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. 2021 Dec 15;79(1):1.
doi: 10.1007/s00018-021-04071-7.

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis

Maria-Theodora Melissari #  1 , Ana Henriques #  1   2 , Christos Tzaferis  3 , Alejandro Prados  3   2 , Michalis E Sarris  1 , Niki Chalkidi  1 , Dimitra Mavroeidi  1 , Panagiotis Chouvardas  3   4   5 , Sofia Grammenoudi  1 , George Kollias  3   6 , Vasiliki Koliaraki  7
Affiliations

Col6a1+/CD201+ mesenchymal cells regulate intestinal morphogenesis and homeostasis

Maria-Theodora Melissari et al. Cell Mol Life Sci. .

Abstract

Intestinal mesenchymal cells encompass multiple subsets, whose origins, functions, and pathophysiological importance are still not clear. Here, we used the Col6a1Cre mouse, which targets distinct fibroblast subsets and perivascular cells that can be further distinguished by the combination of the CD201, PDGFRα and αSMA markers. Developmental studies revealed that the Col6a1Cre mouse also targets mesenchymal aggregates that are crucial for intestinal morphogenesis and patterning, suggesting an ontogenic relationship between them and homeostatic PDGFRαhi telocytes. Cell depletion experiments in adulthood showed that Col6a1+/CD201+ mesenchymal cells regulate homeostatic enteroendocrine cell differentiation and epithelial proliferation. During acute colitis, they expressed an inflammatory and extracellular matrix remodelling gene signature, but they also retained their properties and topology. Notably, both in homeostasis and tissue regeneration, they were dispensable for normal organ architecture, while CD34+ mesenchymal cells expanded, localised at the top of the crypts, and showed increased expression of villous-associated morphogenetic factors, providing thus evidence for the plasticity potential of intestinal mesenchymal cells. Our results provide a comprehensive analysis of the identities, origin, and functional significance of distinct mesenchymal populations in the intestine.

Keywords: Cell plasticity; Colitis; Fibroblasts; Tissue damage.

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Conflict of interest statement

The authors have no relevant financial or non-financial interests to disclose.

Figures

Fig. 1
Fig. 1
The Col6a1Cre mouse targets predominantly CD34− IMCs in the mouse colon. A FACS sorting strategy for the isolation of Col6a1-GFP+ (GC) and Col6a1-GFP− (TC) mesenchymal cells from the colon. Single cell preparations from the colon (UC) were stained for the Lin+ markers CD45, EpCAM, CD31 and Ter119 and Propidium Iodide (PI) for dead cell exclusion. 3 samples from 4–5 mice each were subsequently analyzed. B Heatmap of differentially expressed genes in GC vs TC and UC samples, corresponding to GO terms related to epithelial proliferation/differentiation and blood vessel regulation. Log2-transformed normalized read counts of genes are shown. Read counts are scaled per column, red denotes high expression and blue low expression values. C Representative FACS analysis of CD34 expression in Lin- cells in the colon of Col6a1mTmG mice (n = 4–5 mice). D Immuno-histochemical analysis for CD34 expression in the colon of Col6a1mTmG mice (n = 9–10 mice, Scale bar: 50 μm). E Total number and size of intestinal structures after 3 days of co-culture with sorted Col6a1-GFP+ and GFP− colonic IMCs, with and without R-Spondin 1, respectively. Data represent mean ± SEM from one of four experiments performed in quadruplicates. *p < 0.05, **p < 0.01, ***p < 0.001. F Representative bright-field images of intestinal organoids co-cultured with Col6a1-GFP+ and GFP− IMCs at day 3, in the absence of R-Spondin 1 (Scale bar: 100 μm). G Mean expression (z-score) of genes signatures extracted from the different intestinal mesenchymal clusters identified in Kinchen et al. [14] in Col6a1-GFP+ bulk RNA-seq samples. MF, myofibroblasts; SMC, smooth muscle cells. H Heatmap of the top 50 differentially expressed genes in the Str2 population (Kinchen et al. [14]) and their relative expression in the GC and TC samples
Fig. 2
Fig. 2
GFP+/CD201+ mesenchymal cells comprise distinct subsets in the mouse colon. A Immunohistochemistry for CD201 in the colon of Col6a1mTmG mice (n = 4 mice, Scale bar: 50 μm). The dotted line delimitates the epithelial surface towards the lumen. B Representative FACS analysis of CD201 expression in Lin− cells in the colon of Col6a1mTmG mice (n = 10 mice). C Gene expression analysis of selected genes in FACS-sorted GFP+CD201+, GFP+CD201− and GFP− colonic mesenchymal cells from Col6a1mTmG mice. Expression is measured in relation to the Hprt housekeeping gene (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001. D) tSNE plots showing the expression of CD34, CD201, PDGFRα and αSMA in Lin− colonic mesenchymal cells using FACS analysis and E quantification of CD34+ and CD201+ cells in PDGFRα+ subsets (n = 8 mice). F Immunohistochemistry for αSMA and CD31 in the colon of Col6a1mTmG mice, showing their localization around blood vessels (white arrow) (Scale bar: 50 μm). The bottom of crypts is shown. G Immunohistochemistry for PDGFRα and CD34 in the colon of Col6a1mTmG mice. Different planes are shown. White arrows indicate PDGFRαhi (upper panel) and PDGFRαlo (lower panel) mesenchymal cells (Scale bar: 50 μm). The dotted line delimitates the epithelial surface towards the lumen. H Confocal imaging of GFP+ cells at the top of Col6a1mTmG colonic crypts (Scale bar: 10 μm). I Immuno-histochemical analysis of αSMAlo cells in the colon. White arrows indicate co-localization with GFP along the crypt’s length. (Scale bar = 10 μm) (n = 4 mice). J Quantification of Col6a1Cre-GFP+ cells in PDGFRα+ (top) and PDGFRα- (bottom) mesenchymal subsets using FACS analysis (n = 6 mice). K Immunohistochemistry for CD31 in the colon of Col6a1mTmG mice (n = 3 mice, Scale bar: 10 μm)
Fig. 3
Fig. 3
The Col6a1Cre mouse targets mesenchymal cell aggregates during development, which are necessary for intestinal morphogenesis. Confocal images showing GFP and PDGFRα expression in A the small intestine and colon at E15.5 (Scale bar = 50 μm) and B the small intestine and colon of Col6a1mTmG mice at the indicated developmental stages (Scale bar = 50 μm), (n = 3 mice per developmental stage). C Confocal images showing GFP and αSMA expression in the small intestine and colon of Col6a1mTmG mice at the indicated developmental stages (Scale bar = 50 μm), (n = 3 mice per developmental stage). D FACS analysis of Col6a1-GFP+ intestinal mesenchymal cells at E18.5. E FACS-based quantification of GFP+ cells in PDGFRαhi and PDGFRαlo cells in E16.5 and E18.5. F FACS analysis of GFP+ cells in E18.5, showing that they all are CD201+PDGFRαhi cells (n = 3–4 mice per developmental stage in all FACS analyses). G Schematic representation of DT administration. Pregnant females received two injections of diphtheria toxin (DT) (5 μg) at E14.5 and E15.5, which was followed by ex vivo culture of the intestine from E16.5 to E18.5. H Lightsheet imaging (maximum projection, Scale bar = 100 μm) and confocal images showing GFP, PDGFRα and αSMA expression (Scale bar = 50 μm) in the small intestine of Col6a1DTR and control mice. I) quantification of villi/nm in the presence of DT (n = 7). All (GFP+ and GFP−) and only GFP− villi are presented in DT treated mice. ***p < 0.001, **p < 0.01
Fig. 4
Fig. 4
Col6a1-cre lineage cell depletion leads to deregulated epithelial cell differentiation and proliferation during homeostasis. A Schematic representation of DT administration in homeostasis. Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice received 3 daily intra-rectal administrations of DT (20 ng/g body weight) and mice were sacrificed after 5 days. B Confocal images of GFP expression in Col6a1mTmG and Col6a1DTR mice (Scale bar = 50 μm). C FACS analysis and quantification of GFP+ cells in the colon of Col6a1mTmG and Col6a1DTR mice after DT administration (n = 9). D FACS analysis and quantification of PDGFRα+ subsets in GFP+ cells in Col6a1mTmG and Col6a1DTR mice (n = 3–5). E FACS analysis and quantification of CD201+PDGFRαhi cells in Col6a1mTmG and Col6a1DTR mice (n = 3–4 mice). F FACS analysis of PDGFRα+ subsets in Col6a1mTmG and Col6a1DTR mice (n = 4–10). G H&E staining of Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice (Scale bar: 100 μm). H Expression analysis of the indicated genes in colon samples from Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice. Expression is measured in relation to the B2m housekeeping gene (n = 9–14). I Immuno-histochemical-based quantification of differentiated epithelial cell types per crypt in Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice (n = 5–13). J Expression analysis of the indicated genes in colon samples from Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice. Expression is measured in relation to the B2m housekeeping gene (n = 5–15). K Representative BrdU staining and L quantification of the ratio of BrdU+ epithelial cells in the top/bottom of the colonic crypts of Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice (Scale bar: 100 μm), (n = 6–7). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
Loss of Col6a1-cre lineage IMCs is followed by CD34+ mesenchymal cell plasticity. A Immuno-histochemical and B FACS analysis of CD34 and PDGFRα expression in the colon of Col6a1DTR and Col6a1mTmG mice (Scale bar = 50 μm) (n = 6 mice). A zoomed-in view of the crypt top is shown in the merged image. C Immuno-histochemical analysis and D quantification of Ki67+CD34+ mesenchymal cells in the colon of Col6a1DTR and Col6a1mTmG mice (Scale bar = 50 μm) (n = 5 mice). Both broad and zoomed-in views of the crypt top are shown. E FACS-based quantification of BrdU+CD34+ cells in the colon of Col6a1DTR and control mice following three consecutives injections with BrdU at days 3–5 of the protocol (n = 8–10 mice). F Immuno-histochemical analysis of αSMA expression in the colon of Col6a1DTR and control mice (the top of the crypt is shown) (n = 5). G FACS analysis and H quantification of αSMA+ cells in the CD34+ subset (n = 4 mice). I Gene expression analysis of isolated CD34+ cells from Col6a1DTR and control mice (n = 5–6). *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 6
Fig. 6
Col6a1-cre lineage cells retain their properties and topology during colitis but are dispensable for tissue regeneration. A FACS sorting strategy for the isolation of Col6a1-GFP+ (GDS) and Col6a1-GFP− (TDS) mesenchymal cells from the colon at the end of the acute DSS protocol. 3 samples from 4–5 mice each were subsequently analyzed. B Heatmap of differentially expressed genes in GDS vs TDS and UDS, as well as the respective untreated samples, corresponding to GO terms related to epithelial proliferation/differentiation, blood vessel regulation and inflammatory response. Log2-transformed normalized read counts of genes are shown. Read counts are scaled per column, red denotes high expression and blue low expression values. C Confocal images of GFP expression in Col6a1mTmG mice at day 8 of the acute DSS protocol, Scale bar = 50 μm. D Mean expression (z-score) of genes signatures extracted from the different intestinal mesenchymal clusters identified in Kinchen et al. [14] during DSS colitis in Col6a1-GFP+ bulk RNA-seq samples. MF, myofibroblasts. E Schematic representation of DT administration during acute colitis and regeneration. Mice received 2.5% DSS for 5 days, followed by regular water for 16 days. 100 μl DT (20 ng/g body weight) was administered intrarectally at days 4, 5 and 6 of the regime. F Quantification of GFP+ cells in the colon of control and Col6a1DTR mice after DT administration (n = 5–7 mice), G H&E staining and H) histopathological score of Col6a1DTR and control (Col6a1mTmG, iDTRf/f/) mice at the end of the protocol (Scale bar = 100 μm) (n = 10–11 mice). Immuno-histochemical analysis of I CD34 and PDGFRα expression and J CD34 and αSMA expression in the colon of Col6a1DTR and control mice (Scale bar = 50 μm) (n = 4 mice)
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