Enteric glial cell diversification is influenced by spatiotemporal factors and source of neural progenitors in mice

Abstract

Previously focused primarily on enteric neurons, studies of the enteric nervous system (ENS) in both health and disease are now broadening to recognize the equally significant role played by enteric glial cells (EGCs). Commensurate to the vast array of gastrointestinal functions they influence, EGCs exhibit considerable diversity in terms of location, morphology, molecular profiles, and functional attributes. However, the mechanisms underlying this diversification of EGCs remain largely unexplored. To begin unraveling the mechanistic complexities of EGC diversity, the current study aimed to examine its spatiotemporal aspects in greater detail, and to assess whether the various sources of enteric neural progenitors contribute differentially to this diversity. Based on established topo-morphological criteria for categorizing EGCs into four main subtypes, our detailed immunofluorescence analyses first revealed that these subtypes emerge sequentially during early postnatal development, in a coordinated manner with the structural changes that occur in the ENS. When combined with genetic cell lineage tracing experiments, our analyses then uncovered a strongly biased contribution by Schwann cell-derived enteric neural progenitors to particular topo-morphological subtypes of EGCs. Taken together, these findings provide a robust foundation for further investigations into the molecular and cellular mechanisms governing EGC diversity.

Keywords: cell lineage tracing; enteric glial cells; enteric nervous system; myenteric plexus; postnatal development; schwann cell precursors; submucosal plexus; topo-morphological subtypes.

FIGURE 1

Analysis of EGC diversity in the maturing postnatal myenteric plexus of the distal ileum. (A,B) Immunofluorescence analysis of the myenteric plexus (A) and associated circular muscle (B) in the distal ileum of wild-type FVB mice, at indicated postnatal ages (P1, P5, P10 and P20). Intestinal tissues were immunolabeled with antibodies against SOX10 for EGCs (cyan) and βIII-Tubulin for neuronal fibers (gray). Displayed images are z-stack projections representative of observations made from N = 3 mice per time point. Scale bar, 70 μm. (C,D) Quantitative analysis of the relative proportions of EGC Type I to IV, using images such as those displayed in panels (A,B) (N = 3 mice per time point; n = 3 to 5 60x fields of view per animal). (E) Quantitative analysis of indicated morphometric parameters (ganglionic surface area, extraganglionic surface area, interganglionic fiber surface area and circular muscle thickness) in the distal ileum as a function of age during the early postnatal period. (F) Correlation analysis (excluding 0% and 100% values; incompatible with proportion calculations) between Type I proportion and ganglionic area, Type II proportion and interganglionic fiber area, Type III proportion and extraganglionic area, and Type IV and circular muscle thickness. Colored symbols correspond to the indicated time points. r is Pearson’s correlation coefficient. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; One-Way ANOVA and Tukey’s multiple comparison test.

FIGURE 2

Analysis of EGC diversity in the maturing postnatal myenteric plexus of the distal colon. (A,B) Immunofluorescence analysis of the myenteric plexus (A) and associated circular muscle (B) in the distal colon of wild-type FVB mice, at indicated postnatal ages (P1, P5, P10 and P20). Intestinal tissues were immunolabeled with antibodies against SOX10 for EGCs (cyan) and βIII-Tubulin for neuronal fibers (gray). Displayed images are z-stack projections representative of observations made from N = 3 mice per time point. Scale bar, 70μm. (C,D) Quantitative analysis of the relative proportions of EGC Type I to IV, using images such as those displayed in panels (A,B) (N = 3 mice per time point; n = 3 to 5 60x fields of view per animal). (E) Quantitative analysis of indicated morphometric parameters (ganglionic surface area, extraganglionic surface area, interganglionic fiber surface area and circular muscle thickness) in the distal colon as a function of age during the early postnatal period. (F) Correlation analysis (excluding 0% and 100% values; incompatible with proportion calculations) between Type I proportion and ganglionic area, Type II proportion and interganglionic fiber area, Type III proportion and extraganglionic area, and Type IV and circular muscle thickness. Colored symbols correspond to the indicated time points. r is Pearson’s correlation coefficient. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; One-Way ANOVA and Tukey’s multiple comparison test.

FIGURE 3

Analysis of EGC diversity in the maturing postnatal submucosal plexus of the distal ileum. (A) Immunofluorescence analysis of the submucosal plexus in the distal ileum of wild-type FVB mice, at indicated postnatal ages (P1, P5, P10 and P20). Intestinal tissues were immunolabeled with antibodies against SOX10 for EGCs (cyan) and βIII-Tubulin for neuronal fibers (gray). Displayed images are z-stack projections representative of observations made from N = 3 mice per time point. Scale bar, 70 μm. (B,C) Quantitative analysis of the relative proportions of EGC Type I to IV, using images such as those displayed in panel A (N = 3 mice per time point; n = 3 to 5 60x fields of view per animal). (D) Quantitative analysis of indicated morphometric parameters (ganglionic surface area, extraganglionic surface area, and interganglionic fiber surface area) in the distal ileum as a function of age during the early postnatal period. (E) Correlation analysis (excluding 0% and 100% values; incompatible with proportion calculations) between Type I proportion and ganglionic area, Type II proportion and interganglionic fiber area, and Type III proportion and extraganglionic area. Colored symbols correspond to the indicated time points. r is Pearson’s correlation coefficient. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; One-Way ANOVA and Tukey’s multiple comparison test.

FIGURE 4

Analysis of EGC diversity in the maturing postnatal submucosal plexus of the distal colon. (A) Immunofluorescence analysis of the submucosal plexus in the distal colon of FVB WT mice, at indicated postnatal ages (P1, P5, P10 and P20). Intestinal tissues were immunolabeled with antibodies against SOX10 for EGCs (cyan) and βIII-Tubulin for neuronal fibers (gray). Displayed images are z-stack projections representative of observations made from N = 3 mice per time point. Scale bar, 70μm. (B-C) Quantitative analysis of the relative proportions of EGC Type I to IV, using images such as those displayed in panel A (N = 3 mice per time point; n = 3 to 5 60x fields of view per animal). (D) Quantitative analysis of indicated morphometric parameters (ganglionic surface area, extraganglionic surface area, and interganglionic fiber surface area) in the distal colon as a function of age during the early postnatal period. (E) Correlation analysis (excluding 0% and 100% values; incompatible with proportion calculations) between Type I proportion and ganglionic area, Type II proportion and interganglionic fiber area, and Type III proportion and extraganglionic area. Colored symbols correspond to the indicated time points. r is Pearson’s correlation coefficient. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; One-Way ANOVA and Tukey’s multiple comparison test.

FIGURE 5

Global analysis of the SCP contribution to enteric gliogenesis. (A,B) Immunofluorescence-based analysis of the distribution of YFP+ SCPs in the myenteric plexus of the distal ileum (A) and distal colon (B) from Dhh-Cre;Rosa26^{[FloxedSTOP]YFP} mice at P5. Intestinal tissues were immunolabeled with antibodies against GFP/YFP for SCP-derived cells (green) and βIII-Tubulin for neuronal fibers (grey). Displayed images are z-stack projections representative of observations made from N = 3 mice per time point. Scale bar, 200 μm. (C) Quantitative analysis of the global SCP contribution to enteric gliogenesis in the myenteric plexus/muscular layer (black dots) and submucosal plexus (white dots) of the distal ileum and distal colon from Dhh-Cre;Rosa26^{[FloxedSTOP]YFP} mice at P20. Each dot represents the percentage of YFP+ SOX10+ EGCs over the total of SOX10+ EGCs in a single 60x field of view for N = 3 mice (n = 3–5 fields of view per tissue for the myenteric plexus/muscular layer; n = 6–10 fields of view per tissue for the submucosal layer). (D-F) Immunofluorescence analysis of the circular muscle layer (D), the myenteric plexus (E) and the submucosal plexus (F) of the distal colon from Dhh-Cre;Rosa26^{[FloxedSTOP]YFP} mice at P20. Tissues were immunolabeled with SOX10 (cyan), GFP/YFP (green) and βIII-Tubulin antibodies (gray). Scale bar, 70 μm. ***P ≤ 0.001, ****P ≤ 0.0001; Two-Way ANOVA and Tukey’s multiple comparison test.

FIGURE 6

Detailed analysis of the SCP contribution to EGC diversity. (A–D) Quantitative analysis of the SCP contribution to enteric gliogenesis as a function of EGC Types I to IV, in the myenteric plexus/muscular layer of the distal ileum (A) and the distal colon (B), as well as in the submucosal plexus of the distal ileum (C) and the distal colon (D). EGC subtypes were quantified using images such as those displayed in Figures 5D–F (N = 3 mice; n = 3–5 fields of view per tissue for the myenteric plexus/muscular layer; n = 6–10 fields of view per tissue for the submucosal layer). Size of sub-bars corresponds to the average of the percentage of YFP+ SOX10+ EGCs among all SOX10+ EGCs, in the indicated tissue layers and bowel segments.