Reduced Neurog3 Gene Dosage Shifts Enteroendocrine Progenitor Towards Goblet Cell Lineage in the Mouse Intestine

Abstract

This study reports the transcriptome profiles for Neurog3 þderived cells isolated from mouse duodenum with 2 or 1 Neurog3 alleles. Deletion of 1 Neurogenin3 allele resulted in reduced enteroendocrine cells, increased goblet cell number, and altered gene expression profile, indicating that Neurog3 gene dosage determined intestinal Neurog3þ progenitor fate specification. BACKGROUND & AIMS:Transient expression of Neurog3 commits intestinal secretory progenitors to become enteroendocrinebiased progenitors and hence drive enteroendocrine differentiation. Loss of Neurog3 in mouse resulted in the depletion of intestinal enteroendocrine cells (EECs) and an increase in goblet cells. Earlier studies in developing mouse pancreas identified a role of Neurog3 gene dosage in endocrine and exocrine cell fate allocation. We aimed to determine whether Neurog3 gene dosage controls fate choice of enteroendocrine progenitors. METHODS:We acquired mutant Neurog3 reporter mice carrying 2, 1, or null Neurog3 alleles to study Neurog3 gene dosage effect by lineage tracing. Cell types arising from Neu-rog3þ progenitors were determined by immunohistochemistry using antibodies against intestinal lineage-specific markers. RNA sequencing of sorted Neurog3 þ/þ , Neurog3 þ/-, or bulk intestinal cells were performed and differentially expressed genes were analyzed. RESULTS:We identified 2731 genes enriched in sorted Neu-rog3 þ/þ -derived cells in the Neurog3 þ/þEYFP mouse intestine when compared with bulk duodenum epithelial cells. In the intestine of Neurog3 þ/-EGFP heterozygous mouse, we observed a 63% decrease in EEC numbers. Many Neurog3-derived cells stained for goblet marker Mucin 2. RNA sequencing of sorted Neurog3 þ/cells uncovered enriched expression of genes characteristic for both goblet and enteroendocrine cells, indicating the mixed lineages arose from Neurog3þ progenitors. Consistent with this hypothesis, deletion of both Neurog3 alleles resulted in the total absence of EECs. All Neurog3 þderived cells stained for Mucin 2. CONCLUSIONS:We identified that the fate of Neurog3 þ enteroendocrine progenitors is dependent on Neurog3 gene dosage. High Neurog3 gene dosage enforces the commitment of secretory progenitors to an EE lineage, while constraining their goblet cell lineage potential. Transcriptome profiling data was deposited to Gene Ontology omnibus, accession number:

Cells of the human intestinal tract mapped across space and time

“…LEC2 cells emerge during the second trimester (15)(16)(17) and are present in gut regions as well as fetal mLNs (Supp. Figure 7J).…”

“…3B). NEUROG3, a transcription factor that mediates epithelial cell commitment to EEC lineage 16 , and the absence of mature neuropeptides suggests that the NEUROG3+ EECs may represent early committed EECs. M cells, which specialise in transferring luminal antigen through the epithelial lining of the gut were very rare and seen only in pediatric and fetal samples within this dataset (Figure 2B & Supplementary Fig.…”

“…To infer cell-cell communication and screen for ligand-receptors involved we applied CellPhoneDB v2.0 python package 83,84 on the normalised raw counts and fine cell type annotations from the second trimester intestinal samples (12)(13)(14)(15)(16)(17). We use default parameters and set subsetting to 5000 cells.…”

MTG16 (CBFA2T3) regulates colonic epithelial differentiation, colitis, and tumorigenesis by repressing E protein transcription factors

“…Instead, we observed attenuation of the goblet cell lineage and upregulation of the enteroendocrine lineage. Neurog3, a bHLH transcription factor required for enteroendocrine cell differentiation 86,[104][105][106] , was consistently upregulated in Mtg16 -/colon. Using GSEA with gene sets for homeostatic epithelial cell types, including cells at different stages of enteroendocrine cell differentiation, we determined that Mtg16 -/colon demonstrated enrichment of all enteroendocrine cell progenitor signatures except the earliest enteroendocrine precursor in which Neurog3 expression has not yet been induced 86 .…”

“…Finally, Mtg16 -/colon exhibited increased Neurog3 + enteroendocrine progenitor cells by RNAscope. Although Neurog3 + progenitor cells in the SI may still differentiate into goblet and Paneth cells 102,106 , they are thought to be fully committed to the enteroendocrine lineage in the colon 102 . Thus, MTG16 functions as a nexus of cell fate control in colonic enteroendocrine:goblet lineage assignment.…”