss-of-function variants in the patients with NEUROG3-associated syndrome. [ABSTRACT FROM AUTHOR]

During mouse pancreas organogenesis, endocrine cells are born from progenitors residing in an epithelial plexus niche. After a period in a lineage-primed Neurog3 LO state, progenitors become endocrine committed via upregulation of Neurog3 We find that the Neurog3 LO transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through Neurog3 HI transition is associated with distinct stages of an epithelial egression process: narrowing the apical surface of the cell, basalward cell movement and eventual cell-rear detachment from the apical lumen surface to allow clustering as nascent islets under the basement membrane. Apical narrowing, basalward movement and Neurog3 transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through Neurog3 -expressing states. NmMyoII is necessary for apical narrowing, basalward cell displacement and Neurog3 upregulation, but all three are limited by ROCK activity. We propose that ROCK-nmMyoII activity, Neurog3 gene-dose and Notch signaling integrate endocrine fate allocation with epithelial plexus growth and morphogenesis, representing a feedback control circuit that coordinates morphogenesis with lineage diversification in the endocrine-birth niche., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

In the context of type 1 diabetes research and the development of insulin-producing β-cell replacement strategies, whether pancreatic ductal cells retain their developmental capability to adopt an endocrine cell identity remains debated, most likely due to the diversity of models employed to induce pancreatic regeneration. In this work, rather than injuring the pancreas, we developed a mouse model allowing the inducible misexpression of the proendocrine gene Neurog3 in ductal cells in vivo. These animals developed a progressive islet hypertrophy attributed to a proportional increase in all endocrine cell populations. Lineage tracing experiments indicated a continuous neo-generation of endocrine cells exhibiting a ductal ontogeny. Interestingly, the resulting supplementary β-like cells were found to be functional. Based on these findings, we suggest that ductal cells could represent a renewable source of new β-like cells and that strategies aiming at controlling the expression of Neurog3, or of its molecular targets/co-factors, may pave new avenues for the improved treatments of diabetes., Competing Interests: Author MC was not employed by Evotec at the time of this study; however, she is currently employed by Evotec International GmbH. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

During pancreatic development, proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3), exit the cell cycle, and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183, which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle, NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G 1 /S cell-cycle checkpoint. Using models of mouse and human pancreas development, we show that lengthening of the G 1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum, these studies demonstrate that progenitor cell-cycle G 1 lengthening, through its actions on stabilization of NEUROG3, is an essential variable in normal endocrine cell genesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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 β-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. [ABSTRACT FROM AUTHOR]

Many pancreatic transcription factors that are essential for islet cell differentiation have been well characterized; however, because they are often expressed in several different cell populations, their functional hierarchy remains unclear. To parse out the spatiotemporal regulation of islet cell differentiation, we used a Neurog3-Cre allele to ablate Nkx2.2 , one of the earliest and most broadly expressed islet transcription factors, specifically in the Neurog3 + endocrine progenitor lineage ( Nkx2.2 △endo ). Remarkably, many essential components of the β cell transcriptional network that were down-regulated in the Nkx2.2 KO mice, were maintained in the Nkx2.2 △endo mice - yet the Nkx2.2 △endo mice displayed defective β cell differentiation and recapitulated the Nkx2.2 KO phenotype. This suggests that Nkx2.2 is not only required in the early pancreatic progenitors, but has additional essential activities within the endocrine progenitor population. Consistently, we demonstrate Nkx2.2 functions as an integral component of a modular regulatory program to correctly specify pancreatic islet cell fates., Competing Interests: The authors declare that no competing interests exist.

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9(+) bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3(TA.LO) cell population, defined as Neurog3 transcriptionally active and Sox9(+) and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9(+) Neurog3(TA.LO) progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3(HI) cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9(+) Neurog3(TA.LO) progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9(+) Neurog3(TA.LO) endocrine-biased progenitors feed production of Neurog3(HI) endocrine-committed cells during pancreas organogenesis., (© 2016 Bechard et al.; Published by Cold Spring Harbor Laboratory Press.)

In human, mutations in bicaudal C1 (BICC1), an RNA binding protein, have been identified in patients with kidney dysplasia. Deletion of Bicc1 in mouse leads to left-right asymmetry randomization and renal cysts. Here, we show that BICC1 is also expressed in both the pancreatic progenitor cells that line the ducts during development, and in the ducts after birth, but not in differentiated endocrine or acinar cells. Genetic inactivation of Bicc1 leads to ductal cell overproliferation and cyst formation. Transcriptome comparison between WT and Bicc1 KO pancreata, before the phenotype onset, reveals that PKD2 functions downstream of BICC1 in preventing cyst formation in the pancreas. Moreover, the analysis highlights immune cell infiltration and stromal reaction developing early in the pancreas of Bicc1 knockout mice. In addition to these functions in duct morphogenesis, BICC1 regulates NEUROG3+ endocrine progenitor production. Its deletion leads to a late but sustained endocrine progenitor decrease, resulting in a 50% reduction of endocrine cells. We show that BICC1 functions downstream of ONECUT1 in the pathway controlling both NEUROG3+ endocrine cell production and ductal morphogenesis, and suggest a new candidate gene for syndromes associating kidney dysplasia with pancreatic disorders, including diabetes. [ABSTRACT FROM AUTHOR]

The development of the nervous system is critically dependent on the production of functionally diverse neuronal cell types at their correct locations. In the embryonic neural tube, dorsoventral signaling has emerged as a fundamental mechanism for generating neuronal diversity. In contrast, far less is known about how different neuronal cell types are organized along the rostrocaudal axis. In the developing mouse and chick neural tube, hindbrain serotonergic neurons and spinal glutamatergic V3 interneurons are produced from ventral p3 progenitors, which possess a common transcriptional identity but are confined to distinct anterior-posterior territories. In this study, we show that the expression of the transcription factor Neurogenin3 (Neurog3) in the spinal cord controls the correct specification of p3-derived neurons. Gain- and loss-of-function manipulations in the chick and mouse embryo show that Neurog3 switches ventral progenitors from a serotonergic to V3 differentiation program by repressing Ascll in spinal p3 progenitors through a mechanism dependent on Hes proteins. In this way, Neurog3 establishes the posterior boundary of the serotonergic system by actively suppressing serotonergic specification in the spinal cord. These results explain how equivalent p3 progenitors within the hindbrain and the spinal cord produce functionally distinct neuron cell types. [ABSTRACT FROM AUTHOR]

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 Insm1GFPCre 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 typespecific 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 programand activating genes essential for RNA splicing, cell migration, controlled cellular proliferation, vasculogenesis, extracellular matrix and hormone secretion. [ABSTRACT FROM AUTHOR]

Background: NEUROG3 is a key regulator of pancreatic endocrine cell differentiation in mouse, essential for the generation of all mature hormone producing cells. It is repressed by Notch signaling that prevents pancreatic cell differentiation by maintaining precursors in an undifferentiated state. Results: We show that, in zebrafish, neurog3 is not expressed in the pancreas and null neurog3 mutant embryos do not display any apparent endocrine defects. The control of endocrine cell fate is instead fulfilled by two basic helixloop- helix factors, Ascl1b and Neurod1, that are both repressed by Notch signaling. ascl1b is transiently expressed in the mid-trunk endoderm just after gastrulation and is required for the generation of the first pancreatic endocrine precursor cells. Neurod1 is expressed afterwards in the pancreatic anlagen and pursues the endocrine cell differentiation program initiated by Ascl1b. Their complementary role in endocrine differentiation of the dorsal bud is demonstrated by the loss of all hormone-secreting cells following their simultaneous inactivation. This defect is due to a blockage of the initiation of endocrine cell differentiation. Conclusions: This study demonstrates that NEUROG3 is not the unique pancreatic endocrine cell fate determinant in vertebrates. A general survey of endocrine cell fate determinants in the whole digestive system among vertebrates indicates that they all belong to the ARP/ASCL family but not necessarily to the Neurog3 subfamily. The identity of the ARP/ASCL factor involved depends not only on the organ but also on the species. One could, therefore, consider differentiating stem cells into insulin-producing cells without the involvement of NEUROG3 but via another ARP/ASCL factor. [ABSTRACT FROM AUTHOR]

Delineating gene regulatory networks that orchestrate cell-type specification is a continuing challenge for developmental biologists. Single-cell analyses offer opportunities to address these challenges and accelerate discovery of rare cell lineage relationships and mechanisms underlying hierarchical lineage decisions. Here, we describe the molecular analysis of mouse pancreatic endocrine cell differentiation using single-cell transcriptomics, chromatin accessibility assays coupled to genetic labeling, and cytometry-based cell purification. We uncover transcription factor networks that delineate β-, α-, and δ-cell lineages. Through genomic footprint analysis, we identify transcription factor-regulatory DNA interactions governing pancreatic cell development at unprecedented resolution. Our analysis suggests that the transcription factor Neurog3 may act as a pioneer transcription factor to specify the pancreatic endocrine lineage. These findings could improve protocols to generate replacement endocrine cells from renewable sources, like stem cells, for diabetes therapy. [ABSTRACT FROM AUTHOR]

Neurog3 (Neurogenin 3 or Ngn3) is both necessary and sufficient to induce endocrine islet cell differentiation from embryonic pancreatic progenitors. Since robust Neurog3 expression has not been detected in hormone-expressing cells, Neurog3 is used as an endocrine progenitor marker and regarded as dispensable for the function of differentiated islet cells. Here we used 3 independent lines of Neurog3 knock-in reporter mice and mRNA/protein-based assays to examine Neurog3 expression in hormone-expressing islet cells. Neurog3 mRNA and protein are detected in hormone- producing cells at both embryonic and adult stages. Significantly, inactivating Neurog3 in insulin-expressing β cells at embryonic stages or in Pdx1-expressing islet cells in adults impairs endocrine function, a phenotype that is accompanied by reduced expression of several Neurog3 target genes that are essential for islet cell differentiation, maturation, and function. These findings demonstrate that Neurog3 is required not only for initiating endocrine cell differentiation, but also for promoting islet cell maturation and maintaining islet function. [ABSTRACT FROM AUTHOR]

NEUROGENIN3+ (NEUROG3+) cells are considered to be pancreatic endocrine progenitors. Our current knowledge on the molecular program of NEUROG3+ cells in humans is largely extrapolated from studies in mice. We hypothesized that single-cell RNA-seq enables in-depth exploration of the rare NEUROG3+ cells directly in humans. We aligned four large single-cell RNA-seq datasets from postnatal human pancreas. Our integrated analysis revealed 10 NEUROG3+ epithelial cells from a total of 11,174 pancreatic cells. Noticeably, human NEUROG3+ cells clustered with mature pancreatic cells and epsilon cells displayed the highest frequency of NEUROG3 positivity. We confirmed the co-expression of NEUROG3 with endocrine markers and the high percentage of NEUROG3+ cells among epsilon cells at the protein level based on immunostaining on pancreatic tissue sections. We further identified unique genetic signatures of the NEUROG3+ cells. Regulatory network inference revealed novel transcription factors including Prospero homeobox protein 1 (PROX1) may act jointly with NEUROG3. As NEUROG3 plays a central role in endocrine differentiation, knowledge gained from our study will accelerate the development of beta cell regeneration therapies to treat diabetes.

Spermatogenesis relies on coordinated differentiation of stem and progenitor spermatogonia, and the transcription factor STAT3 is essential for this process in mammals. Here we studied the THY1+ spermatogonial population in mouse testes, which contains spermatogonial stem cells (SSC) and non-stem cell progenitor spermatogonia, to further define the downstream mechanism regulating differentiation. Transcript abundance for the bHLH transcription factor Neurog3 was found to be significantly reduced upon transient inhibition of STAT3 signaling in these cells and exposure to GDNF, a key growth factor regulating self-renewal of SSCs, suppressed activation of STAT3 and in accordance Neurog3 gene expression. Moreover, STAT3 was found to bind the distal Neurog3 promoter/enhancer region in THY1+ spermatogonia and regulate transcription. Transient inhibition of Neurog3 expression in cultures of proliferating THY1+ spermatogonia increased stem cell content after several self-renewal cycles without effecting overall proliferation of the cells, indicating impaired differentiation of SSCs to produce progenitor spermatogonia. Furthermore, cultured THY1+ spermatogonia with induced deficiency of Neurog3 were found to be incapable of differentiation in vivo following transplantation into testes of recipient mice. Collectively, these results establish a mechanism by which activation of STAT3 regulates the expression of NEUROG3 to subsequently drive differentiation of SSC and progenitor spermatogonia in the mammalian germline.

The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology.

Aim: Type 2 diabetes (T2D) is a complex polygenic disorder. Genetic predisposition may vary in different ethnic groups. A potential candidate gene for T2D is Neurogenin 3 (Ngn3, NEUROG3), which lies on chromosome 10 in a region with several potential linkage signals to T2D in various population studies. The goal of this study was to establish whether NEUROG3 gene variants are contributing to T2D in an Italian T2D cohort., Methods: We genotyped AFMa210xh1 macrosatellite marker in 202 Italian T2D families/sib-pairs. We performed two-point linkage analysis in the late- and early-onset dataset. For the case control study, we selected families with a positive logarithm of odds (LOD) score. Then, we screened NEUROG3 in the selected 61 single unrelated T2D patients and 101 Italian controls and performed association studies., Results: Several variants were identified: a new 152ntC/G, and 44-45delCA, Gly167Arg, Ser 199Phe single nucleotide polymorphisms (SNPs) and 2 new 5'UTR variations (-nt498G/T and nt367C/T) and a new Gly167fsinsCAE Arg167X234 mutation. The variants 44-45delCA/ Ser199Phe and Gly167Arg/Ser199Phe show significant linkage disequilibrium. The haplotype CCCAGT/A/C shows association to T2D in our cohort, while the allele 167Arg, the haplotypes CCCAGT/A and A/C and the diplotype LL/GA/TC show a trend towards association to disease. The 5'UTR and frameshift variants are absent in the controls. Nonparametric linkage analysis within NEUROG3 variants in 9 early-onset T2D families shows a nonparametric LOD score=2.49 (P=0.006)., Conclusions: The biological impact of NEUROG3 might be due to the presence of either CCCAGT at 44-45nt, 167Arg, 199Ser or by a haplotype combination of these 3 or 2 of them.

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. [ABSTRACT FROM AUTHOR]