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

Abstract

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.

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.

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.

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.

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.

Figure 5

Expression profiling along the intestinal crypt axis. (A) GFP-positive cells derived from the small intestine of Lgr5-EGFP-ires-CreERT2 knock-in mice were sorted arbitrarily in five different fractions, ranging from lowest (1+) to highest (5+) GFP expression. (B–F) A gradient plot of the expression along the crypt was generated by plotting the log₂ ratio of the 5+ fraction versus the four lower fractions. (B) Gradient plots of the known CBC stem cell markers Lgr5 and Olfm4. (C–F) Gradient plots of the proposed quiescent/+4 marker genes Hopx, Lrig1, Bmi1 and Tert. Dotted lines are based on ratios from the arrays and continuous lines are based on ratios calculated from qPCR analyses. The ratios of Lgr5 are plotted in grey for comparison in (C–F).

Figure 6

Single molecule transcript counting of intestinal stem cell markers. The top images are representative crypts, the bottom figures show average expression profiles (patches are standard error of the means). At least 30 crypts were analysed. Numbers on the y axis denote the average number of transcripts per crypt cell.

Figure 7

Protein expression pattern of Bmi1 and Hopx. Immunohistochemistry was performed on small intestine of wild-type mice. Bmi1-stained cells at equal levels throughout the crypt. Control staining (insert i) was performed on small intestine of Bmi1-knockdown mice. A representative crypt is shown in insert (ii). Hopx showed nuclear staining in the bottom half of the crypt. A representative crypt is shown in the insert. Olfm4 expression was restricted to the crypt bottom.

Figure 8

Bmi1 marks individual cells irrespective of position along the crypt villus axis. (A–D) LacZ staining of Bmi1-CreER/R26R-LacZ mice 20 h after induction (p.i.). Positive cells were present at various positions along the crypt-villus axis. (E–H) Confocal imaging of Bmi1-CreER/R26R-Confetti mice 20 h p.i. Crypt outline is shown by a white dotted line. Bmi1-tracing cells are shown in yellow or red and bright field image is shown in grey. (I–J) 3D representations of (E) and (F). Bmi1+ cells are marked by Confetti-YFP (yellow), Paneth cells are visualized by lysozyme staining (purple). Crypt outline is shown by a white dotted line. (K) Quantification of the number of marked cells at each position along the crypt axis.

Figure 9

Functional classification of genes from the intestinal stem cell signature. Genes comprising the intestinal stem cell signature were functionally classified with PANTHER (www.pantherdb.org) by molecular function. Annotations were manually checked and, when applicable, re-assigned to a different category based on literature. The figure represents 279 genes for some of the most functionally relevant categories. The complete list containing all 510 genes enriched in stem cells can be found in Supplementary Table S12.