There is a well-established structural program that generates the different cells that make up the crypt villus of the small intestine. At the base of the crypts are LGR5+ epithelial stem cells that continuously self-renew and differentiate into the progenitor cells that propagate the rest of the structure. As these progenitor cells migrate towards the villus tip they differentiate into the functional cells of the villus, such as mucus secreting cells, goblet cells, tuft cells, absorbtive enterocytes and enteroendocrine cells. Differentiated cells are functional for approximately three days as they migrate to the villus tip before being shed. Contrary to previous thoughts that these cells were terminally differentiated, recent studies have demonstrated that they constantly change their functional state by expressing different genes depending upon their position along the villus. A number of theories have been generated to account for this spatial heterogeneity, including a zonated response to spatial gradients, or an internal clock where epithelial cells are pre-programmed to express different functional genes.
In this study the authors investigate a third mechanism, where the adjacent stroma provides the signalling molecules that determine epithelial cell functionality. They used spatially-resolved transcriptomics to analyse the stromal genes expression in the different zones of the villus. They used various NGS methods, including single cell sequencing in combination with laser capture microdissection.
Four stromal cell zones were isolated from the jejunum crypt-villus axis of five mice. RNA‑seq analysis of these zones identified a number of ligand/receptor pathways, for example, in the crypt stroma the BMP pathway inhibitor Gremlin 1 and IL18 receptor had higher levels of expression.
IL18 has previously been demonstrated to be part of an anti-bacterial gene module expressed by enterocytes in lower villi zones. Single molecule FISH (smFISH) analysis of the stromal zones demonstrated staining of IL18r1 in stroma where innate lymphoid cells were present in the villus suggesting zonated enterocyte IL18 signalling.
Towards the villus tip, Bmp4, a protein implicated in epithelial proliferation and enteroendocrine cell differentiation was expressed in the stroma cells of this zone. Wnt4 and Egf were also detected in the tip zonal regions. Egfr has been demonstrated to have higher expression in the villus tip enterocytes, consistent with another zonal stroma/villus interaction.
LGR5, normally associated with the epithelial stem cells at the base of the crypt, was one of the most highly expressed transcripts in the villus tip stroma. smFISH identified LGR5 expression in PDGFR+ telocytes coexpressing Bmp4.
Villus tip telocytes (VTTs) expressed LGR5 in similar levels as the basal crypt epithelial stem cells. VTTs also expressed the Lgr5 ligand Rspo324, mainly along telopodes, extensions of the telocyte that extend toward the lamina propria.
Mouse knock-in LGR5 models confirmed that LGR5 specifically labels telocytes in the villus tip.
PDGFRa+ cells, which includes telocytes, were isolated by FACS sorting and analysed using the MARS-seq protocol for single cell transcriptomic analysis. 329 pure mesenchymal cells were isolated clustering into four separate populations. From the markers identified in the clusters, and the LCM transcriptomic data, clusters were annotated as crypt telocytes, enteric mesothelial fibroblasts, myofibroblasts and villus tip telocytes (VTTs).
smFISH validation of the markers for each cluster demonstrated Grem1 and Sfrp1 expression was elevated in crypt telocytes and Ly6a and Angptl4 expression was elevated in VTTs. VTTs also had elevated expression of Bmps 2 and 4.
ECM components Col3a1, Col1a1 and Timp2 were elevated in both VTTs and crypt telocytes. VTTs also expressed elevated Mfap5, Emilin2 and Fbn1, encoding microfibrillar proteins.
VTTs expressed higher levels of Wnt5a compared to crypt telocytes suggesting a switch to the non-canonical Wnt signalling pathway and consistent with this Axin2 was expressed throughout the villi epithelial cells but downregulated at the villus tip.
Using a mouse model of VTT ablation, expression of enterocyte genes at the villus tip were investigated with smFISH. Egfr, Cdh1, Klf4, Fos, Ada, Nt5e and Slc28a2 all had reduced expression demonstrating the importance of VTTs as regulators of enterocyte spatial expression.
In this study the authors used spatially-resolved transcriptomics to investigate the gene expression in the associated stroma along the crypt-villus axis. They identified four mesenchymal cell populations that included a sub-population of telocytes localised at the villus tip that expressed Lgr5, a gene normally associated with crypt stem cells. VTTs were shown to regulate the enterocytes as they formed the villus tip and were subsequently shed. Bmp signalling and a switch to the non-canonical WNt signalling pathway may facilitate these changes. This study also highlights the use of laser capture microdissection combined with RNA-seq and single cell sequencing to unravel spatial interactions within complex tissues and similar approaches could be used for zonal interactions in other structured tissues.
Bahar Halpern, K., Massalha, H., Zwick, R.K. et al. Lgr5+ telocytes are a signaling source at the intestinal villus tip. Nat Commun 11, 1936 (2020)