Unbiased single-cell profiling of murine colonic cells

To gain a deeper understanding of the heterogeneity and complexity of the colonic epithelium and mesenchyme, as well as the associated signaling networks, we performed scRNAseq of murine colonic tissue. We dissociated epithelial and mesenchymal fractions individually into single cells and used fluorescence-activated cell sorting (FACS) to further purify epithelial (EpCAM+, CD45−) and non-epithelial cells (EpCAM−, CD45−), while concurrently depleting lymphocytes (EpCAM−, CD45+) (Fig 1A and 1B). Overall, we observe a colonic cellular composition consisting of 91.2% epithelial cells and 8.8% of non-epithelial cells. In contrast to previous studies, we next pooled the sorted epithelial (EpCAM+, CD45−) and non-epithelial (EpCAM−, CD45−) cells originating from both isolation protocols at equal fractions (1:1, epithelial:non-epithelial) to avoid epithelial overrepresentation and obtain sufficient data to describe both epithelial and non-epithelial cells in their full spectrum. Following quality control, scRNAseq results in a combined dataset of 7,395 cells with a mean transcript count of 3,243 per cell. Graph-based clustering reveals 16 individual clusters, which can be classified into three major groups: Epithelium (Epcam⁺, Krt19⁺), stroma (Vim⁺, Col1a1⁺), and endothelium (Pecam1⁺) (Fig 1C, S1A Fig).

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Fig 1. Unbiased analysis of murine colon landscape reveals complexity and heterogeneity of epithelial and mesenchymal cells.

(A) Schematic representation of a colonic crypt with surface markers used to sort epithelial (green: EpCAM+, CD45−) and mesenchymal/non-epithelial cells (red: EpCAM−, CD45−). (B) Flow cytometry analysis using EpCAM and CD45 on single-cell suspension of colonic epithelial (left, raw data: S2 Data) and mesenchymal cell isolation (right, raw data: S3 Data) prior to the sort. (C) Left: three major groups (epithelial, stromal, and endothelial) clustering into 16 distinct cluster revealed by UMAP analysis. Right: Epithelial cells show higher proliferative activity (cells in S-G2M) than non-epithelial cells (UMAP, single cells are colored according to their cluster annotation (left) or cell cycle phase (right)). (D) Characterization of cluster identity using relative expression of marker genes. (Dot plot, size, and color of the dot represent the percentage of cells which express the transcript and the average expression level within a cluster, respectively). EE, enteroendocrine; IESC, intestinal epithelial stem cell; TA, transit-amplifying cell; UMAP, Uniform Manifold Approximation and Projection.

https://doi.org/10.1371/journal.pbio.3001032.g001

We could annotate most known epithelial cell types within our dataset with the exception of tuft cells. This designation is based on a combination of published marker genes [10,11,13] (Fig 1D) and differentially expressed genes (S1B Fig). Clusters 11 and 5 represent IESC and transit-amplifying cells (TA), respectively, based on the expression of Lgr5, Axin2, and Mki67 (Fig 1D) in combination with their high proliferative activity (Fig 1C). Clusters 9 and 10 delineate the secretory lineage (Atoh1⁺), the former consisting of classic goblet cells (Muc2⁺) and the latter the recently described deep crypt secretory cells (Reg4⁺) [14]. Indeed, these cells appear to be mainly responsible for the activation of Notch signaling in intestinal stem cells as they express both ligands: Dll1 and Dll4 (S1D Fig) [15]. An absorptive lineage composed of clusters 6 and 8 (Guca2a⁺, Alpi⁺, Aldh1l1⁺) (Fig 1D, S1B Fig) consists of enterocytes strongly enriched for Gene Ontology (GO) terms such as “Generation of precursor metabolites and energy” and “Anion transport” (S1C Fig). The absorptive and secretory cells express a variety of Egf family ligands (Egf, Tgfa, Areg, and Hbegf) and ephrins (Efnb1 and Efnb2) (S1D Fig). Finally, cluster 14 comprises enteroendocrine cells (Chga⁺, Chgb⁺, and Neurod1⁺) which express high levels of Ihh—the main hedgehog ligand secreted by the intestinal epithelium (S1D Fig) [16].

We observed four stromal clusters (clusters 1, 2, 3 and 4), which all express typical fibroblast markers, such as Vim and Col1a1. In addition, we found clusters 12 and 15, which are pericytes (Rgs5⁺) and glia (S100b⁺), respectively.

Taken together, these data represent a comprehensive, high-quality dataset of murine colonic cell diversity, comprised of matching epithelium and mesenchyme.

Characterization of crypt-associated fibroblasts

We next subset and reanalyzed the four stromal clusters (stromal 1 to 4, Fig 1C) to gain insight into distinct subpopulations of crypt-associated fibroblasts and their individual niche potential in more detail (Fig 2A). Cluster S4 cells are likely smooth muscle cells (SMCs) due to the expression of Acta2 and Myh11 (see Fig 2D) in combination with GO term enrichment for muscle cell-specific functions, e.g., “Regulation of skeletal muscle contraction by calcium ion signaling” and “Myofibril assembly” (S2A Fig). However, we cannot exclude that this cluster also contains myofibroblasts.

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Fig 2. CTFs (Pdgfra^high) and CBFs (Pdgfra^low) mark distinct functional colonic mesenchymal cell populations that control stem cell proliferation and epithelial differentiation in the murine colon.

(A) Heterogeneity of colonic mesenchymal cells (UMAP plot of the reanalyzed stromal subset, single cells are colored according to cluster annotation). (B) Relative expression of niche marker genes (UMAP, single cells are colored according to transcript expression). (C) Pdgfra expression across stromal clusters (violin plot, raw data: S1A Data). (D) Relative expression of fibroblast identity marker genes (Vim, Col1a1, Acta2, and Myh11), secreted factors resulting in stem cell maintenance (Wnt2, Wnt2b, Rspo3, and Grem1) and secreted factors resulting in epithelial differentiation (Wnt5a, Bmp3, and Bmp7) (violin plots, raw data: S1B Data). (E) Wnt5a is expressed by CTFs at the top of the colonic crypts (single-molecule RNA in situ hybridization (Scale bar = 20 μm, 5μm (1,2)). (F) GO enrichment terms for CTFs. (G) Heatmap of genes that are differentially expressed among the mesenchymal clusters (0.95 quantile) show strong similarities between CBFs1 and CBFs2 and clear distinction of CTFs and SMC. Bmp, bone morphogenetic protein; CBF, crypt-bottom fibroblast; CTF, crypt-top fibroblasts; GO, Gene Ontology; Pdgfra, platelet-derived growth factor receptor A; SMC, smooth muscle cell; UMAP, Uniform Manifold Approximation and Projection.

https://doi.org/10.1371/journal.pbio.3001032.g002

We interrogated the expression of markers previously used to demarcate the cells of the murine stem cell niche (Fig 2B). The hedgehog transcription factor Gli1 [6] shows ubiquitous expression in all stromal clusters (S1 to S4). Cd34 [5] and Cd90 [8] display a strong enrichment in clusters S1 and S2. However, Cd90 is also expressed in SMC and Cd34 is additionally expressed in endothelial cells. Foxl1 [7,17] is expressed specifically in clusters S3 and S4. Interestingly, while Pdgfra [4] is expressed in clusters S1, S2, and S3, it displays a strong difference in transcript levels. Indeed, cluster S3 shows a high expression of Pdgfra and clusters S1 and S2 show a low expression of Pdgfra (Fig 2C). Due to differences in spatial localization of Pdgfra-positive cells in regards to the colonic crypt (see below, Fig 3B and 3C), we henceforth refer to cluster S3 as CTFs and to clusters S1 and S2 as CBFs (CBFs1 and CBFs2, respectively). In accordance with their localization at the bottom of the crypt in close proximity to the intestinal stem cells, CBFs express factors important for stem cell maintenance, such as canonical Wnt ligands (Wnt2 and Wnt2b), Wnt signaling potentiators (Rspo3), and Bmp signaling antagonists (Grem1) (Fig 2D, S2B Fig). In contrast, CTFs express factors resulting in the induction of epithelial differentiation, such as Bmp ligands (Bmp2, Bmp3, Bmp4, Bmp5, and Bmp7), noncanonical Wnt ligands (Wnt5a), and inhibitors of canonical Wnt signaling (Dkk3) (Fig 2D and 2E, S2B Fig).

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Fig 3. CTFs (Pdgfra^high) and CBFs (Pdgfra^low) are localized in a distinct manner along the colonic crypt axis.

(A–C) Cryosections of Pdgfra-H2B-eGFP mice. (A) High Pdgfra protein expression correlates with the level of Pdgfra transcript in Pdgfra-H2B-eGFP reporter mouse line. (Red: Pdgfra protein, green: GFP, blue: DAPI) (Scale bar = 40 μm), (1,2) Insets of crypt top and crypt bottom respectively (Scale bar = 5 μm). (B) CTFs (Pdgfra^high) are localized at the top of the colonic crypt, whereas CBFs (Pdgfra^low) are at the bottom. (Red: EpCAM, green: GFP, blue: DAPI) (Scale bar = 40 μm). (3,4) Insets of crypt top and crypt bottom, respectively, arrowheads point out Pdgfra^high (GFP high) cells and arrows point out Pdgfra^low (GFP low) cells. (Scale bar = 5 μm) (C) Fluorescence intensity measurement of GFP positive nuclei. n_Bottom = 159, n_Top = 100, **** = p < 0.0001 (Unpaired t test with Welch correction) (Raw data: S1C Data) (D) Flow cytometry analysis for GFP on mesenchymal cells isolated from Pdgfra-H2B-eGFP mice identifying GFP⁻, GFP-low, and GFP-high cells (Raw data: S4 Data). (E) Relative mRNA expression of Pdgfra, Wnt2, Wnt2b, Wnt5a, Rspo3, and Grem1 in CTFs (GFP high) and CBFs (GFP low) isolated from Pdgfra-H2B-eGFP reporter mouse line. (RT-qPCR, normalized to Hprt expression levels, FC levels in CBFs set to 1) (Raw data: S1D Data). (F) CBFs1, CBFs2, and CTFs populations determined by scRNAseq from colonic Pdgfra-positive cells (Pdgfra-H2B-eGFP reporter mouse line). (UMAP, single cells are colored according to cluster annotation). (G) Stromal clusters from wt and Pdgfra⁺ colonic scRNAseq analysis display high similarity. (Spearman correlation). CBF, crypt-bottom fibroblast; CTF, crypt-top fibroblasts; FC, fold change; GFP, green fluorescent protein; Pdgfra, platelet-derived growth factor receptor A; RT-qPCR, quantitative reverse transcription PCR; scRNAseq, single-cell RNA sequencing; UMAP, Uniform Manifold Approximation and Projection; wt, wild-type.

https://doi.org/10.1371/journal.pbio.3001032.g003

GO term enrichment recapitulates the molecular difference between crypt-associated fibroblast populations, with CTFs showing an enrichment for “Bmp signaling pathway”, “Focal adhesion,” and “Prostaglandin Synthesis and Regulation” (Fig 2F), while CBFs show enrichment for terms such as “response to vascular endothelial growth factor signaling” and “regulation of extracellular matrix” (S2C and S2D Fig).

In sum, the analysis of differentially expressed genes exposes a high similarity in marker expression between the clusters CBFs1 and CBFs2 and a clear demarcation of CTFs and SMC (Fig 2G). However, we find interesting smaller differences between CBFs1 and CBFs2. CBFs1 (Fig 2G) show higher, but not exclusive, expression of the canonical Wnt ligands Wnt2 and Wnt2b (S3B Fig). In addition, they express Igfbp3, Igfbp5, Adam23, and Edil1 (Fig 2G), pointing toward potent integrin/extracellular signal-regulated kinase (ERK) signaling regulation [18]. CBFs2 (Fig 2G) show higher, but not exclusive, expression of the Bmp antagonist Gremlin 1 and the marker Cd81 (Fig 2G, S3B Fig), displaying high similarity to the recently described small intestinal trophocytes [9].

Furthermore, we employed the CellphoneDB [19] algorithm to infer possible communication (ligand–receptor interactions) between CBFs1, CBFs2, CTFs, and epithelial cells (S3A Fig). We were able to confirm expected crosstalk between populations, such as Wnt5a (from CTFs) potentially signaling to the noncanonical Wnt receptors Ror1 (expressed in cells of cluster Enterocyte 1) and Ror2 (expressed in IESC, Goblet, Reg4+ secretory, and enteroendocrine cells). Similarly, Wnt2 (from CBFs1, CBFs2) would signal to canonical Wnt receptor Fzd3 (expressed in TA, Goblet, and enteroendocrine) (S3A Fig). In addition, we uncovered some interesting new potential crosstalk, such as between Fgf2 (from CBFs1 and CBFs2) and Cd44 (expressed in IESC and TA cells) and Fgfr2 (expressed in IESC, TA, Goblet, Reg4+ secretory, and enteroendocrine cells) (S3A Fig). Intriguingly, CBFs2 express the ligands Igf1, Efna2, Efna5, and Ptn whose receptors Igf1r (Igf1), Epha4 (Efna2, Efna5), and Ptprs (Ptn) are expressed on epithelial cells (S3A Fig). The relevance of this crosstalk needs to be explored.

Intriguingly, different Wnt signaling antagonists (Sfrp1, Sfrp2, Frzb, Sfrp4, and Dkk3) are expressed in distinct crypt-associated fibroblasts. This may serve to sharpen the gradient of canonical Wnt signaling activity (S2B Fig).

We also performed gene regulatory network analysis using single-cell regulatory network inference and clustering (SCENIC) [20]. This revealed cluster specific enrichment for transcription factor regulons. CTFs show an enrichment for the regulon activities of Lef1, Runx1, Nkx2-3, and Foxl1 (S2E Fig). The transcription factor Nkx2-3 had previously been found to control Bmp2/4 expression in the small intestine [21]. Lef1 regulon activity in CTFs may hint at a possible explanation for the presence of the recently discovered small intestinal Lgr5⁺ villus tip telocytes [22], given that Lgr5 is canonical Wnt targe gene [23], and Lef1 is both a canonical Wnt target gene and a Wnt/b-catenin transcription factor [24]. Intriguingly, this would also point to a difference in transcriptional regulation of Lgr5⁺ mesenchymal cells compared to Lgr5⁺ intestinal stem cells, which have been shown to mainly depend on TCF family member Tcf4 [25].

In conclusion, we identified two molecularly distinct colonic crypt-associated fibroblast populations, which are defined by their potential to either support the maintenance of epithelial stem cells or to induce the differentiation of the intestinal epithelium.

Spatial localization of crypt-associated fibroblasts

Finally, we sought to identify the position of the molecularly defined crypt-associated fibroblast populations along the colonic crypt axis. We analyzed colonic tissue sections of Pdgfra-H2B-eGFP reporter mice [26]. These display differences in nuclear green fluorescent protein (GFP) intensity closely correlated with the Pdgfra transcript levels and perfectly recapitulate differences in Pdgfra protein expression in the murine colon, as exemplified by the co-localization of nuclei with high GFP levels and cells exhibiting high intensity Pdgfra antibody staining (Fig 3A). All cells with GFP positive nuclei show subepithelial localization and co-expression with the pan-fibroblast maker Vimentin (S4A Fig). Fluorescence intensity quantification of GFP positive nuclei revealed a clear association between the localization of fibroblasts and nuclear GFP intensity. CTFs displayed high nuclear GFP signals (mean + SD: 117.1 + 37.9), and CBFs displayed low nuclear GFP signals (mean + SD: 39.5 +14.7) (Fig 3B and 3C). To further investigate the morphology of Pdgfra-expressing cells in the colon, we examined colon from mice carrying the Pdgfra-Cre^ERT2 driver [27] in combination with the cytoplasmic fluorescent reporter mouse line Ai14 (LSL-tdTomato) [28] (single tamoxifen injection, analysis 1 day post injection). As has been previously observed in telocytes of the small intestine [22], we find that Pdgfra-expressing cells have elongated processes and small telopode portrusions (S4B Fig). The morphology is revealed by the distribution of tdTomato.

To validate the gene expression patterns found in our scRNAseq (Fig 2), we isolated colonic mesenchymal GFP-high and GFP-low cells from the Pdgfra-H2B-eGFP mouse line (Fig 3D, S4C Fig). Quantitative real-time PCR confirmed higher expression of Wnt2, Wnt2b, Rspo3, and Grem1 in GFP-low cells compared to GFP-high cells, recapitulating the differences observed between CBFs and CTFs in our scRNAseq (Fig 3E). As expected, Wnt5a and Pdgfra expression was higher in the GFP-high cells (Fig 3E).

In order to further corroborate our findings, we performed an independent scRNAseq analysis of GFP positive colonic mesenchymal cells of the Pdgfra-H2B-eGFP mouse line. Graph-based clustering revealed three main clusters (Fig 3F). Based on the expression of Pdgfra and various niche factors (Wnt2/2b, Grem1, Rspo3, and Wnt5a), we designated cluster P3 as CTFs and cluster P1 and P2 as CBFs1 and CBFs2 (S4D and S4E Fig). SMCs (possibly including myofibroblasts) were not found in this dataset. Based on the top 2,000 differentially expressed genes in each cluster, CBFs1, CBFs2, and CTFs originating from the Pdgfra-H2B-eGFP scRNAseq analysis show very high similarity (Spearman correlation) to the three Pdgfra positive clusters of the same name in our initial scRNAseq experiment (Fig 3G).

Taken together, these data confirm that CTFs and CBFs correspond to Pdgfra high and low expressing cells, respectively and are differentially localized along the crypt axis.

Murine colonic crypt-associated fibroblast identities are conserved in human healthy colon

In order to see whether the molecular identity of murine CBFs and CTFs are evolutionarily conserved, we compared our findings to human colonic stromal cell populations. To do so, we took advantage of the recently published dataset (GSE114374), in which human colonic stroma has been analyzed in inflammatory disease progression versus healthy state at single-cell level [12]. We performed a bioinformatic reanalysis of Kinchen’s healthy colonic stromal dataset. We find a total of 8 clusters, which we annotated, based on published markers [12] as fibroblasts, plasma cells, glia, endothelial cells, SMCs, and pericytes (Fig 4A). We interrogated the expression of previously published murine niche marker genes within the fibroblast clusters (Fig 4B). A very similar picture emerges to that seen in mouse (Fig 2B), with the exception of CD34, which is expressed solely in endothelial cells in the human colon.

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Fig 4. Murine CTFs and CBFs populations are conserved in healthy human colon.

(A) Reanalysis of the healthy colonic stromal dataset from GSE114374 reveals landscape similar to murine colon. (UMAP, single cells are colored according to cluster annotation). (B) Conserved niche markers determine analogous cell populations in human colonic mesenchyme (UMAP, single cells are colored according to transcript expression). (C) Relative expression of PDGFRA separates human mesenchymal cells into three PDGFRA⁺ populations: CTFs, CBFs 1, and CBFs 2. (Violin plot, raw data: S1E Data) (D) Relative expression of factors involved in WNT and BMP pathways. (Violin and dot plots, size, and color of the dot represent the percentage of cells which express the transcript and the average expression level within a cluster, respectively; raw data: S1F Data). (E) Schematic model of conserved colonic crypt-associated fibroblasts. PDGFRA^high CTFs secrete BMP ligands and noncanonical WNT ligands, resulting in epithelial differentiation. PDGFRA^low CBFs secrete canonical WNT ligands, WNT potentiators, and BMP inhibitors to maintain stem cells (blue). BMP, bone morphogenetic protein; CBF, crypt-bottom fibroblast; CTF, crypt-top fibroblast; PDGFRA, platelet-derived growth factor receptor A; UMAP, Uniform Manifold Approximation and Projection.

https://doi.org/10.1371/journal.pbio.3001032.g004

Interestingly, reinforcing our observations in the murine colon, the strength of PDGFRA expression can be used to demarcate distinct crypt-associated fibroblast populations in the human colon as well (Fig 4C). Taken together with the expression pattern of components of the WNT and BMP signaling pathways (Fig 4D), we identify clusters HC1 and HC3 as PDGFRA^low CBFs1 and CBFs2, respectively, that express canonical WNT ligands (WNT2B), WNT signaling potentiators (RSPO3), and BMP inhibitors (GREM1, GREM2, and CHRD). In addition, using the R package matchSCore2 [29], we compared the murine mesenchymal populations with the human mesenchymal populations on the level of the entire transcriptome. We found high interspecies overlap between murine and human CBFs, murine and human CTFs, and murine and human SMCs (S5 Fig).

However, diverging from the expression patterns observed in the murine colon, the canonical WNT ligand WNT2B is expressed not only in CBFs, but also additionally in SMCs (Fig 4D). This finding is consistent with a recent observation that embryonic SMCs express WNT2B during human intestinal development [30]. Moreover, we identify cluster HC2 as PDGFRA^high CTFs, expressing a broad spectrum of BMP ligands (e.g., BMP2), and noncanonical WNT ligands (e.g., WNT5A) (Fig 4D). Interestingly, just like in the murine colon, we also find expression of various canonical WNT signaling inhibitors (SFRP1, SFRP2, FRZB, and DKK3) in all the fibroblast clusters in the human colon; however, compared to mouse, the positions of DKK3 and FRZB expression seems to be reversed in human CBFs and CTFs (Fig 4D and S2B Fig).

Future studies of this highly specific spatial localization and molecular regulation might reveal deeper mechanistic insights into how canonical WNT ligands and antagonists interact to create a canonical WNT signaling gradient optimal for intestinal stem cell maintenance and proliferation.

Ethics statement

All mouse procedures were performed in accordance with Swiss Guidelines and approved by the Cantonal Veterinary Office Zürich, Switzerland (ZH156/2015 and ZH169/2016).

Mice

C57BL/6 wild-type mice were purchased from Charles River, Germany (Strain code: 027). Pdgfra^H2BeGFP [26], Pdgfra-Cre^ERT2 [27], and Ai14 [28] mice were purchased from Jackson Laboratories, United States of America (Stock numbers: 007669, 032770, and 007914). Cells were isolated from female mice at age 8 to 12 weeks.

Epithelial and mesenchymal cell isolation and cell sorting

Colonic epithelial and mesenchymal cells were isolated inspired by previous publications [42,43] and modified as described below. Colonic tissue was harvested, minced into small pieces (2 mm), and subsequently washed with PBS. Tissue pieces were then incubated in Gentle Cell Dissociation Reagent (STEMCELL Technologies, Germany) for 30 minutes at room temperature to detach the epithelium. The epithelial fraction was then filtered through a Falcon 70-μm cell strainer (Corning, Switzerland), washed with plain ADMEM/F12 and incubated for 5 minutes at 37°C in prewarmed TrypLE express (Gibco, Thermofisher, Switzerland), followed by single-cell dissociation using the m_intestine program on the gentleMACS Octo Dissociator (Miltenyi Biotec, Switzerland). The epithelial single-cell suspension was then filtered through a Falcon 40-μm cell strainer (Corning) and stored on ice in ADMEM/F12 (supplemented with 10% FBS). After the detachment of the epithelium, the remaining tissue pieces were digested for 1 hour at 37°C under 110 revolutions per minute (rpm) shaking conditions in DMEM supplemented with 2 mg/mL collagenase D (Roche) and 0.4 mg/mL Dispase (Gibco). The mesenchymal fraction was then filtered through a Falcon 70-μm cell strainer (Corning), washed with plain ADMEM/F12, and subsequently filtered through a Falcon 40-μm cell strainer (Corning). Both epithelial and mesenchymal cells were stained for 30 minutes on ice with anti-CD45-PE (1:500, eBioscience/Thermofisher, Switzerland) and anti-CD326(EpCAM)-FITC (1:500, eBioscience/Thermofisher, Switzerland) in ADMEM/F12 (supplemented with 10% FBS). Prior to cell sorting, all cells were stained for 5 minutes on ice with Zombie Violet Fixable Viability Kit in PBS (1:1000, Biolegend, Thermofisher, Switzerland). In the case of mesenchymal cell isolation from Pdgfra^H2BeGFP, mice only staining with Zombie Violet Fixable Viability Kit was performed. Cells were detected and sorted at the Cytometry Facility at the University of Zürich using a FACSAria III cell sorter (gates visible in corresponding figures) (BD Biosciences, Switzerland).

Single-cell RNA sequencing

Colonic epithelial and mesenchymal cells (Fig 1) were isolated from three female C57BL/6 mice as described above and mixed at a ratio of 1:1. Using the Single Cell 3’ V3 assay, 16,000 cells were loaded on a Chromium Controller (10x Genomics, USA). Colonic mesenchymal Pdgfra-positive cells (Fig 3) were isolated as described above from three female Pdgfra^H2BeGFP mice. Using the Single Cell 3’ V2 assay, 16,000 cells were loaded on a Chromium Controller (10x Genomics). In both cases reverse transcription, cDNA synthesis/amplification and library preparation were carried out in accordance with the manufacturer’s recommendations by the Functional Genomics Center Zürich (FGCZ). scRNA libraries were sequenced on a NovaSeq 6000 instrument (Illumina, Switzerland).

Tissue isolation, cryosectioning, and immunohistochemistry

Colonic tissue was harvested from female Pdgfra^H2BeGFP mice, fixed for 1 hour at room temperature in 4% paraformaldehyde in PBS (ChemCruz/Santa Cruz Biotechnology, USA), and incubated O/N in 30% sucrose in PBS at 4°C. Tissue was then kept in a 1:1 mix of 30% sucrose and optimal cutting temperature (OCT) (TissueTek/Biosystems Switzerland) for 30 minutes at 4°C, before embedding in OCT and being cooled to −80°C. OCT-embedded tissue was cryosectioned using a Microm HM560 Cryostat (Thermo Fisher Scientific, Switzerland) at 5 μm, dried for 2 hours at room temperature before either being directly used for immunohistochemistry, or stored at −80°C. Standard immunohistochemical protocols were performed with the following primary antibodies (1:100 dilution): mouse-anti-Acta2 (Sigma, Germany), goat-anti-Pdgfra (R&D Systems, USA), chicken-anti-Vimentin (Millipore, USA), and rabbit-anti-EpCAM (Abcam, UK). Secondary antibodies (1:400 dilution) were anti-rabbit, anti-mouse, anti-goat antibodies conjugated with Alexa Dyes (A555, A598, or A647) (Thermo Fisher Scientific, Switzerland). Sections were counterstained with DAPI, mounted with FluorSave reagent (Sigma), imaged on a Leica SP8 laser scanning confocal microscope (Leica, Switzerland), and subsequently processed using ImageJ (FIJI).

In situ RNA hybridization

mRNAs were localized in specific cells using the RNAscope method (Advanced Cell Diagnostics, Germany) on colonic tissue sections according to manufacturer’s instructions (RNAscope Fluorescent Multiplex Assay). Probe sets for Wnt5a was designed by Advanced Cell Diagnostics. Images were obtained on a Leica SP8 laser scanning confocal microscope.

RNA isolation, cDNA synthesis, and quantitative real-time PCR

Total RNA from Pdgfra-high and Pdgfra-low cells (Fig 4) was isolated using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized using the RNA to cDNA EcoDry synthesis kit (TaKaRa/Thermo Fisher Scientific, Switzerland). Quantitative real-time PCR reactions were performed with technical triplicates using the SYBR Green Kit (Applied Biosystems/Thermo Fisher Scientific, Switzerland) and monitored by the QuantStudio3 system (Applied Biosystems). Sequences of the used primers are: Hprt: AAG CTT GCT GGT GAA AAG GA, TTG CGC TCA TCT TAG GCT TT; Pdgfra: TCC TTC TAC CAC CTC AGC GAG, CCG GAT GGT CAC TCT TTA GGA AG; Wnt2: CTG AGT GGA CTG CAG AGT GC, ACA ACG CCA GCT GAA GAG AT; Wnt2b: CAC CCG GAC TGA TCT TGT CT, TGT TTC TGC ACT CCT TGC AC; Wnt5a: CAG GGT GAT GCA AAT AGG CAG, AGC CAT AGT CGA TGT TGT CTC C; Rspo3: ACT ACA GCA TCC TTC AGC C, TTT TCG TTT TCT CTC TCT TCC C; and Grem1: ACC CAG AGT ACC GTG GT, GTG TAT GCG GTG CGA TTC A.

Computational analysis for scRNA-seq

Quantification of GFP-positive nuclei

Images acquired on the Leica SP8 confocal microscope were imported into FIJI [51] in the form of Z-stacks and processed by Z-projection (maximum projection). In order to quantify the GFP fluorescence, intensity of individual GFP positive nuclei a threshold (Huang algorithm, auto settings) was applied to the GFP channel. The result was further binarized and improved by filling holes and applying a watershed filter. The nuclei were segmented using the analyze particle function (settings: 8-Infinity, circularity: 0–1). The segmentation was overlaid on the DAPI channel and all nuclei of low quality (e.g., partially sectioned/dim nuclei that were not fully captured in the Z-stack) or of inconclusive position in regards to their crypt-associated position (top, bottom) were excluded. Average fluorescence intensity of the remaining nuclei in the GFP channel was measured and analyzed using Prism 8 (GraphPad, USA).

Quantitative real-time PCR

For quantitative reverse transcription PCR (RT-qPCR), samples were measured in triplicate and average cycle threshold values were quantified relative to Hprt using the ΔΔCT method.