Scholarly article on topic 'Identification of Lgr5-Independent Spheroid-Generating Progenitors of the Mouse Fetal Intestinal Epithelium'

Identification of Lgr5-Independent Spheroid-Generating Progenitors of the Mouse Fetal Intestinal Epithelium Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Roxana C. Mustata, Gabriela Vasile, Valeria Fernandez-Vallone, Sandra Strollo, Anne Lefort, et al.

Summary Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid transcriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

Academic research paper on topic "Identification of Lgr5-Independent Spheroid-Generating Progenitors of the Mouse Fetal Intestinal Epithelium"

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Identification of Lgr5-Independent Spheroid-Generating Progenitors of the Mouse Fetal Intestinal Epithelium

Roxana C. Mustata,15 Gabriela Vasile,14 Valeria Fernandez-Vallone,14 Sandra Strollo,1 Anne Lefort,1 Frederick Libert,1 Daniel Monteyne,2 David Perez-Morga,23 Gilbert Vassart,1* and Marie-Isabelle Garcia1,*

11nstitut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire (IRIBHM), Faculty of Medicine, Université Libre de Bruxelles ULB, Route de Lennik 808, 1070 Brussels, Belgium

2Laboratory of Molecular Parasitology, IBMM, Université Libre de Bruxelles, 12 rue des Professeurs Jeener et Brachet, 6041 Gosselies, Belgium

3Center for Microscopy and Molecular Imaging (CMMI), Université Libre de Bruxelles, 8 rue Adrienne Bolland, B-6041 Gosselies, Belgium 4These authors contributed equally to this work

5Present address: Wellcome Trust and Medical Research Council, Cambridge Stem Cell Institute, Tennis Court Road, Cambridge, CB2 1QR, UK

'Correspondence: gvassart@ulb.ac.be (G.V.), mgarcia@ulb.ac.be (M.-I.G.) http://dx.doi.org/10.1016/j.celrep.2013.09.005

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

SUMMARY

Immortal spheroids were generated from fetal mouse intestine using the culture system initially developed to culture organoids from adult intestinal epithelium. Spheroid proportion progressively decreases from fetal to postnatal period, with a corresponding increase in production of organoids. Like organoids, spheroids show Wnt-dependent indefinite self-renewing properties but display a poorly differentiated phenotype reminiscent of incompletely caudalized progenitors. The spheroid tran-scriptome is strikingly different from that of adult intestinal stem cells, with minimal overlap of Wnt target gene expression. The receptor LGR4, but not LGR5, is essential for their growth. Trop2/ Tacstd2 and Cnx43/Gja1, two markers highly enriched in spheroids, are expressed throughout the embryonic-day-14 intestinal epithelium. Comparison of in utero and neonatal lineage tracing using Cnx43-CreER and Lgr5-CreERT2 mice identified spheroid-generating cells as developmental progenitors involved in generation of the prenatal intestinal epithelium. Ex vivo, spheroid cells have the potential to differentiate into organoids, qualifying as a fetal type of intestinal stem cell.

INTRODUCTION

The adult intestinal epithelium is one of the most rapidly self-renewing tissues in adult mammals. The steady-state maintenance and self-repairing ability of this tissue are ensured by a hierarchy

of stem cells present in the crypts of Lieberkhun (Barker et al., 2012). Crypt base columnar cells (CBCs) are rapidly dividing stem cells, expressing the specific marker Lgr5, responsible for the constant production of transit-amplifying (TA) cells, while simultaneously maintaining their own population in steady state (Barker et al., 2007). Upon leaving the crypts, TA cells differentiate into postmitotic enterocytes, Goblet cells, enteroendocrine cells, and Tuft cells, which populate the intestinal villi before being shed at their tip. Other rarely dividing adult intestinal stem cells have been described, located just above the Paneth cells in the "+4 position.'' These "label-retaining cells'' are characterized by the expression of several marker genes, including Bmi1, Hopx, Tert, and Lrigl (Sangiorgi and Capecchi, 2008; Yan et al., 2012; Takeda et al., 2011; Montgomery et al., 2011; Wong et al., 2012). The possibility of interconversion between slow and rapidly cycling LGR5+ intestinal stem cells has recently been demonstrated in healing processes after tissue injury or in ex vivo intestinal organoids (Tian et al., 2011; Takeda et al., 2011; Roth et al., 2012, Buczacki et al., 2013).

Embryonic development of the murine intestine has been well characterized morphologically, and recent studies have provided important information regarding the inductive cues and transcription factors implicated in the differentiation of the gut (Walton et al., 2012; Kim et al., 2007; Verzi et al., 2011; Noah and Shroyer, 2013; Spence et al., 2011). Despite these progresses, there is still limited understanding about the origin of the complex adult stem cell pool during development. Fetal progenitors are believed to originate from the region between the newly formed villi, around embryonic day 15-16 (E15-E16). We previously showed that Lgr5-expressing cells are detected in the ileal epithelium of E15 embryos and then become exclusively localized to the intervillus region in the late fetal intestine, before being restricted to CBCs in the crypts (Garcia et al., 2009). Earlier studies with chimeric mice have suggested the existence of multiple progenitors in intervillus regions preceding crypt formation

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(Wong et al., 2002), but their relation with adult stem cells is still unclear.

In the present study, we took advantage of the ex vivo culture system of intestinal epithelium, successfully used to characterize the adult CBCs (Sato et al., 2009), to culture fetal intestine at different developmental stages. Self-renewing progenitor cells were identified that could be cultured indefinitely as undifferentiated hollow spheroids. Spheroid transcriptome differed markedly from that of intestinal organoids, with low or absent expression of intestinal differentiation genes and CBC markers, and upregulation of several genes among which the Trop2/ Tacstd2 and Gja1/Cx43/Cnx43 genes (hereafter referred to as Trop2 and Cnx43, respectively). Ex vivo, spheroid cells demonstrated their ability to generate minigut-forming adult intestinal stem cells. Finally, results from lineage-tracing experiments

Figure 1. Ex Vivo Culture of Fetal Small Intestine Generates Mixed Populations of Spheroids and Organoids

(A and B) Ex vivo culture of embryonic E16.5 small Intestine (A) and replating of selected spheroids and organoids from a primary culture at day 7 (B) were performed as reported in the scheme. Selected fields were followed and photographed at the times indicated. (C) Quantification of the percentage of spheroids and organoids in cultured small intestine obtained at different embryonic and postnatal stages (mean ± SEM). For each time point, the number of embryos/mice and total counted elements is as follows: E14 (3, 67), E15 (7, 2476), E16 (5, 982), E17 (9, 1288), E18 (10, 923), P1 (10, 492), P4 (4, 2150), P5 (5, 2682), P15 (5, 383). Scale bars represent 200 mm. See also Figure S1.

showed that the intestinal epithelium in mice is generated in two successive waves relying on different kinds of progenitors: a transient, fetal wave relies on Cnx43-positive cells, whereas the postnatal epithelium is generated from Lgr5-positive precursors of CBCs.

RESULTS

Ex Vivo Culture of Fetal Small Intestine Identifies a Population of Immortal Spheroids

The ex vivo culture system described by Sato et al. (2009) allows indefinite propagation of organoid structures containing all differentiated cell types present in normal intestinal epithelium when adult intestinal crypts are used as starting material. When E16.5 intestine was used, as described in Figure 1A, we observed that, in addition to adult-type minigut organoids, a proportion of hollow spheres (hereafter referred to as "spheroids") were generated (Figure 1A). Upon serial replating, both structures "bred true'': i.e., spheroids generated spheroids and organoids generated organoids (currently for >50 generations or 10 months) (Figure 1B). Among the supplements present in the ex vivo culture medium, spheroids required Rspondin1 for growth and Noggin for efficient replating, whereas EGF did not appear essential (Figure S1). Of note, fetal spheroids were grown in total absence of Wnt ligand supplementation, making them different from the spheroid-like structures generated from normal adult tissue-derived organoids in presence of Wnt3a (Satoetal., 2011).

The relative proportion of spheroids and organoids generated from cultured small intestine was studied at different developmental stages. Whereas E14-E15 intestines generated almost exclusively spheroids, their proportion progressively decreased during late fetal development, representing 60% at E16 and less than 4% at postnatal day 5 (P5). Intestinal crypts harvested at P15 generated exclusively organoids (Figure 1C).

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A Spheroid Organoid B Spheroid

Figure 2. Spheroids Are Composed of a Polarized Epithelium of Intestinal Origin

(A) Frozen sections of spheroids and organoids were immunostained for E-cadherin, ZO-1, Villin, and CD44.

(B) Scanning electron microscopy of spheroids showed a monostratified epithelium. Higher magnification of the rectangle is shown on the right image of the panel.

(C) Transmission electron microscopy of spheroids and organoids; Lu indicates lumens; insets show a higher magnification of the microvilli at the apical membrane.

(D) Spheroid and organoid sections were stained with EdU and TUNEL for proliferative and apoptotic cells, respectively. Scale bars represent 20 mm (A and D), 5 mm (C), and 100 and 10 mm (B, left and right panels, respectively). See also Figure S2.

This observation demonstrated that the relative abundance of spheroid-generating cells is clearly linked to the developmental stage, these cells being predominant in the period preceding villogenesis.

Spheroids Are Made of a Proliferating Polarized Epithelium of Intestinal Origin

Basolateral labeling of E-cadherin and apical staining of the tight-junction marker ZO-1 in both spheroids and organoids demonstrated the epithelial nature and polarized state of the two structures (Figure 2A). Expression of villin on the luminal surface of the spheroids provided evidence for their intestinal origin (Figure 2A). Electron microscopy further showed that spheroids were formed by a monostratified layer of cells with a high degree of interdigitations and exhibiting microvilli sparser and shorter than in organoids (Figures 2B and 2C). With regard to cell proliferation, the overall rate was similar in both kinds of structures

when computed as percentage of living cells, but, contrary to organoids showing cycling cells restricted to crypt-like protrusions only, spheroids displayed proliferating cells all over their surface (Figures 2D and S2A). Apoptotic cells were rarely detected in the epithelium and lumen of spheroids, whereas the lumen of organoids appeared full of dead cells (Figure 2D).

Spheroid Transcriptome Is Radically Different from that of Both Organoids and CBCs

We compared global gene expression of spheroid/organoid pairs obtained from four different embryos coming from three different litters (two at E16, one at E18, and one at P0) (see Experimental Procedures). Significance analysis of microarrays (SAM) followed by further selection of transcripts with a 2-fold up- or downregulation and a q value <0.054 allowed identification of 317 upregulated and 179 downregulated genes (Table S1). Among the most strongly downregulated genes

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Figure 3. Spheroid and Organoid Transcriptomes Are Different

(A) List of the 33 genes most up- or downregulated in spheroids versus organoids. Data were generated from four independent pairs of spheroid/organoids samples.

(B) qRT-PCR analysis of transcripts from spheroids and organoids. Six pairs of spheroids/organoids were used. Bars represent mean ± SEM.

(C) GSEA analysis of microarray data versus CBC signature of Muhoz et al. (2012).

(D) GSEA analysis of microarray data versus Cdx1 KO/Cdx2KO list of upregulated genes (left panel) or downregulated genes (right panel). NES, normalized enrichment score.

See also Figures S3 and S4 and Tables S1, S2, and S3.

were differentiation markers corresponding to the four main intestinal cell types (Figure 3A and Table S1). Loss of differentiation markers was confirmed by quantitative RT-PCR (qRT-PCR) and immunofluorescence (Figures 3B and 4A).

Interestingly, compared to organoids, spheroids also showed low expression levels of several adult intestinal stem cell markers (Lgr5/Gpr49, Smoc2, Axin2, Cdx1) (Figure 3B and Table S1). Gene set enrichment analyses (GSEA) comparing our microarray results with the set of CBC-enriched genes (Murioz et al., 2012)

confirmed the downregulation of the CBC signature in spheroids (Figure 3C). These data were validated by qRT-PCR and extended to additional adult stem cell markers: whereas expression of Tert and Olfm4 were also downregulated in spheroids, Bmi1 and Ascl2 were expressed at similar levels in spheroids and organoids (Figure 3B). The uncoupling between Lgr5 and Ascl2 gene expression was unexpected as both genes are markers for adult CBCs (van der Flier et al., 2009). In accordance with qRT-PCR data, expression of Lgr5 and the Wnt reporter

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marker Axin2 appeared strong in the protrusions of organoids containing adult stem cells but undetectable or very low in the paired spheroids (Figure 4B). Indeed, 99.9% ± 0.1% of the organoids and 3.8% ± 2.6% (mean ± SEM) of the spheroids were Xgal positive in Axin2LacZ/+ samples, suggesting different Wnt stimulatory tones in both kinds of elements (Figures 4B and S2B). Noteworthy, the Lgr4 receptor was highly and similarly expressed in both spheroids and organoids when analyzed by qRT-PCR and Xgal staining on Lgr4LacZ/+ samples (Figures 3B and 4B), whereas Lgr6 was undetectable in any in vivo or ex vivo intestinal sample (Figure S3A). The role of the Lgr4 and Lgr5 receptors in self-maintenance of spheroids was investigated. As demonstrated earlier for P0 intestine (Mustata et al.,

Figure 4. Compared Expression of Differentiation, Stem/Progenitor Markers, and the Wnt Stimulatory Tone in Paired Spheroids and Organoids

(A) Immunofluorescence detection of Paneth (lysozyme staining) and enteroendocrine cells (serotonin) in orga-noids and spheroids. The arrow and arrowhead point to an enteroendocrine and a Paneth cell, respectively.

(B) X-gal staining of Lgr5LacZ/+ or Lgr4LacZ/+ orAx/n2LacZ/+ spheroids and organoids. Quantification of the relative proportion of Xgal-positive (pos) and Xgal-negative (neg) elements among Axin2LacZ/+ spheroids and organoids. Fifty spheroids and 250 organoids were counted per sample (n = 4 independent samples coming from cultured P0 intestine stained at day 4 after the second replating). Morphological classification of the counted elements is detailed on the right side.

(C) Inhibition of porcupine activity affects growth of spheroids. Spheroids were grown for 4 days in the presence of IWP2 1 mM supplemented or not with recombinant Wnt3a at final concentration of 100 ng/ml. A mean of 20 elements were measured per condition and sample (n = 4/5 independent experiments performed on three different samples). Spheroid size is graphed relative to control conditions (mean ± SEM). Significance was computed from paired t test: Ctrl versus IWP2, p = 0.0055; IWP2 versus IWP2 + Wnt3a, *p = 0.027. Scale bars, 20 (A) and 200 (B and C) mm. See also Figures S2-S4.

2011), Lgr4 was essential for growth of both spheroids and organoids as no explants could be maintained from the small intestine of Lgr4-deficient embryos (n = 11 embryos analyzed between E16 and birth) (Figure S3B). The role of Lgr5 and Lgr5-expressing cells in spheroids was addressed using Lgr5-deficient mice (Morita et al., 2004) and the Lgr5-DTR mouse line (Tian et al., 2011), respectively. Contrary to Lgr4, stable spheroids were readily cultured from Lgr5 KO embryos (Figure S3C). Similarly, specifically killing Lgr5--expressing cells by administration of diphteria toxin to explants cultured from heterozygous Lgr5-DTR embryos was without effect on spheroid growth. In contrast, the toxin severely affected initial formation of protrusions in the paired organo-ids (Figure S3D). Together with the low level of Lgr5 expression in spheroids, these data indicate minor if any contribution of the Lgr5 receptor to spheroid maintenance.

Among the most upregulated genes in spheroids versus organoids (Figure 3A), several have been associated with stem/progenitor cells (Cnx43, Trop2, and Ly6a/Sca1) (Todorova et al., 2008; Goldstein et al., 2008; Holmes et al., 2007) or reported to be expressed in malignant tumors (Spp1, Trop2, and Clu) (Cao et al., 2012; Trerotola et al., 2013; Rizzi and Bet-tuzzi, 2010). Also, some of the upregulated genes are reportedly involved in tissue regeneration and/or development (Ctgf, Trop2, Clu, and Vsigl) (Gunasekaran et al., 2012; Goldstein et al., 2008; Lee et al., 2011; Oidovsambuu et al., 2011) (Table S1). The

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preferential expression of Cnx43 and Trop2 in spheroids was confirmed by qRT-PCR and immunofluorescence on matched pairs of spheroids/organoids (Figures 3B and 5A).

Before reaching a mature state with multiple crypt-like protrusions, organoids display a transient spheroid-like appearance (Sato et al., 2009) (Figure S4A). The possibility that organoids would pass through a less differentiated state similar to that of the spheroids described here was ruled out by qRT-PCR analysis. Indeed, at their spheroid-like stage, organoids already exhibited low Trop2, Cnx43, and Ccndl transcript levels, with high expression of the differentiation markers (Crypt4, Muc2, Chga, and Si) (Figure S4A). Of note, the gene expression profiles of spheroids and organoids were stable, with no substantial changes of their ratio after ten replatings (Figure S4B).

Given the striking morphological similarity of fetal spheroids with those reported from cultured adult Apcm'n adenomas, we compared our microarray data to the short list of 38 genes upregu-lated in adenomas and their related organotypic cultures (Farrall et al., 2012). Twelve genes appeared commonly upregulated in both kinds of spheroid cultures, with the Trop2 marker ranking in

Figure 5. Tissue Expression of Spheroid Markers Ex Vivo and during Fetal Intestinal Development

(A) Immunofluorescence images of Cnx43 and Trop2 coexpression in spheroids and organoids. Individual channels for visualizing Cnx43 and Trop2 expression in spheroids are depicted below the corresponding merged image.

(B) Immunohistochemical detection of Cnx43 and Trop2 in duodenal sections at E14, E16.5, and P0. Arrow shows cells expressing high Trop2 at E16.5, empty arrowheads evidence epithelial-expressing Cnx43 at E16.5 and arrowheads points to mesen-chymal expression of Cnx43.

Scale bars represent 20 mm (A and B). See also Figure S5.

the top list, pointing to b-catenin-dependent activation of partially overlapping genetic programs in the two cases (Table S2). Coherent with this view, spheroid growth was inhibited in a dose-dependent manner by the porcupine inhibitor IWP2 and partially restored by Wnt3a addition to the culture medium (Figures 4C and S4C). This suggests dependence of spheroids on autocrine production of Wnt ligand for their survival. Unexpectedly, only two genes (Trex2 and Foxq1) out of the 80 making the intestinal Wnt/TCF signature reported by Van der Flier et al. (2007) were found to be upregulated in fetal spheroids. Together with the low expression of additional Wnt target genes in fetal spheroids (Lgr5 and Axin2), this suggests that Wnt would act differently, depending on the epigenetic status of the Apcmin and fetal spheroids.

From these observations, we concluded that spheroids are made of poorly differentiated intestinal cells with progenitor/stem cell characteristics different from those of adult CBCs.

Spheroids Cells Correspond to Incompletely Caudalized Progenitors

Further inspection of the list of genes upregulated in spheroids pointed to gastric and esophageal genes such as Gkn1,2,3, Invo-lucrin, Vsigl, and Krt4 (Figure 3A and Table S1). Together with the strong downregulation of the caudal type homeobox 1 gene Cdx1 observed in spheroids (Figure 3B), this prompted us to compare by GSEA our microarray results to the list of genes up- and downregulated in the intestine of Cdx1/Cdx2 double knockouts, which are known to lose intestinal differentiation and acquire expression of gastric markers (Verzi et al., 2011). A clear positive relation was observed between the spheroid versus organoids and the Cdx1/Cdx2 knockout intestine data sets (Figure 3D), suggesting a lack of caudal differentiation in spheroid cells. Surprisingly, this phenotype is observed despite low but detectable expression of Cdx2 in spheroids (Figure 3B), suggesting that the reported redundancy of Cdx genes in the

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intestine (Beck and Stringer, 2010) is not effective in the context of spheroids.

Trop2 and Cnx43 Are Expressed in the Fetal Epithelium of the Mouse Intestine

The Trop2 molecule is highly expressed in several types of tumors, including colorectal cancer, but its expression pattern in the developing intestine has not yet been studied (Ohmachi et al., 2006). As reported in Figures 5B and S5A, Trop2 was expressed at high levels in all epithelial proliferating progenitors of the E14 duodenum. At E15.5, strong membrane staining was observed in cells present at the tip of the newly formed villi with lower but still detectable signal, in the intervillus zone (Figure S5A). Between E16.5 and birth, these strongly labeled cells progressively disappeared, having likely been shed from the villi (Figure 5B). Similar staining was observed in the ileum (Figure S5B). When sorted from fetal intestine and cultured ex vivo, the epithelial Trop2+ cells generated spheroids, demonstrating a direct filiation between in vivo Trop2+ cells and ex vivo spheroids (Figure S5C). Of note, several transcripts found to be enriched in spheroids displayed lower or absent expression in Trop2+ cells sorted from the E15 intestine, indicating an effect of the ex vivo culture conditions on spheroid phenotype (Table S3).

Intestinal expression was also studied for the gap junction protein Cnx43 (second position in the microarray list). At E14, Cnx43 was expressed in the lateral intercellular epithelial membranes and at the basal pole of epithelial cells at the mesenchymal interface (Figure 5B). At E16.5, Cnx43 was found in a more restricted pattern in the epithelium, being almost exclusively detected in cells of the intervillus region and showing a characteristic punctuate staining (Figures 5B and S5D). In addition, Cnx43 was also detected in rare mesenchymal cells, many of them located underneath the epithelium of the intervillus region and within the mesenchymal bud of nascent villi. At P0, Cnx43 was almost exclusively expressed in mesenchymal cells surrounding the epithelium, often organized into clusters (Figures 5B and S5D).

Cnx43-Expressing Cells Contribute to Prenatal Villus Formation

We used lineage tracing to explore the respective contribution of Cnx43+ and Lgr5+ cells to generation of the fetal and postnatal intestine. Cnx43-Cre/Rosa26R and Lgr5-Cre/Rosa26R embryos were pulsed with tamoxifen at the onset of villogenesis (E15), and the labeling patterns were assessed at several time points pre-and postnatally. One day post pulse (dpp), Cnx43-Cre/Rosa26R embryos displayed numerous labeled cells in newly formed villi and intervillus regions, mainly as single cells but also sometimes as small groups of two to three cells, whereas 3 dpp the number of labeled cells had decreased (Figure 6A). At a later time, most epithelial positive cells disappeared from the intestine, with labeling confined to villus extremities and only sparse intervillus regions around birth (6 dpp, Figure S6A) and very rare ribbons of crypt/villus units later (2 weeks pp, Figure 6A). In contrast, in E15-pulsed Lgr5-Cre/Rosa26R embryos, only very rare labeled cells were observed after 1 and 3 dpp, which persisted postnatally (2 weeks pp), suggesting a minor contribution of Lgr5+ cells to prenatal villus formation (Figure 6A). Low lineage tracing due to

potential variegation of the Lgr5-Cre knockin allele was unlikely considering the number of embryos analyzed giving similar results (see Experimental Procedures). When a long pulse was performed during the neonatal period via the lactating female (from P5 to P8), Cnx43-dependent recombination yielded only mesenchymal labeling and virtually no epithelial labeling, in agreement with the observed loss of Cnx43 epithelial expression around birth (Figure 6B). In contrast, Lgr5-Cre/Rosa26R intestine displayed abundant labeling of crypt/villus units, compatible with the tracing of immediate CBC precursors (Figures 6B and S6B). Quantification of lineage tracing confirmed these observations (Figure 6C). Together, these results suggest that Cnx43+ cells function as progenitors of prenatal villi, their offspring being lost by shedding in the lumen asTrop2+ cells (Figure S5B). Only a small proportion of them, likely those gaining expression of Lgr5 in the forming intervillus region, may function as progenitors of adult crypt-villus units. In agreement with this view, the rare postnatal ribbons of cells generated from E15 Cnx43-positive progenitors express Lgr5 in the crypts (Figure S6C).

Fetal Spheroids Can Differentiate into Organoids

The ability of spheroid Cnx43+ cells to potentially convert into adult Lgr5+ CBCs was investigated in the ex vivo culture system. After 7-8 days of culture, despite the stability of the phenotype, a small proportion of the grown elements generated dark spheres, with intraluminal accumulation of dead cells, and some of these emitted protrusions similar to the crypt-like domains of organo-ids (~1.4%) (Figures 7A and 7D). As expected for miniguts, the newly formed organoids demonstrated strong expression of Lgr5 restricted to the protrusions and exhibited expression of differentiation markers from the absorptive and secretory lineages (Figure 7C). Interestingly, the proportion of spheroids displaying morphological differentiation toward an organoid-like phenotype increased to 11.5% when spheroids were grown in a medium containing the gamma secretase inhibitor DAPT (1 mM) (Figures 7B and 7D). As observed in the case of spontaneous differentiation, after replating in normal medium, the organoid-like elements generated in the presence of DAPT gave rise to stable organoids. These results indicate that fetal spheroids have the potential to generate adult-type CBCs.

DISCUSSION

Using the minigut culture system, we have isolated from fetal intestine self-renewing cells that generate immortal epithelial "spheroids." Spheroids display genetic commitment to intestinal differentiation but express low levels of intestinal markers in comparison to organoids and their gene expression profile differs radically from that of adult CBC stem cells. Several lines of evidences suggest that spheroid cells represent a "frozen state" of progenitors found in the epithelium before the onset of villogenesis in vivo (i.e., around E14). Afirst strong argument in favor of this hypothesis is given by the inverse relation between the proportion of spheroids obtained from fetal tissue explants and the developmental stage: whereas E14-E15 tissue generates close to 100% spheroids, the ratio progressively decreases during late fetal development, approaching zero when crypts start to form (P5 onward). Second, we have shown that all epithelial cells

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of the E14 intestine express Trop2 and Cnx43, two among the most upregulated genes identified in spheroids by microarray. In addition, Trop2+ cells sorted from fetal intestine generate similar spheroids structures when cultured ex vivo. Finally, in agreement with spheroids being generated from intestinal cells preceding villogenesis, the global gene expression of intestinal spheroids was found to display similarities with that of double Cdx1/Cdx2 knockout intestine, a pattern consistent with an intestinal dedifferentiation-like phenotype characterized

Figure 6. Lineage-Tracing Experiments Performed on Cnx43-CreER/Rosa26R and Lgr5-CreERT2JRosa26R Embryos and Neonatal Mice

(A) E15 embryos pulsed with tamoxifen were sacrificed 1 day, 3 days, and 2 weeks post pulse (pp). Arrows point to single recombined Lgr5-positive cells.

(B) P5-old mice were tamoxifen-pulsed for 4 consecutive days (via maternal milk) and sacrificed at P10 (2 days pp). Full arrowheads point to re-combined Cnx43-positive cells localized in the mesenchyme. Immunohistochemical detection of b-gal or YFP-positive cells (see Experimental Procedures).

(C) Quantification of lineage-tracing experiments reported as the number of positive clones counted per 200 villus-intervillus/crypt units (mean ± SEM) in E15-pulsed embryos harvested after 3 days or 2 weeks and in P5-P8-pulsed mice harvested at P10. The numbers of embryos/mice used for quantification were as follows: three and five for Cnx43-Cre and Lgr5-Cre lines at E15 + 3 days pp and two for both lines at E15 + 2 weeks pp; two and three for Cnx43-Cre and Lgr5-Cre lines at P5-P8 + 2 days pp, respectively. A mean of 200 villus-intervillus/crypt units were analyzed for each sample.

Scale bars represent 200 and 20 mm (A, left and right panels, respectively) and 50 mm (B). See also Figure S6.

by expression of anterior foregut markers (Verzi et al., 2011). The overall transcrip-tomes of stomach and intestine epithelia are very similar at E14.5 (Li et al., 2009), with only the latter displaying striking changes between E14.5 and E16.5, after emergence of villi. This process was coined "intestinalization" by Gumucio and colleagues (Li et al., 2009) and corresponds to the induction of the differentiation markers of the three intestinal lineages present at birth. Of note, despite sharing similar morphology with embryonic spheroids described here, the cystic vesicles generated by ex vivo culture of Cdx2 knockout intestinal crypts appeared different because these do not survive replating (Stringer et al., 2012). We conclude that our spheroids likely correspond to immortalization of progenitors in a state just preceding the "intestinalization" process.

The status of spheroids regarding Wnt signaling displays contradictory features. On the one hand, several arguments point to a role for activation of a b-catenin-dependent transcription program in fetal spheroids; genes upregulated in spheroids show significant overlap with those of Apcm'n adenomas (Farrall et al., 2012); spheroid growth and maintenance require the presence of the Wnt coactivator Rspondin, one of its receptor Lgr4,

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Figure 7. Spheroids Can Generate Intestinal Organoids in Culture

(A and B) Spheroids were cultured in control medium (A) or In presence of DAPT 1 mM (B). Arrows point to crypt-like structures.

(C)X-gal staining of organoid-like structure having spontaneously differentiated from culture of Lgr5LacZ/WT spheroids shows Lgr5+ cells in crypt-like structures (left panel). Newly formed organoid-like structures express high levels of cell lineage differentiation markers as compared to the surrounding spheroids from the same well (right panel).

(D) Quantification of spheroid's conversion to organoids after 7 days of culture.

Data represent the mean ± SEM of four independent experiments. *p < 0.016. Scale bars represent 200 mm.

and autocrine Wnt production. On the other hand, only two out of 80 genes making the intestinal Wnt signature (Van der Flier et al., 2007) are upregulated in spheroids, and Axin2 and Lgr5, two prototypical Wnt targets highly expressed in CBCs, are virtually not expressed in spheroids. This suggests that activation of the Wnt pathway causes upregulation of different sets of genes in Apcmin adenomas, CBCs, and fetal spheroid cells possibly as a result of their different epigenetic status, or crosstalk with different concurrent regulatory cascades. Of interest, the dramatic effect of Lgr4 deficiency on self-renewing ability of spheroids ex vivo compared with the mild intestinal phenotype in vivo (Mustata et al., 2011) parallels the situation described for Lgr4 deficiency in organoids versus postnatal crypts (Mustata et al., 2011). The absence of detectable levels of the Lgr6 paralog in the intestine suggests that fetal extraepithelial signals, rather than potential in vivo redundancy of Lgr receptors, compensate for the default of Lgr4 function.

Expression of Trop2 and Cnx43 is clearly associated with the normal development of prenatal intestinal epithelium in vivo. The Trop2 molecule, highly expressed in the trophoblast and several organs during development, marks subpopulations of adult prostatic stem cells with regeneration capabilities and is upregu-

lated in a wide series of malignant tumors including colorectal cancer (Yagel et al., 1994; Tsukahara et al., 2011; McDougall et al., 2011, Goldstein et al., 2008, Trerotola et al., 2013). This justified its qualification as a possible therapeutic target (Cubas et al., 2009). Recently, it has been proposed thatTrop2 enhances stem-like properties of tumor tissue via b-catenin-dependent mechanisms (Stoyanova et al., 2012).

Connexins, and, in particular, Cnx43, are responsible for the establishment of cell-to-cell communication, involving transfer of small soluble molecules (for a review, see Kar et al., 2012). Expression of Cnx43 in progenitors has been reported in human fetal bulge stem cells of the hair follicle (Arita et al., 2004) and in the embryonic brain where it appears crucial to prevent premature neuronal differentiation (Santiago et al., 2010). Whether Trop2 and Cnx43 are simply markers of fetal spheroids or contribute functionally to their stem cell phenotype will need to be addressed in future studies.

Although spheroid cells and progenitors sorted from fetal intestinal epithelium share expression of a series of genes, a few transcripts showing high expression in spheroids (e.g., loricrin, Sca1) were barely detected in the E15 intestinal epithelium. This suggests that the ex vivo culture conditions, although

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allowing survival of progenitors as spheroids, cause some distortion of gene expression profile. A similar observation has been reported when culturing adult normal and Apcmin adenoma tissues (Farrall et al., 2012). Nonetheless, the strikingly different gene expression profiles displayed by fetal spheroids and orga-noids generated from the same intestine, when exposed to the same culture conditions, highlight the different nature of the self-renewing cells from which they originate. The fundamental difference between CBCs and spheroid-generating cells is further attested by their different Lgr5 status. Whereas CBCs are currently defined as Lgr5-positive cells, fetal spheroids express low level of this gene and experiments with Lgr5-DTR embryos demonstrate that spheroids do thrive in the absence of Lgr5-expressing cells.

The progressive decrease in the number of Cnx43+ cells in fetal intestinal epithelium during the E14-E18 period parallels the spheroid-generating ability of intestinal explants and fits with the lineage-tracing experiments of Cnx43+ cells performed in utero and during the neonatal period. Together, these observations suggest that Trop2/Cnx43+ cells act as transient stem cells responsible for the generation of fetal intestine in an environment characterized by low Wnt and high Bmp stimulatory tones prevailing at this period of intestinal development (Karls-son et al., 2000; Li et al., 2009; Kim et al., 2007; Korinek et al., 1998). In contrast, the majority of Lgr5+ cells are generated later as precursors of adult CBCs. Ex vivo experiments showing the capacity of Cnx43+ cells to convert to organoids suggest that a fraction of these cells would be the precursors of Lgr5+ cells present in the intervillus region during late gestation (Garcia et al., 2009), whereas the vast majority of them are lost at the tip of prenatal villi. Only very rare E15 Cnx43+ cells contribute directly to the postnatal epithelium, being likely those that already expressed, or gained expression, of Lgr5 in a higher Wnt environment (Li et al., 2009). The boosting effect on spontaneous conversion of spheroids to organoids observed ex vivo in the presence of DAPT suggests that Notch pathway contributes to maintenance of spheroid progenitor cells in an undifferentiated state, as it contributes to proliferation of adult intestinal stem cells (Noah and Shroyer, 2013).

Reminiscent of a switch from Cnx43+ progenitors of the early intestinal epithelium to Lgr5+ stem cells, establishment of the definitive intestinal epithelium adapted to digestion of adult food type is known to be a two-step process in batrachians (for a review, see Ishizuya-Oka and Hasebe, 2013). During metamorphosis of Xenopus laevis, the intestinal epithelium of the tadpole is totally replaced by a novel, definitive epithelium generated from rare Lgr5+ cells. Similarly, our results suggest that the intestinal epithelium in mammals is generated in two waves relying on different kinds of stem cells: a transient, fetal wave relies on Cnx43-positive progenitors, whereas the postnatal epithelium is generated from Lgr5-positive precursors of CBCs.

Altogether, our data establish a relationship between transient progenitors responsible for generation of fetal intestinal epithelium and immortal spheroid-generating cells having the capability to "differentiate" into CBCs. This is of particular interest in the recently documented context of interconversion of the various adult intestinal stem cell types, in situations of epithelial regeneration (Takeda et al., 2011; Tian et al., 2011; Parry et al.,

2013; Yan et al., 2012; Roth et al., 2012; Murioz et al., 2012; Montgomery et al., 2011). Considering that most spheroid cells are mitotically active and display an early, poorly differentiated intestinal phenotype, grafting of spheroids cells could be particularly efficient in regeneration of injured gut epithelium.

EXPERIMENTAL PROCEDURES

Animal Experiments and Tissue Processing

Animal procedures complied with the guidelines of the EU and were approved by the Local Ethical Committee. Mice strains were CD1 (Charles River Laboratories), Lgr5/LacZ-NeoR knockin (Morita et al., 2004), Lgr5-DTR knockin (Tian et al., 2011), Lgr4/Gpr48AGt (Leighton et al., 2001), Cnx43-KI-Cre-ER(T) (EMMA), Rosa26R-LacZ, Rosa26R-YFP, Lgr5-Cre-ERT2, and Axin2-lacZ (Jax mice). The day the vaginal plug was observed was considered as E0.5.

For lineage-tracing experiments, tamoxifen (Sigma-Aldrich) was dissolved in a sunflower oil (Sigma-Aldrich)/ethanol mixture (9:1) at 10 mg/ml and used in all experiments at a dose of 0.1 mg/g of body weight. For in utero induction, pregnant mothers were injected intraperitoneally at E15. When required, cesarean sections were performed for delivery, and newborn mice were nursed by adoptive lactating females. For neonatal induction, lactating mothers were injected intraperitoneally once a day for 4 consecutive days (from P5 to P8). The Rosa26R-LacZ background was used in all Lgr5-CreERT2 experiments. For the experiments using Cnx43-CreERT, the Rosa26R-LacZ background was used for in utero pulse + 1 and 6 dpp, and the Rosa26R-YFP context was used in the embryonic pulse + 3 dpp and + 2 weeks pp as well as in neonatal pulse + 2 dpp. The number of embryos analyzed for each time point were as follows: lineage tracing with Cnx43-Cre (n = 4, 4, 3, 2, and 2, for E15 + 1 dpp, 3 dpp, 6 dpp, 2 weeks pp and postnatal pulse, respectively); lineage tracing with Lgr5-Cre(n = 10,7,2, and 3, for E15 + 1 dpp, 3dpp, 2 weeks pp and postnatal pulse, respectively).

Tissue processing, histological protocols, and immunofluorescence/histochemistry experiments were carried out as previously described (Garcia et al., 2009). The primary antibodies used for staining were mouse anti-E-cadherin, rat anti-CD44, mouse antibromodeoxyuridine, all from BD Biosciences, goat anti-Villin (Santa Cruz Biotechnology), mouse antiserotonin and rabbit antilysozyme from Dako, goat anti-Trop2 (R&D Systems), rabbit anti-Cnx43 (Cell Signaling), rabbit anti-ZO-1 (Invitrogen), and chicken anti-p-gal and anti-YFP (Abcam). EdU staining (Invitrogen) and TUNEL assays (Roche) were performed according to the manufacturer's instructions. Samples were visualized with Zeiss Axioplan2 (immunohistochemistry) or Zeiss Observer Z1 microscope (immunofluorescence).

Ex Vivo Culture

Small intestinal tissue was dissociated, and epithelial samples were cultured as previously described (Mustata etal., 2011). Specifically, the culture medium was composed of Advanced-DMEM/F12 medium supplemented with 2 mM L-glutamine, gentamycin, penicillin-streptomycin cocktail, and 2% fetal bovine serum (Gibco). The only growth factors added to the culture medium were at a final concentration of 50 ng/ml EGF (PeproTech), 100 ng/ml Noggin (PeproTech), and 500 ng/ml R-spondin1 (R&D Systems). Culture medium was changed every other day, and, after 5-7 days in culture, spheroids and organo-ids pairs were harvested, mechanically dissociated, and replated in fresh Matrigel.

Diphteria toxin, IWP2, or DAPT compounds (all from Sigma-Aldrich) were added together with fresh medium. In experiments with Trop2+-sorted cells, 1 mM JAG-1 (Anaspec) was added to the Matrigel, and the culture medium was supplemented with 10 mM Y-27632 (Sigma). For Xgal staining experiments, ex vivo cultures were prefixed for 15 min at room temperature before proceeding to staining as described (Garcia et al., 2009). Pictures were acquired with a Moticam Pro camera connected to Motic AE31 microscope or with a Leica DFC 420C camera using the Leica Application Suite V3.8 software. For electron microscopy studies, spheroids and organoids cultured in Matrigel were layered onto a nitrocellulose filter, and samples were processed as described in Supplemental Experimental Procedures. Fluorescence-activated cell sorting (FACS) isdetailed in Supplemental Experimental Procedures.

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Microarray Experiments

Two-channel microarray experiments were performed from spheroid/orga-nold pairs isolated each from a given embryo. Specifically, spheroid/organoid pairs were obtained as reported in Figure 1A. Following initial seeding of small intestine from a given embryo/mouse (at E16, E18, or P0), spheroids and organoids were selectively picked up for each animal and replated for three passages to reach sample homogeneity. Hybridization was performed on the four independent pairs with dye-swap, on Mouse ReadyArray MM1100 slides (38,467 70-mer probes; MI-Microarrays), as described (Garcia et al., 2009). SAM analysis was performed using default parameters. A list of 1,982 upregulated and 1,276 downregulated genes was obtained. Unknown and duplicate genes were removed and genes modulated more than 2-fold with q value <0.054 were kept. The resulting short list of 317 upregulated and 179 downregulated genes is provided in Table S1.

Quantitative Real-Time PCR

Quantitative real-time PCR was performed on total RNA as reported (Garcia et al., 2009). Expression levels were normalized to that of the house keeping genes (HPRT, RPL13, and TBP). Each sample was run in duplicate. Primer sequences were previously reported (Mustata et al., 2011) or are listed in Supplemental Experimental Procedures.

Statistical Evaluation

Statistical analyses were performed with GraphPad Prism. All experimental data are expressed as mean ± SEM. The significance of differences between groups was determined by unpaired nonparametric (Mann-Whitney) test or paired t test analysis.

ACCESSION NUMBERS

Microarray data sets were deposited in the Gene Expression Omnibus under accession number GSE49803.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures, six figures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2013.09.005.

AUTHOR CONTRIBUTIONS

R.C.M., G. Vasile, V.F.-V., S.S., and M.-I.G. performed the majority of the experiments. A.F. and F.L. performed the microarray experiments and made the related statistical analyses. D.M. and D.P.-M. performed and analyzed the electron microscopy experiments. R.C.M., G.Vassart, V.F.-V., G. Vasile, and M.-I.G. conceived the experiments and analyzed the results. R.C.M., G. Vassart, and M.-I.G. wrote the paper.

ACKNOWLEDGMENTS

We are grateful to William C. Skarnes, Genentech, and Hans Clevers for providing us with Lgr4/Gpr48AGt, Lgr5-DTR, and Lgr5-LacZ-NeoR knockin mice, respectively. We thank Christine Dubois for assistance in cell sorting experiments and Cedric Blanpain, David Communi, and Pierre Vanderhaeghen for critical reading of the manuscript. This work was supported by the Interuni-versity Attraction Poles Programme-Belgian State-Belgian Science Policy (6/14), the Fonds de la Recherche Scientifique Médicale of Belgium, the Walloon Region (program "Cibles"), and the not-for-profit Association Recherche Biomédicale et Diagnostic. The CMMI is supported by the European Regional Development Fund and the Walloon Region.

Received: May 22, 2013 Revised: July 16, 2013 Accepted: September 4, 2013 Published: October 17, 2013

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