Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Intravital imaging of hair follicle regeneration in the mouse

Nature Protocols volume 10, pages 1116–1130 (2015)Cite this article

Subjects

Abstract

Hair follicles are mammalian skin organs that periodically and stereotypically regenerate from a small pool of stem cells. Hence, hair follicles are a widely studied model for stem cell biology and regeneration. This protocol describes the use of two-photon laser-scanning microscopy (TPLSM) to study hair regeneration within a living, uninjured mouse. TPLSM provides advantages over conventional approaches, including enabling time-resolved imaging of single hair follicle stem cells. Thus, it is possible to capture behaviors including apoptosis, proliferation and migration, and to revisit the same cells for in vivo lineage tracing. In addition, a wide range of fluorescent reporter mouse lines facilitates TPLSM in the skin. This protocol also describes TPLSM laser ablation, which can spatiotemporally manipulate specific cellular populations of the hair follicle or microenvironment to test their regenerative contributions. The preparation time is variable depending on the goals of the experiment, but it generally takes 30–60 min. Imaging time is dependent on the goals of the experiment. Together, these components of TPLSM can be used to develop a comprehensive understanding of hair regeneration during homeostasis and injury.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The hair follicle regeneration cycle and anatomy.
Figure 2: Intravital two-photon imaging of the mouse hair follicle.
Figure 3: Pictorial and photographic schematics of mounting stage.
Figure 4: Labeling hair follicle stem cells with inducible Cre reporters.
Figure 5: Strategy for repeated imaging of the same skin region.
Figure 6: Laser ablation of hair follicle cells.
Figure 7: Schematic of the two-photon microscope system used in this protocol.

Similar content being viewed by others

References

  1. Morrison, S.J. & Spralding, A.C. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611 (2008).

    Article  CAS  Google Scholar 

  2. Rompolas, P. & Greco, V. Stem cell dynamics in the hair follicle niche. Sem. Cell Dev. Biol. 25–26, 34–42 (2014).

    Article  Google Scholar 

  3. Lo Celso, C. et al. Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nat. Lett. 457, 92–96 (2008).

    Article  Google Scholar 

  4. Xie, Y. et al. Detection of functional haematopoietic stem cell niche using real-time imaging. Nat. Lett. 457, 97–101 (2008).

    Article  Google Scholar 

  5. Yoshida, S., Sukeno, M. & Nabeshima, Y. A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317, 1722–1726 (2007).

    Article  CAS  Google Scholar 

  6. Uchugonova, A., Hoffman, R.M., Weinigel, M. & Koenig, K. Watching stem cells in the skin of living mice noninvasively. Cell Cycle 10, 2017–2020 (2011).

    Article  CAS  Google Scholar 

  7. Li, J.L., Goh, C.C., Keeble, J.L. & Win, J.S. et al. Intravital multiphoton imaging of immune responses in the mouse ear skin. Nat. Protoc. 7, 221–234 (2012).

    Article  CAS  Google Scholar 

  8. Fuchs, E. Finding one's niche in the skin. Cell Stem Cell 4, 499–502 (2009).

    Article  CAS  Google Scholar 

  9. Fuchs, E. The tortoise and the hair: slow-cycling cells in the stem cell race. Cell Rev. 137, 811–819 (2009).

    Article  CAS  Google Scholar 

  10. Cotsarelis, G. Epithelial stem cells: a folliculocentric view. J. Investig. Dermatol. 126, 1459–1468 (2006).

    Article  CAS  Google Scholar 

  11. Li, L. & Clevers, H. Coexistence of quiescence and active adult stem cells in mammals. Science 327, 542–545 (2010).

    Article  CAS  Google Scholar 

  12. Cotsarelis, G., Sun, R.R. & Lavker, R.M. Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61, 1329–1337 (1990).

    Article  CAS  Google Scholar 

  13. Jahoda, C.A., Horne, K.A. & Oliver, R.F. Induction of hair growth by implantation of cultured dermal papilla cells. Nature 311, 560–562 (1984).

    Article  CAS  Google Scholar 

  14. Muller-Rover, S. et al. A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages. J. Investig. Dermatol. 117, 3–15 (2001).

    Article  CAS  Google Scholar 

  15. Greco, V. et al. A two-step mechanism for stem cell activation during hair regeneration. Cell Stem Cell 4, 155–169 (2009).

    Article  CAS  Google Scholar 

  16. Plikus, M.V. et al. Cyclic dermal BMP signaling regulates stem cell activation during hair regeneration. Nat. Lett. 451, 340–344 (2008).

    Article  CAS  Google Scholar 

  17. Page, M.E., Lomard, P., Ng, F., Gottgens, B. & Jensen, K.B. The epidermis comprises autonomous compartments maintained by distinct stem cell populations. Cell Stem Cell 13, 471–482 (2013).

    Article  CAS  Google Scholar 

  18. Chen, X., Nadiarynkh, O., Plotnikov, S. & Campagnola, P.J. Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure. Nat. Protoc. 7, 654–659 (2012).

    Article  CAS  Google Scholar 

  19. Campagnola, P.J. & Loew, L.M. Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues, and organisms. Nat. Biotechnol. 21, 1356–1360 (2003).

    Article  CAS  Google Scholar 

  20. Tumbar, T. et al. Defining the epithelial stem cell niche in skin. Science 303, 359–363 (2004).

    Article  CAS  Google Scholar 

  21. Rendl, M., Lewis, L. & Fuchs, E. Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol. 3, 1910–1924 (2005).

    Article  CAS  Google Scholar 

  22. Kwan, K.M. Conditional alleles in mice: practical considerations for tissue-specific knockouts. Genesis 32, 49–62 (2002).

    Article  CAS  Google Scholar 

  23. Sauer, B. & Henderson, N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85, 5166–5170 (1988).

    Article  CAS  Google Scholar 

  24. Stemberg, N. & Hamilton, D. Bacteriophage P1 site-specific recombination. I. Recombination between loxP sites. J. Mol. Biol. 150, 467–486 (1981).

    Article  Google Scholar 

  25. Nagy, A. Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99–109 (2000).

    Article  CAS  Google Scholar 

  26. Feil, R. et al. Ligand-activated site-specific recombination in mice. Proc. Natl. Acad. Sci. USA 93, 10887–10890 (1996).

    Article  CAS  Google Scholar 

  27. Feil, R., Wagner, J., Metzger, D. & Chambon, P. Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem. Biophys. Res. Commun. 237, 752–757 (1997).

    Article  CAS  Google Scholar 

  28. Metzger, D., Clifford, J., Chiba, H. & Chambon, P. Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proc. Natl. Acad. Sci. USA 92, 6991–6995 (1995).

    Article  CAS  Google Scholar 

  29. Zhang, Y. et al. Inducible site-directed recombination in mouse embryonic stem cells. Nucleic Acids Res. 24, 543–548 (1996).

    Article  CAS  Google Scholar 

  30. Youssef, K.K. et al. Identification of the cell lineage at the origin of basal cell carcinoma. Nat. Cell Biol. 12, 299–305 (2010).

    Article  CAS  Google Scholar 

  31. Means, A.L., Xu, Y., Zhao, A., Ray, K.C. & Gu, G. A CK19 (CreERT) knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs. Genesis 46, 318–323 (2008).

    Article  CAS  Google Scholar 

  32. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nat. Cell Biol. 449, 1003–1007 (2007).

    CAS  Google Scholar 

  33. Rompolas, P., Mesa, K. & Greco, V. Spatial organization within a niche as a determinant of stem-cell fate. Nature 502, 513–518 (2013).

    Article  CAS  Google Scholar 

  34. Cox, G. et al. 3-dimensional imaging of collagen using second harmonic generation. J. Struct. Biol. 141, 53–62 (2003).

    Article  CAS  Google Scholar 

  35. Madisen, L., Zwingman, T.A. & Sunkin, S.M. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    Article  CAS  Google Scholar 

  36. Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).

    Article  CAS  Google Scholar 

  37. Dassule, H.R., Lewis, P., Bei, M., Maas, R & McMahon, AP. Sonic hedgehog regulates growth and morphogenesis of the tooth. Development 127, 4775–4785 (2000).

    CAS  Google Scholar 

  38. Vasioukhin, V., Degenstein, L., Wise, B. & Fuchs, E. The magical touch: genome targeting in epidermal stem cells induced by tamoxifen application to mouse skin. Proc. Natl. Acad. Sci. USA 96, 8551–8556 (1999).

    Article  CAS  Google Scholar 

  39. Snippert, H. et al. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327, 1385–1389 (2010).

    Article  CAS  Google Scholar 

  40. Vitale-Cross, L., Amornphimoltham, P., Fisher, G., Molinolo, A.A. & Gutkind, J.S. Conditional expression of K-ras in an epithelial compartment that includes the stem cells is sufficient to promote squamous cell carcinogenesis. Cancer Res. 64, 8804–8807 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by The New York Stem Cell Foundation and grants to V.G. by the American Cancer Society, grant no. RSG-12-059-02; Yale Spore Grant National Cancer Institute, US National Institutes of Health (NIH) grant no. 1P50CA121974; and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), NIH grant no. 1R01AR063663-01. C.M.P. is supported by the Human Genetics Training Grant (HGTG) through Yale University. S.P. is supported by the James Hudson Brown-Alexander Brown Coxe Postdoctoral Fellowship. K.R.M. is supported by the NIH Predoctoral Program in Cellular and Molecular Biology, grant no. 5T32 GM007223, and is currently a National Science Foundation (NSF) Graduate Research Fellow. A.M.H. is supported by NIAMS Rheumatic Diseases Research Core Centers, grant no. 5 P30 AR053495-07. P.R. is a New York Stem Cell Foundation Druckenmiller Fellow and is supported by the CT Stem Cell Grant 13-SCA-YALE-20. V.G. is a New York Stem Cell Foundation Robertson Investigator. The authors gratefully acknowledge the laboratory of E. Fuchs (Rockefeller University) for providing the K14H2BGFP mice.

Author information

Author notes

  1. Panteleimon Rompolas

    Present address: Present address: Department of Dermatology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania, USA.,

  2. Cristiana M Pineda and Sangbum Park: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Cell Biology, and Dermatology, Yale Stem Cell Center, Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, USA

    Cristiana M Pineda, Sangbum Park, Kailin R Mesa, Markus Wolfel, Panteleimon Rompolas & Valentina Greco

  2. Department of Laboratory Medicine, Yale School of Medicine, New Haven, Connecticut, USA

    David G Gonzalez & Ann M Haberman

  3. Department of Cell Biology, Yale School of Medicine, New Haven, Connecticut, USA.,

    Valentina Greco

  4. Department of Dermatology, Yale School of Medicine, New Haven, Connecticut, USA.,

    Valentina Greco

  5. Yale Stem Cell Center, Yale School of Medicine, New Haven, Connecticut, USA.,

    Valentina Greco

  6. Yale Cancer Center, Yale School of Medicine, New Haven, Connecticut, USA.,

    Valentina Greco

Authors
  1. Cristiana M Pineda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sangbum Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kailin R Mesa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Markus Wolfel
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. David G Gonzalez
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ann M Haberman
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Panteleimon Rompolas
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Valentina Greco
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.M.P., S.P. and P.R. wrote the manuscript and prepared the figures. K.R.M. conducted experiments for figures. M.W., D.G.G. and A.M.H. helped develop and troubleshoot the TPLSM technique. V.G. oversaw and assisted all aspects of the protocol.

Corresponding authors

Correspondence to Panteleimon Rompolas or Valentina Greco.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Hair follicle regeneration captured in vivo

Serial optical sections of a hair follicle in early growth as seen using a K14H2BGFP reporter. Collagen-rich extracellular matrix can be seen through second harmonic regeneration (SHG; blue channel). See also Figure 2. (MOV 2102 kb)

Cell proliferation captured during hair follicle growth.

Time-resolved intravital imaging demonstrating a magnified view of the lower portion of the hair follicle during the growth (anagen) phase of the hair cycle where epithelial cell migration can be observed using a K14H2BGFP reporter. (MOV 2185 kb)

Cell migration captured during hair follicle growth.

Time-resolved intravital imaging of epithelial cell migration as seen by nuclear displacement using a K14H2BGFP reporter. (MOV 2319 kb)

Cell death captured during hair follicle regression.

Time-resolved intravital imaging demonstrating apoptosis of epithelial cells as seen by nuclear fragmentation using a K14H2BGFP reporter; occurring during the regression (catagen) phase of the hair cycle. (MOV 318 kb)

Laser ablation of the hair follicle mesenchymal niche.

Serial optical sections of a hair follicle before and after laser ablation of the mesenchymal dermal papilla niche as seen using a mesenchymal (Lef1RFP; red channel) and epithelial (K14H2BGFP; green channel) reporter. Collagen-rich extracellular matrix can be seen through second harmonic regeneration (SHG; blue channel). Note the autofluorescent “halo” surrounding the ablation site. See also Figure 6. (MOV 5362 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pineda, C., Park, S., Mesa, K. et al. Intravital imaging of hair follicle regeneration in the mouse. Nat Protoc 10, 1116–1130 (2015). https://doi.org/10.1038/nprot.2015.070

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2015.070

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing