Wnt pathway induced interactions between intestinal crypt stem cells as well as colon cancer cells with their associated mesenchymal niche
Final Report Abstract
In 2007 the laboratory of Hans Clevers in Utrecht, Holland, published the identification of the intestinal stem cell by the marker gene Lgr5. Due to this finding, the focus of the lab shifted away from the intestinal stem cell niche towards the characterization of the intestinal stem cell itself. Being able to sort pure populations of intestinal stem cells allowed the definition of the intestinal stem cell transcriptome. This intestinal stem cell signature was next compared to the signatures derived from other adult stem cells first described by the Clevers lab, the Lgr6 positive stem cell in the hair follicle and the Lgr5 positive pyloric stem cell in the stomach. These signatures, especially their overlap, resulted in the definition of candidate genes for further study. The function of Lrig1, an inhibitor of EGFR signaling, in intestinal stem cells was studied in collaboration with Kim Jensen (Cambridge, UK). Together we could show that the Lrig1 knock‐out mouse has an enlarged stem cell pool in the crypt, which leads to the expansion of all other cell types in the crypt, i.e. transit amplifying and Paneth cells. This phenotype could be reverted by administering Iressa, a specific EGFR antagonist, to the mice. In conclusion, this study showed that negative feedback regulation of EGFR is an important mechanism in the intestine to control the number of stem cells. The gene Tnfrsf19 was found to be expressed in different adult stem cell signatures. We therefore decided to further study this gene and I generated a Tnfrsf19 reporter mouse, using the established GFP_IRES_CREERT2 knock‐in cassette. Indeed, Gfp expression was detected in several tissues in restricted cell populations, including the known stem cell population of the intestine. Crossing the Tnfrsf19 knock‐in mouse line with the Rosa26_Lox‐Stop‐Lox_LacZ line allowed us to perform lineage tracing. This technique uses the Cre enzyme of the knock‐in construct to delete the Lox‐Stop‐Lox roadblock, which leads to LacZ expression in the cell of initial deletion and all daughter cells derived from this cell. This allows to effectively prove stemness of a certain cell. Using lineage tracing, we could show that in addition to the small intestine, Tnfrsf19 marks stem cells in the stomach, liver and brain. Detailed analyses of these stem cell populations are currently performed. One other gene, the transcription factor Cdca7, was found to be expressed in intestinal stem cells. I could confirm the specific expression at the very bottom of crypts by RNA insitu. Due to this specific expression pattern and the interesting function, I generated a floxed Cdca7 allele with a GFP_IRES_CREERT2 cassette fused to the last exon. This mouse model allows for conditional deletion in a tissue specific way when crossed to appropriate CreERT2 lines. In addition, the Gfp fusion to the last exon visualizes the expression of Cdca7 and is at the same time a marker for the behavior of the targeted cells upon deletion of Cdca7. Furthermore, the CreERT2 enzyme can be used for lineage tracing experiments. I could detect as expected nuclear Gfp in intestinal stem cells, indicating that the knock‐in allele is functional. Deletion and lineage tracing studies are currently performed to elucidate the function of Cdca7 in the intestinal stem cell.
Publications
- OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells. Gastroenterology. 2009 Jul;137(1):15‐7
van der Flier LG, Haegebarth A, Stange DE, van de Wetering M, Clevers H
- Prominin‐1/CD133 marks stem cells and early progenitors in mouse small intestine. Gastroenterology. 2009 Jun;136(7):2187‐2194.e1
Snippert HJ, van Es JH, van den Born M, Begthel H, Stange DE, Barker N, Clevers H
- Single Lgr5 stem cells build crypt‐villus structures in vitro without a mesenchymal niche. Nature. 2009 May 14;459(7244):262‐5
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, van Es JH, Abo A, Kujala P, Peters PJ, Clevers H
- The ets‐domain transcription factor Spdef promotes maturation of goblet and paneth cells in the intestinal epithelium. Gastroenterology. 2009 Oct;137(4):1333‐45.e1‐3
Gregorieff A, Stange DE, Kujala P, Begthel H, van den Born M, Korving J, Peters PJ, Clevers H
- Transcription factor achaete scute‐like 2 controls intestinal stem cell fate. Cell. 2009 Mar 6;136(5):903‐12
van der Flier LG, van Gijn ME, Hatzis P, Kujala P, Haegebarth A, Stange DE, Begthel H, van den Born M, Guryev V, Oving I, van Es JH, Barker N, Peters PJ, van de Wetering M, Clevers H
- Expression of an ASCL2 related stem cell signature and IGF2 in colorectal cancer liver metastases with 11p15.5 gain. Gut. 2010 Sep;59(9):1236‐44
Stange DE, Engel F, Longerich T, Koo BK, Koch M, Delhomme N, Aigner M, Toedt G, Schirmacher P, Lichter P, Weitz J, Radlwimmer B
- Indian hedgehog regulates intestinal stem cell fate through epithelial‐mesenchymal interactions during development. Gastroenterology. 2010 Sep;139(3):893‐903
Kosinski C, Stange DE, Xu C, Chan AS, Ho C, Yuen ST, Mifflin RC, Powell DW, Clevers H, Leung SY, Chen X
- Lgr5+ve stem cells drive self‐renewal in the stomach and build long‐lived gastric units in vitro. Cell Stem Cell. 2010 Jan 8;6(1):25‐36
Barker N, Huch M, Kujala P, van de Wetering M, Snippert HJ, van Es JH, Sato T, Stange DE, Begthel H, van den Born M, Danenberg E, van den Brink S, Korving J, Abo A, Peters PJ, Wright N, Poulsom R, Clevers H
- Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science. 2010 Mar 12;327(5971):1385‐9
Snippert HJ, Haegebarth A, Kasper M, Jaks V, van Es JH, Barker N, van de Wetering M, van den Born M, Begthel H, Vries RG, Stange DE, Toftgård R, Clevers H
- Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature. 2011 Jan 20;469(7330):415‐8
Sato T, van Es JH, Snippert HJ, Stange DE, Vries RG, van den Born M, Barker N, Shroyer NF, van de Wetering M, Clevers H
