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