The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent ‘+4’ cell markers

Article12 June 2012free access The Lgr5 intestinal stem cell signature: robust expression of proposed quiescent ‘+4’ cell markers Javier Muñoz Javier Muñoz Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands Search for more papers by this author Daniel E Stange Daniel E Stange Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Arnout G Schepers Arnout G Schepers Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Marc van de Wetering Marc van de Wetering Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Shalev Itzkovitz Shalev Itzkovitz Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Richard Volckmann Richard Volckmann Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Kevin S Kung Kevin S Kung Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Jan Koster Jan Koster Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Sorina Radulescu Sorina Radulescu The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Kevin Myant Kevin Myant The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Rogier Versteeg Rogier Versteeg Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Owen J Sansom Owen J Sansom The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Johan H van Es Johan H van Es Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Nick Barker Nick Barker Institute of Medical Biology, Singapore, Singapore Search for more papers by this author Alexander van Oudenaarden Alexander van Oudenaarden Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Shabaz Mohammed Shabaz Mohammed Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands Search for more papers by this author Albert J R Heck Corresponding Author Albert J R Heck Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands Centre for Biomedical Genetics, Universiteitsweg 100, Utrecht, The Netherlands Search for more papers by this author Hans Clevers Corresponding Author Hans Clevers Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Centre for Biomedical Genetics, Universiteitsweg 100, Utrecht, The Netherlands Search for more papers by this author Javier Muñoz Javier Muñoz Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands Search for more papers by this author Daniel E Stange Daniel E Stange Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Arnout G Schepers Arnout G Schepers Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Marc van de Wetering Marc van de Wetering Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Bon-Kyoung Koo Bon-Kyoung Koo Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Shalev Itzkovitz Shalev Itzkovitz Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Richard Volckmann Richard Volckmann Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Kevin S Kung Kevin S Kung Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Jan Koster Jan Koster Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Sorina Radulescu Sorina Radulescu The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Kevin Myant Kevin Myant The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Rogier Versteeg Rogier Versteeg Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Search for more papers by this author Owen J Sansom Owen J Sansom The Beatson Institute for Cancer Research, Glasgow, UK Search for more papers by this author Johan H van Es Johan H van Es Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Search for more papers by this author Nick Barker Nick Barker Institute of Medical Biology, Singapore, Singapore Search for more papers by this author Alexander van Oudenaarden Alexander van Oudenaarden Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Shabaz Mohammed Shabaz Mohammed Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands Search for more papers by this author Albert J R Heck Corresponding Author Albert J R Heck Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands Centre for Biomedical Genetics, Universiteitsweg 100, Utrecht, The Netherlands Search for more papers by this author Hans Clevers Corresponding Author Hans Clevers Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Centre for Biomedical Genetics, Universiteitsweg 100, Utrecht, The Netherlands Search for more papers by this author Author Information Javier Muñoz1,2,‡, Daniel E Stange3,‡, Arnout G Schepers3,‡, Marc van de Wetering3, Bon-Kyoung Koo3, Shalev Itzkovitz4, Richard Volckmann5, Kevin S Kung4, Jan Koster5, Sorina Radulescu6, Kevin Myant6, Rogier Versteeg5, Owen J Sansom6, Johan H van Es3, Nick Barker7, Alexander van Oudenaarden3,4, Shabaz Mohammed1,2, Albert J R Heck 1,2,8 and Hans Clevers 3,8 1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands 2Netherlands Proteomics Center, Utrecht, The Netherlands 3Hubrecht Institute, KNAW and University Medical Center Utrecht, Utrecht, The Netherlands 4Department of Physics, Massachusetts Institute of Technology, Cambridge, MA, USA 5Department of Oncogenomics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 6The Beatson Institute for Cancer Research, Glasgow, UK 7Institute of Medical Biology, Singapore, Singapore 8Centre for Biomedical Genetics, Universiteitsweg 100, Utrecht, The Netherlands ‡These authors contributed equally to this work *Corresponding authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.:+31 30 253 5871; Fax:+31 30 253 6919; E-mail: [email protected] Institute, KNAW and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. Tel.:+31 30 212 1800; Fax:+31 30 251 6464; E-mail: [email protected] The EMBO Journal (2012)31:3079-3091https://doi.org/10.1038/emboj.2012.166 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 Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent ‘+4’ cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5+ CBC cells. Transcriptional profiling of FACS-sorted Lgr5+ stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell-enriched genes was defined. Spatial expression patterns were further characterized by mRNA in-situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5+stem cells. Lineage tracing using a newly established knock-in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find—and confirm by independent approaches—that the proposed quiescent/‘+4’ stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells. Introduction The epithelium of the small intestine represents a prototypic example of a mammalian stem cell-driven self-renewing tissue. The epithelium consists of luminal protrusions called villi, and pit-like recessions called crypts. A small number of stem cells reside at crypt bottoms. Daughter cells exit the stem cell compartment into the transit amplifying (TA) compartment. TA cells go through 4–5 divisions of unusually short duration, that is, 12 h (Marshman et al, 2002). During this process, the TA cells move towards the crypt-villus junction and differentiate into enterocytes, goblet cells and enteroendocrine cells. These differentiated cells continue to move upwards towards the tip of the villus. Upon reaching the villus tip after 2–3 more days, the differentiated cells undergo apoptosis and are shed into the gut lumen. A fourth cell type, the Paneth cell, also derives from the stem cells, but migrates towards the crypt bottom where it resides for 6–8 weeks (Bjerknes and Cheng, 2006). Recently, we have described that small cycling cells located between the Paneth cells, previously termed as crypt base columnar (CBC) cells (Cheng and Leblond, 1974), specifically express the Lgr5 gene (Barker et al, 2007). By lineage tracing, we demonstrated that these Lgr5+ cells generate all cell lineages of the small intestinal epithelium over the lifetime of the animal. Similar data were published utilizing a Prom1/Cd133-based lineage tracing strategy (Zhu et al, 2009). Deletion of adenomatous polyposis coli (Apc), the first hit in colorectal cancer initiation, leads to adenoma formation specifically in Lgr5+ stem cells and thus these cells can be regarded as the cell-of-origin for intestinal cancer (Barker et al, 2009). As further proof of their stem cell identity, we demonstrated that single Lgr5+ cells can be cultured into ever-growing epithelial organoids, which possess all characteristics of the epithelial tissue in the living animal (Sato et al, 2009). In the colon, hair follicle and stomach, Lgr5 also marks stem cells (Barker et al, 2007, 2010; Jaks et al, 2008). Clonal Lgr5-derived colon organoids can be grafted into recipient mice to yield functionally normal epithelium that persists for >6 months (Yui et al, 2012). The related Lgr6 gene is expressed by a population of multipotent skin stem cells (Snippert et al, 2010b). Potten et al (1974) have previously postulated that a cycling, yet DNA label-retaining cell residing at position +4 relative to the crypt bottom represents a stem cell population. Sangiorgi and Capecchi (2008) have employed lineage tracing based on Bmi1 expression, which reportedly occurred specifically in +4 cells. Long-term lineage tracing was observed with kinetics that were similar to the kinetics obtained in the Lgr5-based tracing experiments. Contrasting with the previous report, we observed that Lgr5+ cells express the highest levels of Bmi1 as determined by cell sorting and qPCR analysis (van der Flier et al, 2009a). Furthermore, single molecule mRNA in-situ hybridization revealed that the Bmi1 transcripts are expressed throughout the entire crypt (Itzkovitz et al, 2011). This broad expression pattern of Bmi1 was also observed in a recent publication analysing in detail the starting position of lineage tracing from the Bmi1 locus (Tian et al, 2011). Three other markers are proposed more recently for the quiescent ‘+4’ cell: Hopx (Takeda et al, 2011), Tert (Montgomery et al, 2011) and Lrig1 (Powell et al, 2012). In an independent study, Lrig1 was found to be expressed highest in CBC cells (Wong et al, 2012). Together, these studies suggest that Lgr5+ cells appear to be the ‘workhorse stem cells’ fuelling the daily self-renewal of the small intestine, while a pool of quiescent ‘reserve’ Lgr5 negative (Lgr5−) stem cells may exist above the crypt base (Li and Clevers, 2010). However, based on the discrepant studies on marker gene expression, it appears of paramount importance to obtain detailed molecular signatures for the two stem cell types before definitive conclusions can be drawn. The availability of a knock-in mouse expressing GFP from the Lgr5 locus allows the isolation of CBC stem cells from the intestine (Barker et al, 2007), providing a unique entry to understand ‘stemness’ (Vogel, 2003) and the in vivo differentiation process of this tissue (Simons and Clevers, 2011). Therefore, we have characterized transcriptomic and proteomic differences between Lgr5+ stem cells and their daughter cells enabling us to define a definitive Lgr5 intestinal stem cell (ISC) signature. Results Transcriptomic profile of Lgr5+ stem cells Transcriptional differences between ISCs and their daughter cells can be explored by use of the Lgr5-EGFP-ires-CreERT2 knock-in (Lgr5-ki) mouse (Supplementary Figure S1A; Barker et al, 2007). In this mouse model, GFP expression is driven from the Lgr5 locus, leading to highest GFP levels in Lgr5+ cells (GFPhigh). Yet, due to the stability of the GFP protein, it is distributed upon cell division to the daughter cells, which form a clearly distinguishable daughter cell population (GFPlow). Previously, we performed a gene expression analysis of intestinal Lgr5+ stem cells, which led to the identification of the transcription factor Ascl2 as a regulator of ISC fate (van der Flier et al, 2009a). Since then, we have systematically optimized the workflow for Lgr5+ cell sorting, resulting in a better separation of different GFP cell fractions and shorter isolation time, minimizing sample manipulation and, ultimately, leading to better RNA quality for transcriptional profiling. Here, two independent microarray platforms (Affymetrix and Agilent) were used to compare ISCs and their daughters (Supplementary Figure S1B). These two expression array platforms were chosen for their distinct configurations (two colours versus one colour) and their ability to complement each other (Patterson et al, 2006). A comparison to our previously published Agilent data set revealed that the average intensity of established stem cell genes (e.g., Lgr5, Ascl2, Olfm4 and Tnfrsf19) in the GFPhigh fraction increased by seven-fold upon using the improved FACS sorting procedure, resulting in a five-fold increase (56 versus 274) in the number of identifiable stem cell-enriched genes. For the Affymetrix platform, ratios for all 20 819 unique genes represented on the arrays were calculated (Figure 1A). Using a combination of statistical significance and fold change (see Materials and methods), a set of 379 stem cell-enriched genes was defined (Figure 1A; Supplementary Table S1). For Agilent arrays, 13 967 unique genes were consistently expressed above background (Figure 1B) from which 291 genes were found to be expressed significantly higher in the stem cells (Figure 1B; Supplementary Table S2). Comparing the two platforms, we found an overall good correlation of r=0.85 (Figure 1C). Nevertheless, a strikingly low overlap (161 genes) was observed when the stem cell signatures of the two platforms were compared (Figure 1D). The subsequent inspection of the non-overlapping genes revealed that a substantial fraction (59 and 164 genes, respectively) was enriched >1.5-fold, although not significantly, in the other platform (Figure 1D). Combining the genes that were significant in both platforms with the genes significant in one and enriched >1.5-fold in the other platform, we could define a high-confidence list of 384 stem cell-enriched transcripts (referred to as the ‘mRNA stem cell signature’; Supplementary Table S3). From the genes that remained to be defined by only one of the two microarray platforms, 72% (51/71) and 57% (31/54) showed a low enrichment (log2 of >0.2 and <0.58) on the other platform (Supplementary Tables S4 and S5). Thus, although the two platforms show a high level of concordance, the necessity to define thresholds for the definition of significantly changed genes is the reason for missing a substantial number of stem cell-enriched genes. Our results demonstrate that Agilent and Affymetrix platforms complement each other and may be used in parallel if a high level of comprehensiveness is desired. Figure 1.The mRNA stem cell signature. (A) Expression levels for 20 819 unique genes were detected with Affymetrix, from which 379 were found to be statistically significant and >2-fold enriched in the stem cells. (B) Likewise, 291 out of 13 697 genes were found significantly enriched in stem cells with Agilent. (C) Correlation plot of both transcriptomic data sets. Well-known intestinal stem cell markers are annotated. (D) Overlap between the significant genes found by each platform. Download figure Download PowerPoint Proteomic profile of Lgr5+ stem cells mRNA levels do not always reflect the abundance of the translated protein (Schwanhausser et al, 2011). Therefore, examination of the actual protein content might give further insight into the molecular stem cell signature. We applied a mass spectrometry (MS)-based proteomics approach to study the protein content of Lgr5+ cells and their daughter cells (Supplementary Figure S1C), confidently identifying 7967 unique protein groups (Supplementary Figure S2; Supplementary Tables S6 and S7). Among them, we obtained an excellent representation of proteins that are known to be expressed at a low-copy number in mammalian cells including 648 transcription factors, 276 protein kinases and 248 signalling molecules. Of note, Lgr5 itself was not identified. The identification of plasma membrane proteins by MS is challenging due to insolubility in standard proteomic sample preparations. Nevertheless, our data set contains 1278 proteins with predicted trans-membrane domains (Krogh et al, 2001), and Gene Ontology analyses detected no underrepresentation of this protein class (plasma membrane; P>0.05). However, Lgr5 encodes a 7-transmembrane (7-TM) receptor expressed at low levels (van der Flier et al, 2009b). Both the high hydrophobicity and low expression probably contributed to its absence in our MS survey. Indeed, Gene Ontology analyses showed a clear underrepresentation of 7-TM proteins in our data set (G-protein coupled receptors; P=8.4E–54). For 92% of the identified proteins, mRNA expression was confirmed in the transcriptomic profiling, demonstrating the confidence of our proteomic data set. Most importantly, 4817 unique proteins were quantified in common in two biological replicates (6075 in total) (Figure 2A; Supplementary Figures S3 and S4; Supplementary Table S8). In all, 278 were found to be enriched >1.5-fold (consistently in both replicates) in stem cells (referred to as the ‘protein stem cell signature’; Supplementary Table S9). Our proteomic data confirmed several proteins previously described to be specific for the Lgr5+ stem cells, such as Ascl2 (van der Flier et al, 2009a), Olfm4 (van der Flier et al, 2009b), Sox9 (Bastide et al, 2007) and Msi1 (Kayahara et al, 2003; Potten et al, 2003) (Figure 2A). Figure 2.Proteomic analysis of Lgr5+ cells and the intestinal stem cell signature. (A) The protein stem cell signature. In all, 4817 proteins were quantified in two independent experiments (Supplementary Table S7). The average ratios (log2) are plotted against protein abundance (log10). The number of peptides used for the quantification as well as the variability (calculated as the relative standard deviation of the peptide ratios) is represented in the plot by the spot size and colour scale, respectively. The histogram of frequencies shows the protein densities per bin (size of 0.5). Using a cutoff of >1.5-fold (±0.58 in log2) in both biological replicas, 278 proteins were found to be more abundant in the stem cells. (B) The intestinal stem cell signature. For each method, a list of significantly changed genes (mRNAs or proteins) was established. Genes significant in one method, but not detected or not found enriched in any other method are highlighted in green. Genes that were found significant in one method and could be confirmed by one or both other methods are highlighted in blue and together constitute the intestinal stem cell signature. Download figure Download PowerPoint Complementary transcriptomic and proteomic profiling define the ISC signature Having established both the mRNA and protein signatures of ISCs, we next asked if post-transcriptional regulation might play an important role in regulating specific protein levels. The overall correlation between the mRNA and protein data was high (r=0.78 for Agilent and r=0.80 for Affymetrix; Supplementary Figure S5). This result, besides authenticating both the mRNA and the proteomic measurements, suggests that the ISC phenotype as well as the early differentiation process is strongly regulated at the transcriptional level. Of the 278 proteins in the ‘protein stem cell signature’, 72 were found in the ‘mRNA stem cell signature’ (Figure 2B). Additionally, 147 proteins were >1.5-fold enriched in either both or one array platform and were added to the combined signature (Figure 2B). Nevertheless, some genes were found enriched at the mRNA level, but not at the protein level and vice versa. For 27 genes of the ‘mRNA stem cell signature’, no enrichment was found in the Lgr5+ stem cells although the protein product was detected by MS (Supplementary Table S10). As proteins are the main mediators of biological functions, these genes are unlikely to play a specific biological role in stem cells and were therefore subtracted from the signature. Conversely, no array platform could detect a significant enrichment on the mRNA level for 59 proteins within the ‘protein stem cell signature’ (Figure 2B). Nevertheless, 78% (46/59) of these proteins were enriched (yet below the significance level) in at least one of the array platforms (Supplementary Table S11). For only five proteins, no enrichment on mRNA level was found. Therefore, post-transcriptional regulation did not appear to represent a major mechanism regulating protein levels of ISC-related genes. Nevertheless, the MS data allowed us to define a set of 147 proteins, which could be added to the signature due to their consistent enrichment in Lgr5+ cells on both protein and mRNA levels. As a result of the combined proteomic and transcriptomic profiling, we were able to define a set of 510 genes with stem cell-enriched expression, which we termed the ‘intestinal stem cell signature’ (Figure 2B; Supplementary Table S12). Expression pattern of novel Lgr5 stem cell-enriched genes within the intestinal crypt Subsequently, we investigated the spatial expression pattern of genes enriched in the intestinal stem cell. Enriched genes may exhibit different expression patterns, that is, they may be entirely CBC restricted or display a more extensive gradient within the crypt (Supplementary Figure S1D). We attempted to perform RNA in-situ hybridization for the 33 genes found in all three data sets (Figure 2B). Of these, no or non-specific staining was obtained for 11 genes, most likely reflecting low-level expression of the pertinent gene. The expression pattern of five genes was already known (Ascl2, Cd44, Msi1, Olfm4 and Sox9). From the remaining 17 genes, 9 showed a gradient within the crypt with highest expression at the crypt bottom (Afap1l1, Agr3, Cnn3, Dach1, Slc12a2, Slco3a1, Sorbs2, Tns3 and Vdr; Supplementary Figure S6). Finally, for eight genes an expression pattern restricted to the very bottom of the crypt in the stem cell zone was observed (Aqp4, Cdca7, Cdk6, Clca4, Kcnq1, Nav1, Smoc2 and Soat1; Figure 3). Thus, these results confirmed the findings derived from the transcriptomic and proteomic screenings and provided additional information on the specificity for Lgr5 stem cells. Figure 3.RNA in-situ hybridization screen. An mRNA in-situ hybridization screen was performed for the 33 signature genes in the central overlap (Figure 2B) to explore their spatial expression pattern. A specific expression signal at the very bottom of intestinal crypts in the stem cell zone was detected for eight genes. Download figure Download PowerPoint Smoc2 is expressed specifically in ISCs in vivo To validate the list, we studied one of the novel CBC marker genes, Smoc2, in more detail. The Xenopus laevis orthologue of Smoc1/2 has been described as a BMP signalling inhibitor (Thomas et al, 2009). BMP signalling is active in the intestinal villus compartment where it inhibits de-novo crypt formation (Haramis et al, 2004), and its inhibition by Noggin is essential to maintain intestinal organoid cultures (Sato et al, 2009). As Noggin is not expressed in the intestinal epithelium, Smoc2 expression by ISCs might be a physiological way to block BMP signalling in the stem cell niche. To confirm the stem cell-specific expression of Smoc2, we generated a Smoc2-EGFP-ires-CreERT2 knock-in (Smoc2-ki) mouse model in analogy to the Lgr5-ki (Figure 4A). Homozygous Smoc2-ki mice, constituting functional Smoc2 null mice, did not show any intestinal nor gross non-intestinal phenotype. As expected, GFP expression was detected in CBC cells (Figure 4B). Similarly to the Lgr5-ki, variegated expression of the transgene was detected throughout the small intestine. Lineage tracing performed in Smoc2-ki mice crossed with the R26R-LacZ Cre reporter strain resulted in typical stem cell tracings events: long-lived ‘ribbons’ spanning the entire crypt-villus axis (Figure 4C). This new stem cell-specific mouse model validated the usefulness of the Lgr5 signature for defining stem cell-related genes in vivo. Figure 4.Smoc2 marks intestinal stem cells in vivo. (A) An EGFP-ires-CreERT2 cassette was inserted at the translational start site of Smoc2 by homologous recombination, followed by excision of the Neo cassette by Cre mediated recombination. (B) Endogenous GFP expression was readily detectable in crypt base columnar cells, the Lgr5+ stem cells of the small intestine. Of note, the expression of GFP was patchy as in the Lgr5-ki mouse, indicating a silencing in the majority of crypts. (C) Lineage tracing in Smoc2-EGFP-ires-CreERT2/R26RLacZ mice showed long-term labelling (>6 month) of intestinal stem cells and revealed typical intestinal stem cell tracing events. Download figure Download PowerPoint Expression pattern of proposed quiescent ‘+4’ marker genes in the intestinal crypt We then interrogated the Lgr5 stem cell signature for the expression behaviour of the quiescent/‘+4’ stem cell markers mentioned above, that is, Bmi1, Tert, Hopx and Lrig1. Of note, all these markers were validated in the initial studies by genetic lineage tracing (Sangiorgi and Capecchi, 2008; Montgomery et al, 2011; Takeda et al, 2011; Powell et al, 2012). Both array platforms detected a slight enrichment of Bmi1 (1.4-fold in Affymetrix and 1.6-fold in Agilent), Tert (1.4-fold and 1.3-fold) as well as of Hopx (1.6-fold and 1.7-fold) in Lgr5+ stem cells. Lrig1 showed a >2-fold enrichment in Lgr5+ stem cells (3.2-fold 2.3-fold). Proteomics did not detect protein expression of Bmi1 and Tert, probably due to their low expression levels. The highly expres