The olfactory receptor Olfr78 promotes differentiation of enterochromaffin cells in the mouse colon

Abstract

The gastrointestinal epithelium constitutes a chemosensory system for microbiota-derived metabolites such as short-chain fatty acids (SCFA). Here, we investigate the spatial distribution of Olfr78, one of the SCFA receptors, in the mouse intestine and study the transcriptome of colon enteroendocrine cells expressing Olfr78. The receptor is predominantly detected in the enterochromaffin and L subtypes in the proximal and distal colon, respectively. Using the Olfr78-GFP and VilCre/Olfr78flox transgenic mouse lines, we show that loss of epithelial Olfr78 results in impaired enterochromaffin cell differentiation, blocking cells in an undefined secretory lineage state. This is accompanied by a reduced defense response to bacteria in colon crypts and slight dysbiosis. Using organoid cultures, we further show that maintenance of enterochromaffin cells involves activation of the Olfr78 receptor via the SCFA ligand acetate. Taken together, our work provides evidence that Olfr78 contributes to colon homeostasis by promoting enterochromaffin cell differentiation.

Keywords: Enteroendocrine Cells; Odorant; Organoids; SCFA; Serotonin.

Figure 1. SCFA receptors exhibit unique expression profiles along the small intestine and colon.

(A) The expression profile of SCFA receptors was analyzed by qRT-PCR on wild-type (WT) mouse intestinal biopsies from duodenum (Duo), jejunum (Jej), ileum (Ile), proximal colon (Pr Co) and distal colon (Di Co). Relative expression levels were arbitrary set to 1 in Pr Co samples. Each symbol indicates the value for a given mouse (n = 6). (B) The expression profile of SCFA receptors was analyzed on WT mouse tissues by RNAscope. Arrows and arrowheads point to epithelial and non-epithelial expressing cells, respectively. (C) Quantification of the number of cells expressing the Olfr78, Olfr558 and Ffar3 SCFA receptors per 100 crypt/villus sections from pictures obtained with RNAscope. Each symbol indicates the value of a given mouse (n = 3 to 6). (D) Distribution of Olfr78, Olfr558, and Ffar3 positive cells along proximal colon crypts (blue: bottom, red: middle, green: top of the crypt, n = 3 mice). Data information: Scale bars = 50 µm (B). Data are represented as mean ± SEM. (A) Kruskal–Wallis tests: ***P = 0.002 (Olfr78), ***P = 0.002 (Olfr558) and n.s = not significant (Ffar3) with Dunn’s multiple comparison tests: *P = 0.0370 Duo vs Pr Co and Duo vs Di Co, **P = 0.045 Jej vs Pr Co and Jej vs Di Co (Olfr78); **P = 0.027 Duo vs Pr Co, **P = 0.0016 Duo vs Di Co, *P = 0.0351 Jej vs Di Co (Olfr558). (C) Kruskal–Wallis tests: ***P = 0.001 (Olfr78), **P = 0.0013 (Olfr558) and n.s = not significant (Ffar3) with Dunn’s multiple comparison tests: *P = 0.0370 Duo vs Pr Co and Duo vs Di Co, **P = 0.045 Jej vs Pr Co and Jej vs Di Co (Olfr78); **P = 0.027 Duo vs Pr Co, **P = 0.0016 Duo vs Di Co, *P = 0.0351 Jej vs Di Co (Olfr558). (D) Two-way ANOVA test (interaction ***p = 0.008) with Tukey’s multiple comparison tests: bottom crypts: **p = 0.0067 for Olfr558 vs Ffar3; top crypts: *p = 0.017 for Olfr78 vs Ffar3 and **p = 0.0031 for Olfr558 vs Ffar3. Source data are available online for this figure.

Figure 2. Olfr78 is expressed in different subtypes of enteroendocrine cells in the colon.

(A) Top: Schematic representation of the Olfr78-GFP knockin/knockout mouse line. Bottom: strategy of isolation and sorting by flow cytometry of Epcam^+ve/GFP^+ve cells. Graphs show 10,000 cells and 90,000 cells in Olfr78^+/+ (WT) and Olfr78^GFP/+ (heterozygous, HE) samples, respectively. (B) Heatmap of differentially regulated genes in colon Epcam^+ve/GFP^+ve cells (each pool obtained from 2 Olfr78^GFP/+ mice) versus Epcam^+ve cells (2 biological replicates from a single WT mouse) showing log₂ fold change < −2 or >2 and false discovery rate ≤ 0.001. (C) Histogram showing fold change of expression of relevant marker genes in Epcam^+ve/GFP^+ve cells vs Epcam^+ve cells. (D) Heatmap of differentially regulated pre-synaptic, synaptic, and neuropod-associated genes in Epcam^+ve/GFP^+ve cells vs Epcam^+ve cells. (E) Heatmap of differentially expressed transcription factors involved in EEC differentiation in Epcam^+ve/GFP^+ve cells vs Epcam^+ve cells. (F) Immunofluorescence showing 5-HT or PYY production in GFP^+ve cells in proximal and distal colon of Olfr78^GFP/+ mice. Crypts are delineated in white. Red and green arrows point to cells expressing only hormones or GFP, respectively. Yellow arrows indicate GFP^+ve cells colocalizing with 5-HT or PYY. Nuclei were counterstained with DAPI. (G) Quantification of GFP^+ve cells expressing 5-HT or PYY in proximal and distal colon (Pr Co and Di Co, respectively) or 5-HT^+ve and PYY^+ve cells expressing GFP. Each symbol indicates the value of a given mouse. Data information: Scale bars = 20 µm (F). Data are represented as mean ± SEM. **P < 0.01, Mann–Whitney test (G). Source data are available online for this figure.

Figure 3. Loss of Olfr78 impairs terminal differentiation into enterochromaffin cells.

(A) Analysis of Olfr78 expression by qRT-PCR in proximal and distal colon from WT and Olfr78-GFP KO mice. Relative expression levels were arbitrary set to 1 in WT samples. Each symbol indicates the value of a given mouse (n = 8 WT, 5 KO). (B) Representative images showing Alcian Blue-Nuclear Fast Red staining to evidence Goblet cell differentiation (left) and immunohistochemistry for Ki67 staining (right) on proximal and distal colon in Olfr78-GFP WT and Olfr78-GFP KO mice. (C) Bottom: strategy of isolation and sorting by flow cytometry of Epcam^+ve/GFP^+ve cells. Graphs show 90,000 cells in Olfr78^GFP/+ (HE) and Olfr78^GFP/GFP (homozygous/knockout, KO) samples, respectively. (D) Transcriptome comparison between colon epithelial Olfr78-GFP KO vs Olfr78-GFP HE GFP^+ve cells by bulk RNA seq. Upper panel: heatmap of differentially expressed genes between epithelial Epcam^+ve/GFP^+ve cells coming from 2 pools of 2 different Olfr78-GFP HE or KO mice. The number of genes differentially modulated is indicated. Lower panel: GSEA-Biological processes for upregulated (red) and downregulated (green) gene lists in Olfr78-GFP KO vs Olfr78-GFP HE GFP^+ve cells. (E) Heatmaps showing expression levels of EEC markers in sorted epithelial Olfr78-GFP HE and Olfr78-GFP KO GFP^+ve pools. (F) Expression of EEC markers analyzed by qRT-PCR on Olfr78-GFP WT and Olfr78-GFP KO proximal and distal colon biopsies. Relative expression levels were set to 1 in WT samples. Each symbol indicates the value for a given mouse (n = 10 WT, 10 KO). (G) Quantification of 5-HT, PYY, and Olfr558 expressing cells in proximal and distal colon of Olfr78-GFP WT and Olfr78-GFP KO mice performed based on IHC staining (5-HT and PYY) or RNAscope (Olfr558). Each symbol indicates the value of a given mouse (n = 3 WT, 4 KO). (H) Upper panel: UMAP of merged epithelial cells from WT (n = 1764 cells) and KO (n = 1500 cells) mice after scRNAseq. The cell cluster showing Chga expression is circled. Lower panel: Umap of WT and KO epithelial cells showing Tph1 expression. (I) Upper panel: UMAPs of WT and KO Chga-expressing EEC cells showing clustering into 3 cell populations. Lower panel: Umap of WT and KO EEC cells showing Tph1 expression. (J) Heatmap showing the expression of various genes in the 3 EEC-associated groups identified in (I). Data information: Scale bars = 100 µm (Alcian Blue) or 50 µm (Ki67) (B). Data are represented as mean ± SEM. (A) Mann–Whitney tests: **P = 0.0016 (Pr Co and Di Co). (F) Unpaired t-tests (Tph1 in Pr Co; Chgb, Tph1, and Pyy in Di Co), unpaired t-tests with Welch’s correction (Chgb, and Olfr558 in Pr Co and Olfr558 in Di Co), Mann–Whitney test (Pyy in Pr Co). n.s = not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (G) Unpaired t-tests (5-HT and PYY in Pr Co), Mann–Whitney test (Olfr558 in Pr Co and 5-HT, PYY and Olfr558 in Di Co). n.s = not significant; *P < 0.05. Source data are available online for this figure.

Figure 4. Terminal differentiation into serotonin-producing cells is regulated by epithelial Olfr78 expression.

(A) Genomic construction of the Olfr78^fx line. The LoxP sites flanking the exon 3 coding for Olfr78 are evidenced. The coding sequence is labeled in blue. (B) Representative pictures of RNAscope staining showing specific epithelial ablation of Olfr78 expression in Vil1^Cre/+-Olfr78^fx/fx (eKO) mice but not in Vil1^Cre/+-Olfr78^+/+ mice. Arrowheads show non-epithelial expression of Olfr78. (C) Analysis of residual Olfr78 expression by qRT-PCR in control or eKO colon biopsies. Relative expression levels were arbitrary set to 1 in control samples. Each symbol indicates the value of a given mouse (n = 12 controls and 11 eKO). Controls corresponded to Vil1^+/+-Olfr78^fx/fx and Vil1^Cre/+-Olfr78^+/+ mice. (D) Representative pictures of Alcian Blue-Nuclear Fast Red staining on proximal and distal colon from Vil1^Cre/+-Olfr78^+/+ or Vil1^Cre/+-Olfr78^fx/fx. (E) Expression of EEC markers was analyzed by qRT-PCR on colon biopsies from controls or eKO mice. Relative expression levels were arbitrary set to 1 in controls. Each symbol indicates the value for a given mouse (n = 12–14 controls, 11 eKO). (F) Quantification of 5-HT and PYY-expressing cells in proximal and distal colon tissues from controls or eKO mice performed based on IHC staining. Each symbol indicates the value of a given mouse (n = 7 controls, 5 eKO). (G) Serotonin levels in stools collected from controls and eKO colon (mice age: between 8 and 27 weeks old). Each symbol indicates the value of a given mouse (n = 17 controls, 11 eKO). Data information: Scale bars: 50 µm (B, large view) or 25 µm (B, inset), 100 µm (D), Data are represented as mean ± SEM. (C) Unpaired t-tests with Welch’s correction, ****P < 0.0001. (E) Unpaired t-tests (Pr Co), Mann–Whitney test (Di Co). n.s = not significant; ***P < 0.001; ****P < 0.0001. (F) Mann–Whitney tests, n.s = not significant, **P < 0.01. (G) Unpaired t-test, td tendency (p = 0.0797). Source data are available online for this figure.

Figure 5. Enterochromaffin cell differentiation involves activation of the Olfr78 receptor via the SCFA ligand acetate.

(A) Design of the experiment and representative pictures of Olfr78-GFP WT and Olfr78-GFP KO colon organoid cultures after 48 h of treatment (acetate 20 mM or propionate 10 mM) or untreated conditions. (B) Expression levels of EEC markers analyzed by qRT-PCR on colon organoids after 48 h of treatment. Data are reported as the relative expression levels in treated and control conditions in WT and KO organoids. Each symbol indicates the individual value of a given organoid line in each experiment (n = 6 WT and 6 KO). Colored lines identify paired samples in the 3 independent experiments. Data information: Scale bars: 1 mm (A). Data are represented as mean ± SEM. (B) Wilcoxon matched-pairs signed rank test, n.s = not significant, *P < 0.05, **P < 0.01. Source data are available online for this figure.

Figure 6. Loss of Olfr78 expression alters colon homeostasis.

(A) Left: Heatmap of differentially expressed genes identified by RNAseq on colon crypts isolated from Olfr78-GFP WT and KO mice and Vil1^Cre/Olfr78^fx controls and eKO mice (n = 2WT, 3 Controls, 5 KO and 2 eKO mice) (FDR ≤ 0.2, Log₂FC <−0.585 or >0.585 from Degust). (B) Multi-dimensional scaling (MDS) plot of RNA-seq datasets on genes identified in (A). (C) Modulated Mol-Sig GSEA Biological processes in the transcriptome of WT/Controls vs KO/eKO crypts. (D) Upper panel: Umap of WT and KO epithelial cells showing 12 different clusters. Lower panel: Umap of WT and KO epithelial cells showing Reg4 or Saa1 expression. Circles identify differentially enriched clusters present in WT and KO colon. (E) Quantification of GFP^+ve cell density in proximal (Pr Co) and distal colon (Di Co) from Olfr78-GFP HE and Olfr78-GFP KO mice performed based on IHC staining. Each symbol indicates the value of a given mouse (n = 7 HE, 4 KO). (F) Histograms showing the relative microbial abundance at the Phylum and Genus levels in Olfr78-GFP mice (n = 7 WT and 4 KO) and Vil1^Cre/Olfr78^fx controls and eKO mice (n = 11 controls and 11 eKO) by metagenome sequencing. (G) Phylum: Ratio of the prevalence of Firmicutes/Bacteroidetes populations obtained from the data in (F). Genus and Species: Prevalence of genus or species in percentage of total bacteria obtained from the data in (F). Each symbol indicates the value of a given mouse (Olfr78-GFP line: n = 7 WT and 4 KO; VilCre/Olfr78-Fx line: n = 11 controls and 11 eKO). Data information: Data are represented as mean ± SEM (E) and as box and whiskers defining minima to maxima and median line (G). (C) Hypergeometric distribution test of overlapping genes over all genes in the universe, (E) Unpaired t test (G, Alistipes) Unpaired t test: *P < 0.05; (all the others in G) Mann–Whitney tests n.s = not significant; td: tendency (p = 0.0727); *P < 0.05; **P < 0.01.

Figure EV1. SCFA receptors exhibit unique expression profiles along the small intestine and colon.

(A) Representative RNAscope pictures of mesenchymal expression of Olfr78 in the gut. (B) Representative RNAscope pictures of mesenchymal expression of Olfr558 in the colon. (C) Representative RNAscope pictures of Ffar3 expression in myenteric plexuses in the gut. (D) Representative RNAscope pictures of mesenchymal expression of Ffar2 in the Ileum. Data information: Scale bars: 100 µm (low views) or 25 µm (insets). Arrowheads identify isolated SCFA-expressing cells.

Figure EV2. Olfr78 is expressed in different subtypes of enteroendocrine cells in the colon.

(A) FACS strategy for initial population selection and doublets exclusion. (B) List of the 20 most up or downregulated genes in Epcam^+ve/GFP^+ve cells as compared to all Epcam^+ve cells, ranked by FDR. (C) List of significantly up and downregulated transcription factors in Epcam^+ve/GFP^+ve cells as compared to all Epcam^+ve cells.

Figure EV3. Loss of Olfr78 impairs terminal differentiation into enterochromaffin cells.

(A) Olfr78 expression in proximal colon of WT, HE and Olfr78-GFP KO mice analyzed by RNAscope. Arrows and arrowheads identify epithelial and mesenchymal Olfr78-expressing cells, respectively. (B) Quantification of cell proliferation (Ki67^+ve cells) in proximal colon crypts of Olfr78-GFP WT and KO mice. Each symbol indicates the value for a given mouse (n = 6 WT, 5 KO). (C) GFP expression in proximal colon of WT, HE and Olfr78-GFP KO mice analyzed by Immunohistochemistry. (D) List of downregulated genes in Olfr78-GFP KO Epcam^+ve/GFP^+ve cells related to GSEA pre- or post-synapse gene lists, ranked by Log₂(Fold Change). (E) Histograms showing the expression of target genes in the bulk RNAseq from Fig. 3D. CP20M: counts per 20 million mapped reads. (F) Upper panel: Quantification of colocalization between 5-HT or PYY and BFP in the newly generated Olfr558-BFP mouse line (BFP cassette replacing the Olfr558 coding region). Each symbol indicates the value of a given mouse (n = 4). Lower panel: Representative pictures of double immunohistochemistry showing 5-HT/BFP and PYY/BFP co-stainings in proximal colon of Olfr558-BFP WT or HE mice. Data information: Scale bars: 50 µm (A, C and F) or 25 µm (A and C, inset). Data are represented as mean ± SEM.; unpaired t-test, n.s = not significant.

Figure EV4. Terminal differentiation into serotonin-producing cells is regulated by epithelial Olfr78 expression.

(A) Left: PCR strategy for loxp sites recombination verification in Vil1^Cre/+-Olfr78^fx/fx. Right: Gel electrophoresis showing WT and recombinant bands in Vil1^Cre/+-Olfr78^fx/fx. (B) Quantification of Ki67^+ve cells in proximal colon crypts of Olfr78-GFP WT and KO mice. Each symbol indicates the value for a given mouse. (C) Histograms showing the transit time of carmine red (left), the number of fecal pellets expelled during 2 h (middle) and the average wet stools weight (right) in Olfr78-GFP WT or KO mice. Each symbol indicates the value for a given mouse (n = 7 WT and 7 KO). (D) Graphs showing the relative expression levels of EEC markers analyzed by qRT-PCR on colon organoids after 48 h of treatment with propionate at 10 mM. Data are reported as the relative expression levels in treated and control conditions in Olfr78-GFP WT and KO organoids. Each symbol indicates the individual value of a given organoid line in each experiment (n = 3–6 WT and 6 KO). Colored lines identify paired samples in 2 independent experiments. (E) Raw cycle threshold values obtained from qPCR experiments performed on Olfr78-GFP WT or KO organoids (mean ± SEM). Data information: Data are represented as mean ± SEM.; (B, C) unpaired t-test n.s = not significant; td: tendency (p = 0.0673), (D) Wilcoxon matched-pairs signed rank test, n.s = not significant, *P < 0.05, **P < 0.01.

Figure EV5. Loss of Olfr78 expression alters colon homeostasis.

(A) Umap of Olfr78-GFP WT and KO colon epithelial cells showing Mpst or Papss2 expression. Circles identify enriched clusters differentially present in WT and KO mice. (B) Left panel: Merged UMAP of colon mesenchymal cells from Olfr78-GFP WT and Olfr78-GFP KO mice. Right panel: Heatmap showing the top 5 markers of each cluster of the UMAP. (C) Umap of Olfr78-GFP WT and KO colon mesenchymal cells showing Cd45 or Pdgfra expression. (D) Weight of adult Olfr78-GFP mice. Each symbol indicates the value for a given mouse (n = 5 WT and 6 KO). (E) Analysis of Turicibacter sanguinis prevalence by qPCR in the fecal microbiota of Olfr78-GFP WT or KO mice at different ages. Each symbol indicates the value for a given mouse (n = 6–8 WT and 5–9 KO). (F) Quantification of fecal SCFA concentrations. Each symbol indicates the value for a given mouse (Olfr78-GFP line: n = 13 WT and 10 KO; VilCre/Olfr78-Fx line: n = 17 controls and 12 eKO). Data information: Data are represented as mean ± SEM.; (D, E, F) Mann–Whitney tests, (E), n.s = not significant; *P < 0.05.