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. 2018 Feb;154(3):624-636.
doi: 10.1053/j.gastro.2017.10.005. Epub 2017 Oct 12.

Transcription and Signaling Regulators in Developing Neuronal Subtypes of Mouse and Human Enteric Nervous System

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Free PMC article

Transcription and Signaling Regulators in Developing Neuronal Subtypes of Mouse and Human Enteric Nervous System

Fatima Memic et al. Gastroenterology. .
Free PMC article

Abstract

Background & aims: The enteric nervous system (ENS) regulates gastrointestinal function via different subtypes of neurons, organized into fine-tuned neural circuits. It is not clear how cell diversity is created within the embryonic ENS; information required for development of cell-based therapies and models of enteric neuropathies. We aimed to identify proteins that regulate ENS differentiation and network formation.

Methods: We generated and compared RNA expression profiles of the entire ENS, ENS progenitor cells, and non-ENS gut cells of mice, collected at embryonic days 11.5 and 15.5, when different subtypes of neurons are formed. Gastrointestinal tissues from R26ReYFP reporter mice crossed to Sox10-CreERT2 or Wnt1-Cre mice were dissected and the 6 populations of cells were isolated by flow cytometry. We used histochemistry to map differentially expressed proteins in mouse and human gut tissues at different stages of development, in different regions. We examined enteric neuronal diversity and gastric function in Wnt1-Cre x Sox6fl/fl mice, which do not express the Sox6 gene in the ENS.

Results: We identified 147 transcription and signaling factors that varied in spatial and temporal expression during development of the mouse ENS. Of the factors also analyzed in human ENS, most were conserved. We uncovered 16 signaling pathways (such as fibroblast growth factor and Eph/ephrin pathways). Transcription factors were grouped according to their specific expression in enteric progenitor cells (such as MEF2C), enteric neurons (such as SOX4), or neuron subpopulations (such as SATB1 and SOX6). Lack of SOX6 in the ENS reduced the numbers of gastric dopamine neurons and delayed gastric emptying.

Conclusions: Using transcriptome and histochemical analyses of the developing mouse and human ENS, we mapped expression patterns of transcription and signaling factors. Further studies of these candidate determinants might elucidate the mechanisms by which enteric stem cells differentiate into neuronal subtypes and form distinct connectivity patterns during ENS development. We found expression of SOX6 to be required for development of gastric dopamine neurons.

Keywords: Gastric Motility; HOX; Neural Crest; Tyrosine Hydroxylase.

Figures

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Figure 1
Figure 1
Transcriptome screen design, verification, and identification of enriched genes. (A) Schematic drawing depicting the transcriptome analysis. ENS populations included SOX10+ progenitors (red boxes) and the whole ENS (blue boxes), each at 2 distinct development stages: E11.5 (S11 and W11) and E15.5 (S15 and W15). W11 contained immature neurons differentiating into, for example, 5-HT+ neurons, whereas W15 included immature neurons differentiating into other subtypes (eg, TH+). Non-ENS control gut tissue (gray boxes) at E11.5 (C11) and E15.5 (C15) was also included. (B) Heat map summarizing differentially expressed genes, compiled from the union of genes with top-10 absolute fold change (and P < .05) in the comparisons of S11vsC11, S15vsC15, W11vsC11, and W15vsC15. Genes and conditions are clustered by their hierarchical similarity. Color intensity represents the mean-centred log2 expression values. (C) Graphs comparing gene ontology (GO) term enrichment, ENS, and gut marker genes in the 4 ENS populations to control populations. (D) Heat map depicting cell cycle or neuronal genes. Genes are clustered by their hierarchical similarity. Color intensity represents the mean-centred log2 expression values. (E) Tables showing the number of transcription factors, signaling factors, and receptors found in pairwise comparisons (absolute fold change >1.2; P < .05) of the transcriptomes. n/a, not analyzed.
Figure 2
Figure 2
Expression dynamics of transcription factors in the developing ENS. (A) Table summarizing IHC expression analysis (Supplementary Figures 3 and 4) of transcription factors in relation to HUC/D+ neurons and SOX10+ progenitor cells in stomach and intestine of mouse and human embryos at different stages. Genes are grouped according to their expression dynamics. (B) Table showing transcription factors ordered according to their DNA binding domain and their onset of expression. Onset time was estimated based on IHC, ISH (Supplementary Figure 2), and/or the transcriptome analysis. (C) Examples of IHC from groups I to IV showing similar gene expression in mouse and human. Yellow arrowheads, expression in progenitors; white arrowheads, expression in neurons. (D) Expression of SOX proteins in the developing ENS together with SOX2/10+ progenitors or HUC/D+ neurons at E15.5 (SOX4 at E12.5). Arrowheads indicate double-positive cells. (E) Expression of Hox genes in the developing ENS. Note localization of Hoxa3, Hoxc5, Hoxb3, and Hoxc4 in HUC/D+ neurons (arrowheads).
Figure 3
Figure 3
Coexpression analysis of transcription factors with enteric neurotransmitters/neuropeptides. (A and B) Tables summarizing IHC coexpression analysis of abundant (A) or selectively expressed (B) transcription factors together with ENS marker genes at E18.5. TH, NPY, 5-HT, and ChAT were analyzed in the stomach, and all other markers in the small intestine. Coexpression with SOX6 could be addressed only in the stomach. (C) Coexpression (arrowheads) between TH and 6 transcription factors in the stomach at E18.5. CALB1, calbindin; CGRP, calcitonin gene-related peptide; ChAT, choline acetyltransferase; 5-HT, 5-hydroxytryptamine; NOS1, nitric oxide synthase 1; NPY, neuropeptide Y; TH, tyrosine hydroxylase; VIP, vasoactive intestinal polypeptide.
Figure 4
Figure 4
Loss of Sox6 results in selective reduction of gastric TH+ neurons and gastric motility. (A) IHC of SOX6 expression in progenitor cells (yellow arrowheads) or neurons (white arrowheads) in the developing stomach but not intestine. (B and C) IHC showing neurotransmitters that are coexpressed (arrowheads) (B), or not coexpressed (C) with SOX6 in enteric neurons at E18.5. (D) Pairwise IHC analysis showing expression of NPY and CALB1 with each other but not with TH in enteric neurons at E18.5. (E) IHC of SOX6 at E18.5 showing expression in ENS (arrowhead) and non-ENS tissue (stars). (F and G) Representative IHC images depicting expression of phenotypic marker proteins in the stomach of control embryos (F) and Sox6 mutant embryos (G). (HJ) Average percentage of neurons expressing specific markers in the stomach of Sox6 mutant and littermate control E18.5 embryos (H and I) or adults (J). n = 3–4. (K) Decreased weight of Sox6 mutant males compared with littermate control mice. n = 3–5. (L) Enlarged stomach with more residual food in a Sox6 mutant (left) in comparison with a control (right) mouse. n = 4. (M) Gastric emptying shown as percentage of administered rhodamine dextran that emptied from the stomach of Sox6 mutant and littermate control male adult mice. n = 3 *P < .05, **P < .01, *** P < .001.
Figure 5
Figure 5
Novel cell-cell communication pathways during ENS development. (A) Table summarizing novel signaling pathways, including the identified ligands and receptors, signaling pathway associated genes (see Supplementary Table 8), and putative functions based on studies in other developing tissue. N/D, not determined; eg, indicates examples of binding partners found in the screen when ligand or receptor are incompletely studied or show promiscuous binding capacities. (B) Expression of ligand-receptor couples found within the ENS using IHC and analysis of ISH images. (C) IHC or ISH depicting ligands and receptors expressed in the ENS without the image of corresponding binding partners. (D) Expression of ligand-receptor couples using IHC and ISH, where ligands are expressed outside the ENS. Arrowheads indicate expression in HUC/D+ (yellow), SOX10+ (white), or ENS (black) cells. *Expression outside ENS. All images are shown in Supplementary Figures 5–7.
Figure 6
Figure 6
Summary of IHC analysis of cell-cell communication components in the developing gut wall. Table summarizing the IHC expression analysis of receptors and ligands in the stomach and intestine at different developmental stages in mouse and human. Column to the right indicates non-ENS expression. n/a, antibody staining inconclusive or incompatible with species.
Figure 7
Figure 7
Summary of genes with putative regulatory functions in the developing ENS. (A) Transcription factors in the developing ENS. Left column includes genes with putative functions in stem cell maintenance versus neurogenesis. Right column includes genes with putative functions in specification and/or differentiation of enteric neurons. (B) Signaling factors in the developing ENS. Ligands expressed in the ENS (left column) or in proximal gut tissue (right column) presented with putative receptors. Ligand/receptor couples are likely involved in proliferation/differentiation or migration/network formation as indicated. See Supplementary Tables 6 and 7 for more information.

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