LGR 5 controls extracellular matrix production by stem cells in the developing intestine

Article28 May 2020Open Access LGR5 controls extracellular matrix production by stem cells in the developing intestine Valeria Fernandez Vallone Valeria Fernandez Vallone Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Morgane Leprovots Morgane Leprovots Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Didac Ribatallada-Soriano Didac Ribatallada-Soriano Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Romain Gerbier Romain Gerbier Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Anne Lefort Anne Lefort Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Frédérick Libert Frédérick Libert Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Gilbert Vassart Gilbert Vassart Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Marie-Isabelle Garcia Corresponding Author Marie-Isabelle Garcia [email protected] orcid.org/0000-0003-2147-7003 Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Valeria Fernandez Vallone Valeria Fernandez Vallone Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Morgane Leprovots Morgane Leprovots Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Didac Ribatallada-Soriano Didac Ribatallada-Soriano Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Romain Gerbier Romain Gerbier Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Anne Lefort Anne Lefort Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Frédérick Libert Frédérick Libert Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Gilbert Vassart Gilbert Vassart Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Marie-Isabelle Garcia Corresponding Author Marie-Isabelle Garcia [email protected] orcid.org/0000-0003-2147-7003 Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium Search for more papers by this author Author Information Valeria Fernandez Vallone1,2, Morgane Leprovots1, Didac Ribatallada-Soriano1, Romain Gerbier1, Anne Lefort1, Frédérick Libert1, Gilbert Vassart1 and Marie-Isabelle Garcia *,1 1Faculty of Medicine, Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Université Libre de Bruxelles ULB, Brussels, Belgium 2Present address: 1 Charité – Universitätsmedizin Berlin, Berlin Institute of Health (BIH), Berlin, Germany *Corresponding author. Tel: +32 2 555 4195; Fax: +32 2 555 4655; E-mail: [email protected] EMBO Reports (2020)21:e49224https://doi.org/10.15252/embr.201949224 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The Lgr5 receptor is a marker of intestinal stem cells (ISCs) that regulates Wnt/b-catenin signaling. In this study, phenotype analysis of knockin/knockout Lgr5-eGFP-IRES-Cre and Lgr5-DTReGFP embryos reveals that Lgr5 deficiency during Wnt-mediated cytodifferentiation results in amplification of ISCs and early differentiation into Paneth cells, which can be counteracted by in utero treatment with the Wnt inhibitor LGK974. Conditional ablation of Lgr5 postnatally, but not in adults, alters stem cell fate toward the Paneth lineage. Together, these in vivo studies suggest that Lgr5 is part of a feedback loop to adjust the Wnt tone in ISCs. Moreover, transcriptome analyses reveal that Lgr5 controls fetal ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. Finally, using the ex vivo culture system, evidences are provided that Lgr5 antagonizes the Rspondin 2-Wnt-mediated response in ISCs in organoids, revealing a sophisticated regulatory process for Wnt signaling in ISCs. Synopsis The stem cell marker Lgr5 is part of a feedback loop allowing fine-tuning of Wnt signaling in intestinal stem cells during late fetal and postnatal development. Its interaction with the Rspondin 2 ligand modulates epithelial extracellular matrix production. Lgr5 deficiency during Wnt-mediated cytodifferentiation results in the amplification of the stem cell pool. Lgr5 deficiency also increases early Paneth cell differentiation during mouse intestinal development. Intestinal Lgr5+ stem cell maturation is associated with progressive reduction of matrisome components. The Rspo2/Lgr5 interaction in intestinal stem cells fine-tunes Wnt signalling and ECM production in the epithelium. Introduction The adult intestinal epithelium is a specialized tissue involved in nutrient absorption and protection against pathogens and environmental toxic agents. Under homeostatic conditions, within few days, this epithelium undergoes rapid and constant renewal supported by a pool of intestinal stem cells (ISCs), also called crypt base columnar cells, identified by the expression of the Lgr5 receptor 1. Restricted to the bottom of the crypts of Lieberkühn, ISCs have the capacity to both self-renew and give rise to transit-amplifying cells, which differentiate along the villus architecture into all the cell lineages of the epithelium, i.e., absorptive enterocytes, mucus-producing goblet cells, hormone-secreting enteroendocrine cells, Paneth cells generating antimicrobial products, and the type 2 immune response-inducer tuft cells 2. Other populations of slowly cycling or label-retaining reserve stem cells have been identified to efficiently regenerate the intestinal epithelium upon loss of Lgr5-expressing stem cells; additional evidences have been provided for coexistence and possible mutual interconversion between these two stem cell populations 3-5. However, the molecular mechanisms associated with emergence and establishment of adult stem cells during development still remain incompletely understood. By using transgenic mouse lines, evidences have been provided that Cdx2 is a master transcription factor required for intestinal specification before the embryonic stage E14 6, 7. Thereafter, the intestinal epithelium undergoes a profound remodeling, in part instructed by the underlying mesenchyme, leading to appearance of separate domains constituted by villus and intermingled intervillus regions 8-10. Coherent with a proximal-to-distal wave of cytodifferentiation along the intestine mediated by the Wnt/b-catenin pathway around E14.5, the Wnt/β-catenin target gene Lgr5 becomes upregulated and identifies cells (ISC precursors) restricted to the intervillus regions that grow as adult-type organoids in the ex vivo culture system 11-14. After birth, concomitant with Paneth cell lineage differentiation, intestinal crypts will be formed by invagination of the intervillus regions into the surrounding mesenchyme, bearing in their bottom the Lgr5-expressing adult ISCs? 15. Despite general consensus on the function of the Lgr5 receptor as a Wnt/β-catenin signaling modulator in stem cells, how it does so remains still controversial. First of all, in vitro, binding of the natural ligands Rspondins to the receptor Lgr5 has been demonstrated to either enhance or inhibit the Wnt pathway depending on the cell type analyzed 16-20. Secondly, in vivo, homozygous Lgr5-LacZNeo knockin/knockout embryos deficient for Lgr5 exhibited an overactivated Wnt/b-catenin signaling pathway at birth associated with precocious Paneth cell differentiation, this suggesting a negative regulatory function of Lgr5 on this cascade 21. However, conditional ablation of the Lgr5 function in adults did not result in significant alteration in Paneth cell differentiation 17. Moreover, the molecular mechanisms associated with Lgr5 function in ISCs are still debated, does this G-protein-coupled receptor simply control Wnt signaling at the extracellular level by trapping the E3 ubiquitin ligase Znrf3/Rnf43 at the cell membrane, or does Lgr5 signal via its transmembrane domains and intracellular tail 17, 22, 23. In the present report, we further investigated the role of the Lgr5 receptor during intestinal development by analyzing the transcriptome of Lgr5-expressing or Lgr5-deficient ISCs just after the onset of the Wnt-mediated cytodifferentiation (E16) and in adult homeostatic tissues. We provided evidences that Lgr5 controls ISC maturation associated with acquisition of a definitive stable epithelial phenotype, as well as the capacity of ISCs to generate their own extracellular matrix. In addition, using the ex vivo culture system, we demonstrate that the Lgr5 receptor/Rspondin 2 ligand interaction negatively regulates the pool of ISCs in organoids, in a process associated with modulation of epithelial extracellular matrix production. Results In utero inhibition of Wnt activity counteracts premature Paneth cell differentiation induced by Lgr5 deficiency in the intestine To clarify the molecular function of the Lgr5 ISC marker in the embryonic intestine, we investigated the potential phenotype of knockin/knockout (KO) homozygous Lgr5 embryos from the Lgr5-GFP-CreERT2 and Lgr5-DTReGFP mouse strains 1, 24. Since Lgr5 KOs generated from both transgenic lines show neonatal lethality associated with ankyloglossia, histological analyses were performed at E18.5 (Fig EV1A). Despite no evidence of gross architectural epithelial alterations, Lgr5 KOs exhibited early differentiation toward the Paneth lineage as revealed by Lendrum's staining (that evidences Paneth cell granules) as well as qRT–PCR analysis of E18.5 tissues (Figs 1A and B, and EV1B, Table EV1). In addition, Lgr5 KOs showed fourfold increased expression of Wnt/β-catenin target genes (Ascl2, Axin2), histologically detected in the intervillus (IV) regions as compared to wild types (WTs) (Fig 1C and D). This was associated with an early expansion of the eGFP-positive (+ve) stem cell pool both at E16.5 and E18.5 in Lgr5 KO embryos as compared to heterozygous (HEs) (Fig 1E). Of relevance, upregulation of the truncated Lgr5 transcript itself was even higher [10-fold versus (vs) WTs], suggesting a negative control of the Lgr5 receptor on its own expression (Fig 1D). Altogether, these data confirm previous studies on other Lgr5-deficient mouse strains 21, 25 and suggest that Lgr5 deficiency generates overactivation of the Wnt/β-catenin pathway in the prenatal small intestine inducing an expansion of ISC precursors and leading to premature Paneth cell differentiation around birth. ISCs co-express the two paralogue receptors Lgr4 and Lgr5 17, 26. Since deficiency for the Lgr4 receptor leads to ISC loss due to insufficient Wnt signaling in cultured crypts, we assessed the long-term growth properties of Lgr5-deficient ISCs in the ex vivo culture system 26. Irrespective of the mouse strain of origin, upon initial seeding, Lgr5 KO E18.5 small intestines generated a threefold to fourfold increase in the absolute number of growing organoids, which exhibited higher complexity as compared to WTs and HEs (Figs 1F and EV1C). As reported earlier, such higher organoid complexity could be explained by the presence of Paneth cells in Lgr5 KO versus control samples at the time of seeding 14. The stemness status of Lgr5 KO ISCs was studied by replating Lgr5-DTReGFP samples for more than 20 passages (Fig EV1D and E). Organoid growth and Wnt target gene expression were maintained over passages in Lgr5 KOs demonstrating that long-term replating of Lgr5 KO organoids is preserved ex vivo. However, the Wnt tone in Lgr5 KO organoids was not sufficient to confer reduced growth requirements as compared to WTs since KO organoids remained dependent on Rspo1 to grow ex vivo (Fig 1G). Click here to expand this figure. Figure EV1. Lgr5 deficiency induces early Paneth cell differentiation and stem cell expansion in the small intestine at E18.5 Sagittal sections of craniofacial region showing the presence of an ankyloglossia in Lgr5-null Cre embryos at E18.5 as compared to the WT littermate. The tongue (T) and mandible (M) are indicated. Paneth cell quantification as number of cells per 10 intervillus regions on Lgr5-Cre duodenum (Duo) and ileum (Ile). Each symbol indicates the value for a given embryo. Quantification showing organoid complexity ex vivo measured by the branching coefficient for wild-type (WT), heterozygous (HE), and KO organoids from 3 different mouse strains: Lgr5-DTReGFP, Lgr5-Cre, and Lgr5-LacZNeo at day 6 of culture upon initial seeding. Each symbol indicates the mean value for a given embryo. Representative pictures of WT and KO Lgr5-DTReGFP organoids at day 5 of passage 22. Gene expression analysis by qRT–PCR of the indicated stem cell markers in Lgr5-DTReGFP WT and Lgr5 KO organoid cultures, each originating from a given embryo (n = 7 WT and 9 KO at passage 2 and passage 12, n = 2 WT and 5 KO at passage 22). Values are normalized to the WTs at passage 2. Data information: Scale bars, 1 mm (A) and 50 μm (D). Data are represented as means ± SEM. *P < 0.05; **P < 0.01 by Kruskal–Wallis test followed by Dunn's multiple comparison test (B, C). Download figure Download PowerPoint Figure 1. Lgr5 deficiency induces early Paneth cell differentiation and stem cell expansion in the small intestine at E18.5 Paneth cell quantification on Lgr5-DTReGFP duodenum (Duo) and ileum (Ile). Left panel: Representative images of Lendrum's staining that evidence Paneth cell granules. Arrows show differentiated Paneth cells. Right panel: Quantification of the Paneth cells per 10 intervillus regions. Each symbol indicates the value for a given embryo. Expression analysis by qRT–PCR of the indicated Paneth cell markers in Lgr5-DTReGFP ileums (n = 8 WT, 12 HE, 10 KO). X-gal staining in Axin2Lac/+-Lgr5-GFP-CreERT2 WT or KO ileums. Gene expression analysis by qRT–PCR of stem cell markers in Lgr5-DTReGFP ileums (n = 3 WT, 6 HE, 6 KO). FACS quantification of the number of eGFP+ve (ISC) cells per small intestine at the indicated developmental stages in Lgr5 HEs and Lgr5 KOs. Each symbol indicates the value for a given embryo. Mean value for each group is depicted on the graph. Plating efficiency of ex vivo cultured E18.5 small intestines quantified 6 days after initial seeding. Each symbol indicates the value for a given embryo. Influence of mouse Rspondin 1 concentration on growth of replated Lgr5-DTReGFP WT and Lgr5 KO organoids after 5 days of culture. Data information: Scale bars, 20 μm (A, C) and 50 μm (G). Data are represented as means ± SEM. *P < 0.05; **P < 0.01, ***P < 0.001; ****P < 0.0001 by Kruskal–Wallis test followed by Dunn's multiple comparison test (A, B, D, F) and two-way ANOVA followed by Tukey's multiple comparisons test (E). Download figure Download PowerPoint In attempts to reduce in vivo the excessive Wnt signaling tone observed in Lgr5 KO embryos, we treated pregnant females (Lgr5-DTReGFP and Lgr5-GFP-CreERT2 strains) with the orally administrable Wnt inhibitor LGK974. This inhibitor of the acyl transferase Porcupine (which alters Wnt ligand secretion) restores normal Wnt levels in tumor-bearing mice without affecting highly proliferative tissues such as the intestine 27. Pilot experiments demonstrated that the compound efficiently crosses the placenta but that its administration before embryonic stage E11.5 can affect normal embryonic development (Fig EV2A). Then, we tested two different administration windows for daily oral gavage (dose of 3 mg/kg/day), i.e., starting before (E13-E15) or during (E15-E17) the onset of Wnt-mediated cytodifferentiation (Fig 2A). Administration of LGK974 between E15-E17, but not earlier, reduced Paneth cell differentiation in Lgr5 KOs as compared to control levels (Figs 2B and EV2B). The treatment reduced expression of Wnt target genes (Axin2, Ascl2, Lgr5 ex1) and the ISC pool (as quantified with the Olfm4 marker) within the IV region without significantly altering expression of other reported stem cell markers (Hopx, Tert) (Figs 2C–E and EV2C). LGK974 administration also induced downregulation of the Paneth cell marker Defa5 but not that of other cell lineages markers (Figs 2E and EV2C and D). In a converse series of experiments, attempts to upregulate Wnt/β-catenin signaling in Lgr5-expressing stem cells between E15-E17 by a genetic approach via deletion of the β-catenin exon 3 (encoding the sequences targeting the protein for proteasome degradation 28) exacerbated the Paneth cell differentiation phenotype in Lgr5 KO embryos as compared to Lgr5 HEs (2.29 ± 0.33 versus 1.15 ± 0.22 Paneth cells/10 intervilli, respectively; P = 0.0317) (Fig EV2E). Together, these rescue experiments further strengthened the notion that the Lgr5 receptor is involved in negative regulation of the Wnt/β-catenin activity at the onset of cytodifferentiation in the embryonic intestine. Click here to expand this figure. Figure EV2. In utero inhibition of Wnt activity counteracts early Paneth cell differentiation induced by Lgr5 deficiency Gestating Lgr5-Cre heterozygous females were vehicle- or LGK974-treated by oral gavage between E9.5 and E15 at the indicated dose. Global morphological analysis of whole embryos was done at E16.5. Gestating Lgr5-DTReGFP heterozygous females were vehicle- or LGK974-treated by oral gavage between E13-E15 or E15-E17 at the indicated dose. Duodenum of treated Lgr5-DTReGFP KO embryos was analyzed at E18.5 for Paneth cell differentiation. Each symbol indicates the value for a given embryo. Gestating Lgr5-Cre heterozygous females were vehicle- or LGK974-treated by oral gavage between E15 and E17 at the indicated dose. Gene expression analysis of stem cell (Axin2) and Paneth differentiation (Defa5) markers was performed at E18.5 in ileums of Lgr5-Cre embryos by qRT–PCR. Each symbol indicates the value for a given embryo. Gene expression analysis of Paneth differentiation markers was performed at E18.5 in ileums of Lgr5-DTReGFP embryos by qRT–PCR (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 6 KO). Gestating females were intra-peritoneally injected with tamoxifen between E15 and E17. Left panel: Recombination of floxed b-catenin exon 3 (Δex3) was verified by PCR on ileums of embryos. Genotypes of embryos for b-catenin (Ctnnb1) and Lgr5 loci are indicated. Right panel: Ileums of embryos were analyzed at E18.5 for Paneth cell differentiation by Lendrum staining. Each symbol indicates the value for a given embryo. Data information: Scale bar, 500 μm. Data are represented as means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by Kruskal–Wallis test followed by Dunn's multiple comparison test (B, E) and two-way ANOVA followed by Tukey's multiple comparison test (C, D). Download figure Download PowerPoint Figure 2. In utero inhibition of Wnt activity counteracts early Paneth cell differentiation induced by Lgr5 deficiency Design of the experiment. Gestating Lgr5-DTReGFP females were vehicle- or LGK974-treated by oral gavage between E13-E15 or E15-E17. Small intestines of treated embryos were analyzed at E18.5. Paneth cell quantification on Lgr5-DTReGFP ileums. Each symbol indicates the value for a given embryo. Immunofluorescence showing Olfm4+ve cells in Lgr5-DTReGFP duodenums. Cell membranes are shown with b-catenin, and nuclei were counterstained with DAPI. Quantification of Olfm4+ve cells per intervillus regions in duodenum and ileum. Each symbol indicates the value for a given embryo. Axin2 expression levels detected by RNAscope on Lgr5-DTReGFP proximal intestine. Gene expression analysis by qRT–PCR of stem cell and differentiation markers in Lgr5-DTReGFP ileums (vehicle-treated: 7 WT and 7 KO; LGK974-treated: 3 WT and 7 KO). Data information: scale bars, 50 μm (C) and 100 μm (D). Data are represented as means ± SEM. ns not significant, *P < 0.05; **P < 0.01; ****P < 0.0001 by two-way ANOVA followed by Tukey's multiple comparisons test (B, C, E). Download figure Download PowerPoint Postnatal Lgr5 ablation in ISCs alters stem cell fate towards the Paneth cell lineage To determine whether the phenotype observed in Lgr5-deficient embryos could be reproduced postnatally (PN) when the Paneth cells normally emerge in control tissues, we generated conditional deficient-Lgr5 mice (cKO). These mice are double heterozygous Lgr5GFP-CreERT2/flox in which cre-mediated deletion of Lgr5 exon 16 causes a frameshift and a null phenotype 1, 17. Following 3 consecutive tamoxifen injections to lactating females (PN days 6–8), we compared the fate of cKO Tom-recombined cells to that of control heterozygous (HE) Lgr5GFP-CreERT2/+/Rosa26R-Tom littermates after 10 days of chase. No significant differences in terms of clone number were observed in Lgr5-ablated Tom+ve tissues as compared to controls, suggesting that stemness was preserved during this period of chase (Fig 3A and B). However, cKO PN18 Lgr5-ablated Tom+ve tissues exhibited a clear bias toward Paneth cell differentiation (Fig 3C). Such phenotype was observed in proximal and distal small intestines (Fig EV3A and B). When Lgr5 ablation was induced in adults already bearing a definitive number of Paneth cells in crypts, such phenotype was not observed, and no significant differences were detected in the proportion of traced clones (Fig 3C). Together, these data are consistent with the fact that loss of Lgr5 function in homeostatic adult tissues is not associated with an overt phenotype 17 whereas the absence of this receptor during prenatal and early postnatal stages impacts on stem cell fate. Figure 3. Postnatal Lgr5 ablation in ISCs alters stem cell fate toward the Paneth cell lineage Schematic representation of the genetic elements for lineage tracing and Lgr5 ablation during postnatal development and adult homeostasis and experimental design. Lactating females were tamoxifen (Tam)-treated between PN 6 and PN 8. Small intestines of treated pups were analyzed 10 days after the last injection (PN 18); adult mice received one tamoxifen intra-peritoneal injection, and intestines were analyzed 10 days later. Representative pictures of lineage tracing in Lgr5-expressing or Lgr5 cKO intestine after 10 days of chase and quantification of the number of traced clones per recombined surface at PN18 (ileum). Each symbol indicates the value for a given mouse. Representative immunofluorescence pictures showing Paneth cells (Plyz+ve) in traced clones (RFP+ve) from control (Lgr5Cre/+) and cKO (Lgr5Cre/flox) PN18 and adult ileums. Arrowheads point to Plyz+ve cells in traced clones. Quantification of the number of Paneth cell number per recombined RFP+ve crypt on control and cKO ileums in PN18. Each symbol indicates the value for a given mouse. Data information: Scale bars: 100 μm (B) and 50 μm (C). Data are represented as means ± SEM. ns, not significant, **P < 0.01 versus controls by unpaired t-test (B left –PN18) Mann–Whitney test (B right—adult, C). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Postnatal Lgr5 ablation in ISCs alters stem cell fate toward the Paneth cell lineage Fate of RFP+ve-traced clones in control (Lgr5-Cre/+) and cKO (Lgr5-Cre/flx) at postnatal day 18 (PN18) after 10 days of chase. The proportion of fully recombined and mosaic RFP+ve crypt/villus units as well as RFP+ve cells only labeling Paneth cells is indicated for the duodenum and ileum of controls and cKOs (n = 5 for each genotype). Representative immunofluorescence picture showing Paneth cells (Plyz marker) in co-staining with the epithelial cell membrane marker b-catenin in a control (Lgr5Cre/+) at PN18 in ileum 10 days after tamoxifen injection. Scale bar, 50 μm. Download figure Download PowerPoint Lgr5 controls extracellular matrix autocrine production in stem cells To investigate the molecular pathways altered by Lgr5 deficiency, we analyzed the transcriptomic profile of fluorescence-activated cell sorting (FACS)-isolated GFP+ve ISC precursors from Lgr5-DTReGFP HEs and KOs at E16.5 [2 independent pools of Lgr5 HEs (n = 7 and 8) and Lgr5 KOs (n = 2 and 6)] (Dataset EV1). At this developmental stage, potential impact of Paneth cells was excluded based on Lendrum's stainings. Moreover, the gating strategy based on doublets exclusion further limited potential contamination of sorted ISCs with any other cell type (Fig EV4A). RNAseq on these samples identified 487 differentially expressed genes (96 upregulated/391 downregulated) in Lgr5 KOs versus HEs (false discovery rate FDR 0.1 and fold change FC above 1.5) (Fig 4A and B). Lgr5 deficiency was strikingly associated with de-enrichment in the Epithelial–Mesenchymal Transition Gene dataset (47 genes modulated out of 200 genes in the EMT dataset, P value 4.e−60) reminiscent of profound reorganization of the matrisome with significant reduction in extracellular matrix (ECM) structural constituents, including collagen fibrils, in Lgr5-deficient ISC precursors (Fig 4B and C). In accordance with ECM playing a role in development 29, these downregulated genes were also associated with tissue development, morphogenesis, and regulation of cell migration (Fig 4C). Coherent with evolution of precursor ISCs toward a more mature stage over time, epithelial expression of ECM components was detected in fetal but not in adult tissues (Fig 4C and D, Table EV1). Note that decreased expression of ECM markers in Lgr5 E16.5 KOs as compared to WTs could not be visualized by RNAscope, likely due to low expression levels in a limited pool of ISCs. To further test this hypothesis, we compared the transcriptomes of E18.5 embryos and adult Lgr5 HE ISCs (Dataset EV2). Stem cell maturation was associated with downregulation of EMT processes with decreased expression of ECM-associated genes (Figs 4B–D and EV4B). Moreover, in agreement with the study of Navis et al 30 investigating transcriptomic changes during suckling-to-weaning transition, maturation also involved metabolic changes in ISCs (Fig EV4B). Click here to expand this figure. Figure EV4. Transcriptome analysis of Lgr5 ISC precursors in adults Representative FACS plots showing gating strategy used for sorting experiments (Lgr5-DTReGFP E16.5). Back gate for eGFP+ sorted cells (visualized as cyan dots) shows high stringency for duplets exclusion (depicted by a red dashed line). ISC (eGFP+ve) cells from Lgr5-DTReGFP embryonic E18.5 and adult stages were sorted by FACS and subjected to RNAseq analysis. Heatmap of differentially regulated genes (fold change × 1.5, FDR 0.1) in E18.5 HE as compared to adult ISCs from duodenum (Duo) and ileum (Ile). Selected genes are evidenced. Highlighted genes: Wnt-related genes (Wnt3, Axin2), differentiation markers (Defa17, Si), extracellular matrix-related genes (Col1a1, Vim), and metabolic-related genes (Lct, Ass1). Graphs showing relative expression levels of the Wnt ligands and Wnt receptors/co-receptors (Fzd, Lrp, Gpc, Sdc) at different developmental stages E16.5, E18.5, and adult HEs based on the RNAseq data. Graph showing relative proportion of the main Wnt ligands expressed in Lgr5 KOs and HEs at E16.5. Expression of the Wnt ligand Wnt5a in normal embryonic and adult small intestines by RNAscope. The arrows show Wnt5a-expressing cells localized in stroma and epithelium in E16.5 embryos. Data information: Scale bar, 100 μm. Biological replicates for RNAseq experiments on ISC (B, C, D): Lgr5-DTReGFP E16.5 independent embryonic pools (n = 2 HE, 2 KO); Lgr5-DTReGFP E18.5 individual embryos (n = 2 HE depicted as S1 and S2 in panel B), and Lgr5-DTReGFP adult individual animals (n = 2 HE depicted as S1