Intravital imaging of hair follicle regeneration in the mouse

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.

Spatiotemporal coordination of stem cell commitment during epidermal homeostasis

“…These results suggest a simple model for stem cell fate without hierarchy or division asymmetry (23) (Fig. 2, B and C; figs.…”

“…We performed this multigenerational analysis in the ear epidermis and detected no mother-daughter bias in fate choice [supplementary theory (ST) S5] or in their lifetimes (Pearson correlation R = −0.11, P = 0.2). Second, we tested whether asymmetric fate divisions are the main mode of self-renewal, as widely suggested from static lineage tracing (10, 11, 15, 23). Asymmetric divisions should result in anticorrelated sister cell fates, but we found that sister cell fates were either independent (ear) or positively correlated (paw).…”

“…Previous work has demonstrated that cells in the basal layer are not constrained to column boundaries, and it has been proposed that cells could differentiate through self-assembling processes (23, 27, 28). To interrogate the mechanism by which cells transit through suprabasal layers, we used a dual membrane fluorescent reporter to differentially label neighboring cells in the ear epidermis (fig.…”

Epithelial Skin Biology

“…An additional confounding problem for analyzing epidermal homeostasis and lineage tracing in the laboratory mouse is their behavioral traits of scratching and grooming, which can inadvertently affect the outcome. At first glance, it may seem that the elegant live-imaging studies recently performed on mouse ear skin (Pineda et al, 2015; Rompolas, Mesa, & Greco, 2013) will offer a better avenue for tracing clonal behavior. Here, however is the issue of compressing the ear between glass plates in order to capture the image.…”

Light‐Patterned RNA Interference of 3D‐Cultured Human Embryonic Stem Cells

“…We are currently applying this technology to generate an in vitro optic cup for retinal tissue growth by spatial control of Wnt signaling to pattern the differentiation of hESC to neural retina and retinal pigment epithelium. [11,40] Moreover, the two photon excitation approach can also be combined with in vivo imaging [41] to become a promising means for investigation of developmental processes in vivo.…”