Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling

Abstract

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Fig. 1. Subepithelial PDGFRa^high cells support the growth of fetal epithelium and possess region-specific transcriptional profiles.

a Detection of EpCAM (red), DAPI (blue) and PDGFRa (green, left and lower panel) or CD29 (green, right) at E16.5. Lower panel is 3D-rendered image of a representative SI. Scale bars, 20 μm (upper panels) and 50 μm (lower panel). b FACS profile showing the distribution of DAPI⁻/CD31⁻/CD45⁻ parenchymal cells according to their EpCAM (left) and PDGFRa/CD29 (right) profile. c Overview of E16.5 SI and the division into proximal (prox), mid and distal (dist) regions. Scale bar, 1 mm. d Detection of EpCAM (red), DAPI (blue) and LAMB1 (green, upper left), LAMA5 (green, upper right and lower right), or NPNT (green, lower left) at E16.5. Scale bars, 50 μm except lower right, where scale bar, 20 μm. e GO-term enrichment analysis showing top 10 terms in the Biological Process category comparing PDGFRa^high and PDGFRa^med cells (intersect of pairwise tests as presented in Supplementary Fig. 2a). f, g Formation of organoids from DAPI⁻/CD31⁻/CD45⁻/EpCAM⁺ cells isolated from the proximal 20% of the E16.5 SI cultured with PDGFRa^med (middle) or PDGFRa^high cells (right) without ENR in basal medium. Percentage of spheres were calculated relative to organoid formation in basal medium with ENR. Scale bars, 100 μm. h RNA-seq of PDGFRa^high cells depicting the 560 differentially expressed genes comparing proximal, mid and distal regions. Results in a–d and f are representative of at least n = 3 independent experiments. h presents RNA-seq data from n = 3 biological replicates collected in independent experiments. Data presented in g are means ± s.d. from n = 6 independent experiments for EpCAM⁺ and EpCAM⁺+PDGFRa^high cultures and n = 3 independent experiments for EpCAM⁺+PDGFRa^med cultures. Statistical significance was assessed using one-way ANOVA test. Figure e presents results from GO enrichment test using goana.

Fig. 2. PDGFRa^high cells-assisted Wnt-signalling gradient is present in E16.5 SI and required for the establishment of region-specific organoid cultures.

a RNA-seq of PDGFRa^high cells showing differentially expressed ligands between proximal, mid and distal regions. Highlighted in red are Wnt-pathway components. b RNA-seq results for Axin2 and Lgr5 from sorted EpCAM cells isolated from proximal, mid and distal regions. The y-axis shows RPKM (reads per kilobase per million mapped reads). c Detection of Top.CFP (white), E-cadherin (red) and DAPI (blue) in proximal, mid and distal regions. Scale bars, 20 μm. d–f Formation of organoids from DAPI⁻/CD31⁻/CD45⁻/EpCAM⁺ cells isolated from proximal, mid and distal regions and grown in the presence of ENR (d, e) or WENR (d, f). Scale bars, 100 μm. Results in c and d are representative of n = 3 independent experiments. In b, e and f data presented are means ± s.d. from n = 3 biologically independent samples. In e and f statistical significance was assessed using two-tailed Welch’s t-test.

Fig. 3. Perturbation of Wnt-signalling has region-specific effects on SI transcription and structure.

a FACS profile showing the reduction of CFP⁺ cells (DAPI⁻/CD31⁻/CD45⁻/EpCAM⁺) in proximal, mid and distal regions upon PORCN inhibition (PORCNi). b RNA-seq data for Axin2 with and without PORCN inhibition. c RNA-seq analysis showing the number of differentially expressed genes comparing control and PORCNi samples for proximal, mid and distal regions. d H&E staining of proximal and distal regions in control and PORCNi samples. Scale bars, 100 μm. e Workflow for machine learning-assisted 3D image analysis. Scale bars, 40 μm. f Surface renderings of Ilastik-processed probability maps depicting villi in control and PORCNi samples. Scale bars, 100 μm. g Quantification of villus count in proximal, mid and distal regions in control and PORCN inhibited samples. h Surface renderings of Ilastik-processed probability maps depicting epithelium in control and PORCNi samples. Scale bars, 100 μm. i Quantification of mesenchymal depth in proximal, mid and distal regions in control and PORCNi samples. Results in a and d are representative of n = 3 independent experiments. Results in f and h are representative of n = 3 biological replicates. Data presented in b are means ± s.d. from n = 4 (Ctrl) or n = 3 (PORCNi) biological replicates. In g and i data presented are means ± s.d. from n = 3 biological replicates and statistical significance was assessed using two-tailed Welch’s t-test.

Fig. 4. Wnt signalling regulates Shh expression in the developing distal SI.

a Heatmap showing enrichment of ChIP-seq tags over the 6238 identified TCF7L2 binding sites (±10 kb). b Euler diagram showing the overlap between the 385 genes upregulated comparing distal versus proximal E16.5 SI EpCAM⁺ cells (see also Supplementary Fig. 2d) and the 1366 genes down-regulated in control-treated relative to PORCN-inhibited distal regions (see also Fig. 3c). c Detection of SHH (white) and DAPI (blue) at E16.5. Scale bars, 20 μm. d ChIP-seq track depicting TCF7L2 coverage at the MACS1 Shh enhancer region (chr5:29211998-29212804, marked in red) in SI EpCAM⁺ cells. e qPCR analysis for Shh using intestinal biopsies from E16.5 embryos. f qPCR analysis for Shh using established organoid cultures from E16.5 distal SI cultured in ENR and treated with Vehicle, 5 μM IWP-2, or 10 μM IWR-1-endo for 24 h. g Fetal whole intestine culture depicting cluster formation in PDGFRaCreER^T2;mTmG mice following DMSO (1), 1 μM SAG (2), 1 μM PORCNi (3) or SAG+PORCNi (4) treatment. Scale bars, 2 mm (overview) and 275 μm (enumerated panels). h Quantification of the number of PDGFRa⁺ clusters. Results in e represent n = 3 biological replicates, and results in f represent n = 3 independent experiments. Results in h are derived from two independent litters of embryos where in total 4 (DMSO), 5 (SAG), 4 (PORCNi) and 6 (SAG+PORCNi) whole intestines were cultured. The data-points represent n = 5 (DMSO), n = 6 (SAG), n = 13 (PORCNi) and n = 7 (SAG+PORCNi) analysed images. Statistical significance was assessed using one-way ANOVA test. Graphs show means ± s.d.