Transformation of intestinal stem cells into gastric stem cells on loss of transcription factor Cdx2

Abstract

The endodermal lining of the adult gastro-intestinal tract harbours stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation programme and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2(null) small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. We show that the intestinal genetic programme is critically dependent on the single transcription factor encoding gene Cdx2.

Figure 1. Isolated Cdx2^null SI SCs form gastric organoids.

(a) Graph summarizing the growth performance (two independent experiments) of Cdx2^null SI SC-derived organoids and control Sto SC-derived organoids (issued from single Sto SCs) in medium dedicated to SI organoids (ENR, last rows of the graph, containing Egf, Noggin and R-Spondin1), in medium dedicated to Sto organoids (ENRWfg, top rows of the graphs, containing in addition to ENR, Wnt3a conditioned medium (W), Fgf10 (f) and Gastrin (g)), in SI medium supplemented with Fgf and Gastrin (ENRfg), SI medium supplemented with Wnt (ENRW), SI medium supplemented with Wnt and Gastrin (ENRWg) and SI medium supplemented with Wnt and Fgf (ENRWf). Black bars, stomach control organoids; dark grey bars, Cdx2^null SI organoids. x-axis, number of passages (one passage in average every 7 or 8 days). Error bars, s.d. (b) Control SI organoids in intestinal and stomach conditions (left two panels); insets, higher magnification of buds; red arrows, Paneth cells; Cdx2^null SI organoids (third panel from left) and control Sto organoids (right panel) in stomach conditions. Bars, 150 μm. med, medium. Images are representative of the results of more than 30 experiments.

Figure 2. Cdx2^null SI organoids lost intestinal and express stomach markers.

(a) Cdx2^null SI organoids grown from single SCs fail to express the intestinal markers Villin, Muc2 and Lyz1, whereas control SI organoids grown in the same stomach conditions (ENRWfg) do, like they do in intestinal (ENR) growth conditions. (b) Cdx2^null SI organoids and control Sto organoids grown for 10 days in intestinal culture medium do not express the intestinal differentiation marker ALP, whereas this marker is expressed in control SI organoids in the same growth conditions. Bar, 25 μm. (c) RT-PCR experiments using RNA from independent clones of control SI SC-derived organoids grown in intestinal and gastric conditions, and from Cdx2^null SI SC-derived organoids grown in gastric conditions; intestinal markers, Olfm4, Cdh17, Muc2, Tff3, Lyz1, Cdx1, Muc13 and Reg4. (d) Organoids growing from single Cdx2^null SI SCs express the gastric markers Gif, Pgc and Gastrin, like control Sto organoids do, but unlike control SI organoids, all samples were grown in the same gastric conditions. (e) Cdx2^null SI organoids and control Sto organoids grown for 10 days in culture medium to allow gastric differentiation of control Sto organoids do express the gastric differentiation markers Tff2, Cldn18 and Muc5AC, Control SI organoids do not. Bar, 25 μm. (f) Expression detected by RT-PCR of the gastric markers Gif, Muc6, Tff2, Muc1, Muc5AC, Claudin18, Shh and H⁺/K⁺ ATPase in Cdx2^null SI organoids, compared with control SI and control Sto organoids. For Gif, Muc6 and Muc1 detection, the culture medium for all three samples was Sto medium; for Tff2, Muc5AC, Claudin18, Shh and H⁺/K⁺ ATPase detection, the medium used for all three samples was stomach differentiation medium allowing gastric differentiation of control Sto organoids; this medium corresponds to SI medium. med, medium. Images are representative of the results obtained in three independent experiments performed on each of two independent clones.

Figure 3. Transcriptome analysis of Cdx2^nullSI SCs.

(a) Heatmap comparison of the transcriptome of Cdx2^null SI SCs with control SI SCs and Sto SCs (two independent samples of each) cultured in the same gastric conditions. Downregulated and upregulated genes considered to build the heatmap are the genes with at least a twofold change in transcription. Genes characterizing the signature of SI and Sto SCs are listed on the right of the heatmap: among the downregulated genes are the intestinal markers Olfm4, Muc2, Cdx1, Cdh17, Tff3, Muc13 and Reg4; among the upregulated genes are the gastric markers Claudin18, Muc1, Muc6, Tff2 and Gif. Hierarchical clustering is shown on the top. (b) Transcriptome analysis of Cdx2^null SI SCs compared with control gastric pyloric and corpus SCs (two independent samples of each category of SCs). Hierarchical clustering is on the top. (c) Pair-wise scatter plot analysis of changes in gene expression (two independent samples each time) showing that Cdx2^null SI SCs exhibit much more gene expression similarity with control Sto SCs (graph on the right) than with control SI SCs (graph in the middle). Each dot (blue or red) in the graph represents a gene present on the array used in the Affymetrix analysis. Dots along the bisector line are similarly expressed in the two samples compared. Dots shown in red correspond to genes with higher expression (Log₂ fold) in the sample indicated on the closest axis; the farther from the bisector, the bigger the difference. The genes the most altered in expression (red dots) correspond to the genes highlighted on the right of the heatmap in Fig. 3a.

Figure 4. SI and Sto SC marker gene expression in Cdx2^null SI organoids.

(a) Expression of the SI SC markers Olfm4, Dach1, Clca4, Smoc2, Cdca7 and Msi1. org, organoids. (b) Expression of the stomach-specific SC markers Gif, Col11a2, and PepsinogenC (Pgc) in control SI organoids (black bar), Cdx2^null SI organoids (dark grey) and control Sto organoids (lighter grey). Along the y-axis, relative RNA amounts measured in three independent samples per condition, normalized for Gapdh expression. Error bars are s.d. values. For each marker, one-way analysis of variance according to the ANOVA test is indicated by the P value underneath the graph. P<0.05 indicates that the differences are significant. Tuckey’s test for multiple comparison was run for pair-wise comparison of the means of the samples, with *** meaning highly significant difference, ** very significant difference and * significant difference (see Methods for more details). org, organoids. (c) Time course of the downregulation of intestinal markers and the upregulation of gastric markers in SI SCs on inactivation of Cdx2. Left panel, RT-PCR for Cdx2, Olfm4, Gif and the house keeping gene Hprt. Right panel, graph representing the quantitative RT-PCR results of two independent time course experiments; light grey, five time points in experiment 1; dark grey, five time points in experiment 2; grey with internal patterns, control SI and control Sto.

Figure 5. Expression of Cdx2 in wild-type Sto organoids fails to fully convert them into SI organoids.

(a) Expression of Cdx2 in whole mounts of control SI organoids, Cdx2^null SI and control Sto organoids and Cdx2 expressing Sto organoids (Cdx2⁺ Sto), all grown in Sto medium. Bar, 25 μm. (b) Left panel, quantitative measurement of transcripts of the intestinal markers Tff3 and Muc2 in Cdx2⁺ Sto compared with control Sto and SI organoids. The graph showing expression of intestinal markers in Cdx2⁺ Sto versus control Sto organoids is blown up in the insets to document the statistically significant small increase in expression (t-test, see Methods). For each marker, two independent experiments were performed, and the ANOVA and Tuckey’s tests were used (See Methods for more details). Asterisks in the inset were calculated using one tail t-test. org, organoids. Right panel, immunostaining for Muc2 in Cdx2⁺ Sto organoids, Cdx2^nullSI organoids and control SI and Sto organoids; a similar proportion of Cdx2⁺ Sto and control SI organoids (about 50%) contained positive cells for the immune reaction. Bar, 25 μm. (c) Quantitative measurement of transcripts (two independent experiments for each gene) of the gastric markers Gif, Muc6, Tff2 and Claudin18 in Cdx2⁺ Sto organoids compared with control Sto organoids and control SI organoids. Error bars, s.d. values. org, organoids. (d) Transcriptome analysis on performing RNA-Seq on Cdx2⁺ Sto organoids and control SI and Sto organoids. Scatter plot of the Log₂ mean r.p.m. values per gene for Cdx2⁺ Sto against the Log₂ mean r.p.m. values per gene for control organoids (four independent samples each time). The highlighted genes in the inset (graph on the right) are intestinal markers that are slightly upregulated (Log₂ fold) in the Cdx2⁺ Sto compared with control Sto organoids. Dots along the bisector line are similarly expressed in the two samples compared. Outer dashed lines indicate the twofold difference boundary. Images in a, b (immunostainings) are representative of the results of three independent experiments each time.