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.

Understanding the origin of pancreatic β cells has profound implications for regenerative therapies in diabetes. For over a century, it was widely held that adult pancreatic duct cells act as endocrine progenitors, but lineage-tracing experiments challenged this dogma. Gribben et al. recently used two existing lineage-tracing models and single-cell RNA sequencing to conclude that adult pancreatic ducts contain endocrine progenitors that differentiate to insulin-expressing β cells at a physiologically important rate. We now offer an alternative interpretation of these experiments. Our data indicate that the two Cre lines that were used directly label adult islet somatostatin-producing ∂ cells, which precludes their use to assess whether β cells originate from duct cells. Furthermore, many labeled ∂ cells, which have an elongated neuron-like shape, were likely misclassified as β cells because insulin-somatostatin coimmunolocalizations were not used. We conclude that most evidence so far indicates that endocrine and exocrine lineage borders are rarely crossed in the adult pancreas.

Sequence variants in cis-acting enhancers are important for polygenic disease, but their role in Mendelian disease is poorly understood. Redundancy between enhancers that regulate the same gene is thought to mitigate the pathogenic impact of enhancer mutations. Recent findings, however, have shown that loss-of-function mutations in a single enhancer near PTF1A cause pancreas agenesis and neonatal diabetes. Using mouse and human genetic models, we show that this enhancer activates an entire PTF1A enhancer cluster in early pancreatic multipotent progenitors. This leading role, therefore, precludes functional redundancy. We further demonstrate that transient expression of PTF1A in multipotent progenitors sets in motion an epigenetic cascade that is required for duct and endocrine differentiation. These findings shed insights into the genome regulatory mechanisms that drive pancreas differentiation. Furthermore, they reveal an enhancer that acts as a regulatory master key and is thus vulnerable to pathogenic loss-of-function mutations.

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.

Congenital enteropathies (CE) are a group of rare inherited diseases with a typical onset early in life. They involve defects in enterocyte structure or differentiation. They can cause a severe condition of intestinal failure (IF). The diagnostic approach is based first on clinical presentation (consanguinity, prenatal expression, polyhydramnios, early neonatal onset, aspect of stools, persistence at bowel rest, associated extra-digestive manifestations….) and histo-pathological analyses. These rare intestinal diseases cause protracted diarrhea that might resolve, for a few, with a dietetic approach. However, protracted or permanent IF may require long term parenteral nutrition and, in limited cases, intestinal transplantation. With the progresses in both clinical nutrition and genetics, many of these CE are nowadays associated with recognized gene mutations. It improved our knowledge and the understanding in the patho-physiology of these diseases, thus, leading potentially to therapeutic perspectives. These review cover most of the early onset CE and excludes the immune related diarrhea.

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.

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.
CONCLUSIONS: 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.

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).
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.
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.
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.

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.

Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 (Ascl1) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMHC) and -dorsomedial (VMHDM) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1-/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3-null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1-null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3.