Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration

Abstract

Tissue development and regeneration depend on cell-cell interactions and signals that target stem cells and their immediate progeny. However, the cellular behaviours that lead to a properly regenerated tissue are not well understood. Using a new, non-invasive, intravital two-photon imaging approach we study physiological hair-follicle regeneration over time in live mice. By these means we have monitored the behaviour of epithelial stem cells and their progeny during physiological hair regeneration and addressed how the mesenchyme influences their behaviour. Consistent with earlier studies, stem cells are quiescent during the initial stages of hair regeneration, whereas the progeny are more actively dividing. Moreover, stem cell progeny divisions are spatially organized within follicles. In addition to cell divisions, coordinated cell movements of the progeny allow the rapid expansion of the hair follicle. Finally, we show the requirement of the mesenchyme for hair regeneration through targeted cell ablation and long-term tracking of live hair follicles. Thus, we have established an in vivo approach that has led to the direct observation of cellular mechanisms of growth regulation within the hair follicle and that has enabled us to precisely investigate functional requirements of hair-follicle components during the process of physiological regeneration.

Figure 1. Cell divisions are spatially regulated in the hair follicle at the beginning of a new growth

a, A hair follicle in quiescence. Distinct populations of cells that participate in hair regeneration, including stem cells, progeny and mesenchyme, reside in defined anatomical compartments of the hair follicle. b, A three-dimensional reconstruction of quiescent live hair follicles from serial optical sections acquired by two-photon laser scanning microscopy. Epithelial nuclei (green) are made visible using the H2B–GFP fusion protein driven by the keratin 14 promoter (K14H2BGFP). c, An example of progeny cell division. A single optical section of a live hair follicle (left panel) and magnified views (right, insets) of three nuclei in the progeny compartment that undergo mitosis in real time (right). d, The locations and axes of cell divisions were quantified from several hair follicles (n = 17) at early growth phase (anagen II). e, Two examples of vertical (left panel) and horizontal (right panel) stem cell divisions. A single optical slice of live hair follicles (left, insets) and magnified views of nuclei in the stem cell compartment (right, insets) undergoing mitosis. Scale bars, 20 µm.

Figure 2. The stem cell progeny compartment undergoes morphological reorganization during growth

a, Downwards extension of follicles during growth. Optical sections at three consecutive time points (3 h apart) of a growing live hair follicle shows the progeny compartment extending downwards (left three panels). The increase in the distances between nuclei, and total cell numbers were quantified within the stem cells and the progeny compartments (approximately anagen II to IIIa) (right panel, data are expressed as mean ± s.e.m., n = 13–20, P < 0.001; see also Supplementary Fig. 5). b, Reorganization of nuclei within the hair follicle. Two optical sections (left panels), corresponding traces and measurements (right panels) at the coronal and transverse planes (xy and xz) of the same follicle at time 0 and 4 h, respectively (approximately anagen II to IIIa) (bottom panels). c, Downwards migration of nuclei in the hair follicle during growth. Single optical sections showing complete (left panel) and lower partial views (top panels) of a single hair follicle at successive time points, 1 h apart. Red arrows in the optical slices and corresponding traces (bottom panels) mark a nucleus that is moving downwards, covering a distance of ~30 µm within 5 h (approximately anagen IIIb). DP, dermal papilla. Scale bars, 20 µm.

Figure 3. Ablation of the mesenchymal dermal papilla impairs initiation of hair regeneration

a, Set-up for the experiment using laser-induced cell ablation of dermal papilla cells to test the requirement of the mesenchyme for hair growth. b, High-magnification optical sections of four time points of live hair follicles whose dermal papilla was ablated (approximately telogen phase). c, Low-magnification optical sections of three time points show a group of follicles in which only a few follicles had the dermal papilla ablated (yellow arrows). b, Low-magnification optical sections of three time points show two follicles of which one had the progeny partially ablated (yellow arrow). e, Quantification of the growth of hair follicles (measured as the total length of the hair follicle) with ablated dermal papilla (top) or partially ablated progeny compartments (bottom) compared to intact control follicles. Data are expressed as mean ± s.e.m. (n = 8–10, P < 0.0001). Scale bars, 50µm.

Figure 4. The cellular mechanisms that participate in hair regeneration

During the initial stages of hair regeneration, the stem cell progeny is the first compartment that starts to proliferate. Cell divisions are also detected within the bulge, although the number of divisions is lower than in the stem cell progeny. Divisions in the progeny compartment are oriented along the axis of hair follicle growth, whereas orientations of divisions in the bulge are more random. The hair follicle undergoes a downwards extension in which the distance between nuclei increases within the progeny but not within the stem cell compartment. The epithelial nuclei surrounding the mesenchymal dermal papilla realign and constrict around the mensenchyme. Ablation of the mesenchyme results in impaired hair follicle growth.