Lineage Tracing in the Intestinal Epithelium

Nick Barker1, Hans Clevers1

1 Hubrecht Institute for Developmental Biology and Stem Cell Research, and University Medical Center Utrecht (UMCU), Utrecht, The Netherlands
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 5A.4
DOI:  10.1002/9780470151808.sc05a04s13
Online Posting Date:  May, 2010
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Abstract

This unit describes the theory and detailed protocols for performing in vivo lineage tracing from Lgr5+ve intestinal stem cells using an Lgr5‐EGFP‐ires‐CreERT2/Rosa26lacZ mouse model. Lineage tracing can be initiated in mice at any age by administering limiting doses of the hormone tamoxifen. This activates the lacZ reporter gene in the Lgr5+ve stem cells, which subsequently transmit this permanent genetic mark to their progeny as they repopulate the epithelium during normal homeostasis. Because the Lgr5+ve cells are long‐lived, self‐renewing stem cells, they continuously generate lacZ progeny, which contribute to tissue renewal over the entire lifetime of the mouse. The same protocols can be applied to performing in vivo lineage tracing from other Lgr5+ve stem cell populations, including those in the hair‐follicle and stomach. Curr. Protoc. Stem Cell Biol. 13:5A.4.1‐5A.4.11. © 2010 by John Wiley & Sons, Inc.

Keywords: Lgr5; in vivo lineage tracing; stem cell; intestine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Fixation of the Intestine
  • Support Protocol 1: Tamoxifen‐Induction of In Vivo Lineage Tracing
  • Basic Protocol 2: β‐Galactosidase (lacZ) Staining to Visualize Intestinal Stem Cells
  • Basic Protocol 3: Whole‐Mount Analysis of lacZ Staining in the Intestine
  • Basic Protocol 4: Detailed Analysis of lacZ Staining on Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Fixation of the Intestine

  Materials
  • Tamoxifen‐treated Lgr5‐ires‐CreERT2/Rosa26‐lacZ mice (see protocol 2)
  • Gluteraldehyde lacZ fixative (see recipe) or paraformaldehyde lacZ fixative (see recipe)
  • Phosphate‐buffered saline lacking Ca2+ and Mg2+ (CMF‐PBS)
  • 3‐ml syringes and 21‐G needles
  • 50‐ml centrifuge tube
  • Rolling platform

Support Protocol 1: Tamoxifen‐Induction of In Vivo Lineage Tracing

  Materials
  • Tamoxifen powder (Sigma, cat. no. T‐5648), stored at least 1 year at 4°C
  • 100% ethanol
  • Sunflower oil (supermarket variety)
  • Adult Lgr5‐iresCreERT2/Rosa26‐lacZ mice (6 to 8 weeks old, ∼25 g; Jackson Laboratory)
  • 37°C incubator
  • 1‐ml syringe and 25‐G needle (BD Microlance)

Basic Protocol 2: β‐Galactosidase (lacZ) Staining to Visualize Intestinal Stem Cells

  Materials
  • Fixed, freshly isolated intestines from tamoxifen‐treated mice (see protocol 1)
  • Equilibration buffer (see recipe)
  • β‐galactosidase (lacZ) substrate (see recipe)
  • Phosphate‐buffered saline lacking Ca2+ and Mg2+ (CMF‐PBS)
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • Rolling platform
  • 50‐ml centrifuge tubes

Basic Protocol 3: Whole‐Mount Analysis of lacZ Staining in the Intestine

  Materials
  • Fixed and stained intestinal sections from tamoxifen‐treated mice (see protocol 1)
  • Phosphate‐buffered saline lacking Ca2+ and Mg2+ (CMF‐PBS)
  • 4% low‐melting‐point agarose (Invitrogen, cat. no. 16520‐100), pre‐warmed to 40°C
  • Glue (Bison, http://www.bison.nl, cat. no. Bi2058)
  • Dissection pins
  • Cardboard
  • Petri dishes
  • Stereo microscope (e.g., Olympus SZX9) linked to a digital camera
  • Plastic basemolds (Klinipath, cat. no. 3051‐P)
  • Scalpel
  • Vibratome (Microm model HM650V)
  • Vibratome knives (Gillette, cat. no. 10)
  • Starfrost microscope slides
  • Coverslips (Menzel‐Gläser)

Basic Protocol 4: Detailed Analysis of lacZ Staining on Tissue Sections

  Materials
  • Intestinal tissue (see protocol 1)
  • Tissue dehydration solutions: 70%, 80%, 96%, and 100% ethanol
  • n‐Butanol (Baker, cat. no. 8017)
  • Liquid paraffin (60°C)
  • De‐wax solvent (xylene; Klinipath, cat. no. 4055‐9005)
  • Tissue rehydration solutions: 100%, 96%, 90%, 80%, 70%, 60%, 50%, and 25% ethanol
  • 0.1% (w/v) Neutral Red in ddH 2O
  • Pertex mounting medium (Histolab)
  • Tissue cassettes (Klinipath)
  • Metal molds on an embedding station
  • Heated forceps
  • Cold plate (−12°C)
  • Microtome
  • 40°C water bath
  • Starfrost microscope slides
  • Hot‐plate (∼55°C)
  • Slide racks (Klinipath)
  • Coverslips (Menzel‐Gläser)
  • Digital camera connected to a standard light microscope
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Figures

Videos

Literature Cited

Literature Cited
   Barker, N. and Clevers, H. 2007. Tracking down the stem cells of the intestine: Strategies to identify adult stem cells. Gastroenterology 133:1755‐760.
   Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., and Clevers, H. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003‐1007.
   Barker, N., van de Wetering, M., and Clevers, H. 2008. The intestinal stem cell. Genes Dev. 22:1856‐1864.
   Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H.J., van Es, J.H., Sato, T., Stange, D.E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P.J., Wright, N., Poulsom, R., and Clevers, H. 2010. Lgr5(+ve) stem cells drive self‐renewal in the stomach and build long‐lived gastric units in vitro. Cell Stem Cell 6:25‐36.
   Bjerknes, M. and Cheng, H. 1981. The stem cell zone of the small intestinal epithelium III. Evidence from columnar, enteroendocrine, and mucosal cells in the adult mouse. Am. J. Anat. 160:77‐91.
   Bjerknes, M. and Cheng, H. 1999. Clonal analysis of intestinal epithelial progenitors. Gastroenterology 116:7‐14.
   Cheng, H. and Leblond, C.P. 1974. Origin, differentiation, and renewal of the four epithelial cell types in the mouse small intestine. V. Unitarian theory of the origin of the four epithelial cell types. Am. J. Anat. 141:537‐561.
   Jaks, V., Barker, N., Kasper, M., van Es, J.H., Snippert, H.J., Clevers, H., and Toftgard, R. 2008. Lgr5 marks cycling, yet long‐lived, hair follicle stem cells. Nat. Genet. 40:1291‐1299.
   Sancho, E., Batlle, E., and Clevers, H. 2004. Signaling pathways in intestinal development and cancer. Annu. Rev. Cell Dev. Biol. 20:695‐723.
   Soriano, P. 1999. Generalized lac‐Z expression with the ROSA26 Cre expression strain. Nat. Genet. 21:70‐71.
   Stappenbeck, T.S., Mills, J.C., and Gordon, J.I. 2003. Molecular features of adult mouse small intestinal epithelial progenitors. Proc. Natl. Acad. Sci. U.S.A. 100:1004‐1009.
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