Abstract
Tissue development and regeneration depend on cell–cell interactions and signals that target stem cells and their immediate progeny1. However, the cellular behaviours that lead to a properly regenerated tissue are not well understood. Using a new, non-invasive, two-photon (intravital instead of two-photon?) imaging approach we study physiological hair-follicle regeneration in real time in live mice. By these means we have monitored the behaviour of epithelial stem cells and their progeny2–4 during physiological hair regeneration and addressed how the mesenchyme5 influences their behaviour. Consistent with earlier studies6, stem cells are quiescent during the initial stages of hair regeneration, whereas the progeny is 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.
Although stem cells and their immediate progeny are critical for tissue regeneration, we still lack knowledge concerning the discrete sequential steps that lead to proper tissue regeneration. Available methods to address these questions during regeneration are largely static and provide only snapshots of this highly dynamic process. An alternative approach is to continuously visualize stem cells and their progeny throughout physiological regeneration. Recent technological advances have enabled stem cell imaging in mammalian tissues in vivo, such as haematopoietic stem cells in the bone marrow7,8. However, these studies are inherently invasive and therefore it is not possible to monitor the stem cells for long periods using these methods.{However, these studies are inherently invasive and therefore it is not possible to monitor the same stem cells for long periods using these methods.}
To study a physiological tissue regeneration process, we established a method to image entire skin hair follicles in real time in live mice. The skin is a suitable tissue for live imaging owing to its accessibility9,10. In addition, hair follicles continuously regenerate through alternating rest (telogen) and growth (anagen) phases (Supplementary Fig. 1), enabling us to study stem cell behaviours during physiological regeneration. Furthermore, hair follicles are anatomically well defined and the location of different cell types is well characterized2,6,11,12. During the quiescent phase, two populations coexist in the lower portion of the hair follicle; the stem cells which are localized in a specialized region called the bulge and their immediate progeny within the hair germ (Fig. 1a, b). The stem cell progeny is in contact with a mesenchymal group of cells—called dermal papilla (Fig. 1a)—that is able to induce hair regeneration (Supplementary Fig. 1)5. Our recent work has shown that signals released by the mesenchyme activate the progeny at the beginning of the hair regeneration cycle6. Thus, hair regeneration initially relies mainly on the expansion of the progeny and not of the stem cells. Subsequently, stem cells in the bulge will slowly contribute to hair regeneration over time6,12–14.
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.
To visualize directly the hair-follicle stem cells and progeny, we used a transgenic mouse line that marks all of the epithelial nuclei in the skin by expressing a fusion protein of histone H2B with green fluorescent protein (GFP) driven by the keratin 14 promoter (K14H2BGFP)3,6. This transgenic mouse provides a strong nuclear signal that can resolve individual cells. Using a two-photon laser scanning microscope we acquired serial optical sections from the skin of anaesthetized 3-week-old mice (Supplementary Fig. 2 and Supplementary movie 1), which enabled us to visualize the entire hair follicle and identify the location of the stem cells and their progeny based on their distinct morphological features (Fig. 1b and Supplementary Fig. 3).
During the first stages of regeneration, proliferation is first initiated in the stem cell progeny, which undergo a rapid downward expansion (Supplementary Fig. 1). We asked whether this asymmetrically organized growth of the hair follicle relies on spatial regulation of processes such as cell divisions. To test this idea, we performed time-resolved imaging of hair follicles at the beginning of the hair regeneration cycle (around postnatal day 22 (P22)) to visualize cell proliferation “in real time over several hours” instead write: over the course of several hours. By these means, we have recorded several mitotic events with clear spatial and temporal resolution (Fig. 1c and Supplementary movie 2). Systematic quantification of the location and axis of divisions from several recordings shows that most divisions occur in the lower part of the follicle at the interface between the progeny and the mesenchyme at the beginning of a new growth (Fig. 1d). At this stage, the axis of division is parallel to the long axis of hair follicle growth. A second, less pronounced, centre of growth was identified in the upper part of the progeny (Fig. 1d). Cell divisions were observed within the bulge even at lower frequencies, (Fig. 1d, e, and Supplementary movies 3 and 4). The axes of these bulge divisions varied, ranging from perpendicular to parallel with respect to the long axis of hair follicle growth (Fig. 1d, e). Hair growth in the mouse is a fairly synchronized process at 3 weeks of age. To test whether this synchronization is the result of temporal regulation of cell divisions within the hair follicle, we quantified the time when cell divisions occurred within a single hair follicle and in relation to neighbouring hair follicles (at approximately P22). In some of our recordings, clustering was observed between cell divisions within one follicle as well as between adjacent follicles. However, these behaviours were not consistent in every recording (Supplementary Fig. 4 and Supplementary movie 2).
During regeneration, the lower part of the hair follicle undergoes a major reorganization as the mesenchyme transitions from being extruded from the epithelium to being encompassed by it15 (Supplementary Fig. 1). This led us to propose that changes in tissue architecture may rely on additional cellular processes besides cell divisions. Analysing several of our recordings we observed that some follicles rapidly stretch downwards during this growth phase (at approximately P23; Fig. 2a, Supplementary Fig. 5, and Supplementary movies 5 and 6). To test whether this tissue extension is the result of a morphological rearrangement of the tissue, we quantified the distance between adjacent nuclei within the stem cell or the progeny compartment. The distance between nuclei increased specifically within the progeny, which may account for the overall downwards extension of the hair follicle (Fig. 2a and Supplementary Fig. 5). This type of behaviour is reminiscent of morphogenetic movements observed during development in classical model organisms such as Drosophila or zebrafish (Danio) embryos16 and provides a previously unidentified mechanism of hair growth in addition to cell division.
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.
To dissect further these novel dynamic processes, we analysed time-resolved optical sections at the coronal plane of the follicles where the epithelium interfaces the mesenchyme. During growth, in vivo recordings revealed a major reorganization of the epithelial stem cell progeny surrounding the mesenchymal dermal papilla. Over 4 h the nuclei transition from a disorganized pattern to a single row aligned around the mesenchyme (Fig. 2b and Supplementary movie 7). In addition, the lower epithelial part of the follicle constricts as it encompasses the mesenchyme (Fig. 2b; 0 h versus 4 h). This major epithelial nuclear reorganization occurs concurrently in adjacent follicles (Supplementary Fig. 6 and Supplementary movie 7). Furthermore, in more advanced growth stages, long-range migrations within the outer most layer (outer root sheath) of the lower hair follicle were observed (Fig. 2c and Supplementary movie 8). Recent data using lineage-tracing approaches have indicated that stem cells can migrate out of the bulge, either downwards towards the progeny or upwards towards the sebaceous gland (Fig. 1a)17,18. We did not observe downwards migrations of the stem cells to the progeny but captured a short upwards migration within the bulge stem cells (Supplementary Fig. 7 and Supplementary movie 9). Based on these data, we suggest that migratory events within the bulge may be temporally regulated or may take place at a much slower pace than we can resolve in the timeframe of our experiments (3–14 h). Taken together, our findings reveal new dynamic cellular processes adopted by the stem cells and their immediate progeny during physiological regeneration that would have been missed by conventional static analysis.
Epithelial–mesenchymal interactions are crucial for the development and regeneration of many tissues such as limb tissue (I would not put tissue, but if something has to be added we would phrased it as follow “regeneration of many organs such as limb”)19. In the hair follicle, the mesenchymal dermal papilla is a key signalling centre, able to induce de novo hair-follicle formation after transplantation5. Moreover, previous work has identified mesenchymal signals, including signalling by FGF7 and FGF10, and BMP inhibitors, as regulators for the initiation of the hair regeneration cycle6,20. These and other data suggest that the mesenchyme is sufficient to induce hair regeneration. However, the requirement of the mesenchyme for initiation of hair regeneration has not been tested. To be able to selectively eliminate the mesenchyme, we set up a laser-induced cell-ablation approach to target fluorescently labelled dermal papilla cells (using a Lef1RFP transgenic mouse (expressing red fluorescent protein under the control of a Lef1 promoter fragment) a pair of bracket should be removed6,21 at the beginning of a new hair growth (at approximately P19; Fig. 3a and Supplementary Fig. 8). Analysis of the tissue immediately after laser ablation showed that the dermal papilla was disrupted, whereas adjacent tissues, such as the progeny or the overlying epidermis, remained undamaged (Supplementary Fig. 8). To assess the long-term effects of dermal papilla ablation on hair regeneration, we revisited the same hair follicles in separate imaging sessions, hours or even days apart (Fig. 3a). When the same hair follicles were observed at 4 and 7 days after ablation, follicles with ablated mesenchymal dermal papillae appeared quiescent (Fig. 3b). In contrast, surrounding follicles with intact mesenchyme grew at a normal pace (Fig. 3c, e). To determine whether the mesenchymal ablation altered the molecular and cellular composition of the hair follicle, skin samples containing mesenchymal ablated follicles were collected and analysed at 7 days post ablation. Histological and immunofluorescence data showed that the stem cells and their progeny maintain their molecular properties after dermal papilla ablation (CD34 ; Pcadherin, Supplementary Fig. 9). Furthermore, the ablated follicles retain the ability to host other tissue cell types such as melanocytes (Trp2; Supplementary Fig. 9). To test whether failure to initiate hair growth was due to dermal papilla ablation rather than to collateral injury of the stem cell progeny, we targeted only cells within the progeny, leaving the dermal papilla intact. Hair follicles in which the progeny was injured recovered and grew at a similar rate to their surrounding uninjured follicles (Fig. 3d,e). These results indicate that the mesenchymal dermal papilla is required for hair growth initiation, and underscores the importance of epithelial–mesenchymal interactions for stem cell activation and regeneration.
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.
In summary, we have imaged hair-follicle stem cells and their progeny in an uninjured living mammal, over a long period of time, with high spatial and temporal resolution. We have shown that hair regeneration involves a range of dynamic cellular behaviours and relies on the presence of the mesenchyme (Fig. 4). Given that components of the stem cell microenvironment are conserved across different tissues, our findings will probably be relevant to other tissues as well. More broadly, understanding how stem cell and progeny behaviour is regulated in physiological conditions may be necessary to advance our use of stem cells in regenerative medicine, and to uncover the cellular mechanisms that go awry in cancer and other diseases.
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.
METHODS
In vivo imaging
Three-week-old mice were anaesthetized with intraperitoneal injection of ketamine and xylazine (this is a cocktail), and the skin around the head region was shaved using a mechanical trimmer and depilatory cream. The mouse was placed on a heated stage, and the head and the ear were supported by a custom-made stage. A glass coverslip was placed against the skin in the junction region between the head and the ear. Image stacks of the skin were acquired with a LaVision TriM Scope II (LaVision Biotec) microscope equipped with a Chameleon Vision II (Coherent) two-photon laser. A laser beam (at 940 nm for GFP and 1040 nm for RFP, respectively) was focused through a ×20 water immersion lens (N.A. 1.0; Olympus) and scanned with a field of view of 0.25 to 0.5 mm2 at 600 Hz. Serial optical sections were acquired in 2–3-µm steps to image a total depth of ~100 µm of tissue in 5-min intervals. Several phases covering the transition from quiescent to growth stages were analysed (telogen to anagen phases). Distinctive inherent landmarks in the skin were used to navigate back to the original field of view and visualize the same follicles in separate experiments. Anaesthesia was maintained throughout the course of the experiment with vaporized isofluorane delivered by a nose cone.
Three-dimensional two-photon laser ablation
Laser ablation was carried out with the same optics as for acquisition, essentially as described previously22. A 900-nm laser beam was used to scan a 10 µm2 area and ablation was achieved using 25% laser power for 1 s. Ablation parameters were adjusted according to the depth of the target (30–80 µm).
Image analysis
Raw image stacks (512 × 512 to 1024 × 1024 pixels in the x–y plane and 2–3-µm voxel depth; typically 30–50 optical sections) were imported into ImageJ (NIH Image) for further analysis. Optical planes from sequential time points were manually realigned to compensate for minor tissue drift during the time course of the acquisition. The contrast was adjusted accordingly and selected optical planes or z-projections of sequential optical sections were used to assemble time-lapse movies. Three-dimensional rendering and cell tracking was performed using the Volocity software package (Perkin Elmer).
Statistical analysis
Data are expressed as mean ± s.e.m. An unpaired Student's t-test was used to analyse data sets with two groups. For all analyses, a P value of less than 0.05 was accepted as indicating a significant difference. Statistical calculations were performed using the Prism software package (GraphPad).
Histology and immunolabelling
Sections frozen in OCT were fixed with 4% formaldehyde for 10 min at room temperature and treated as described previously6. Antibodies were used at the following dilutions: Ncam (Chemicon, rat 1:100), CD34 (eBioscience, rat 1:50), Pcadherin (R&D, goat 1:100) and Trp2 (Santa Cruz, goat 1:100).
Supplementary Material
A time-lapse movie of serial optical sections (2 µm apart), from skin imaging in live mice by means of two-photon microscopy. The sequence (top to bottom) shows the different layers of the epidermis as well as the hair follicles located underneath; several micrometers below the surface of the skin of a transgenic live mouse expressing the GFP-Histone H2B fusion protein under the control of the Keratin 14 promoter (K14H2BGFP). Also see Fig. S2.
A time-lapse movie of a single optical section showing a group of live hair follicles in growth phase (Anagen II). The movie captures several mitotic events (arrows) which appear to be coordinated in both follicles. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes.
Two time-lapse movies of a single optical sections showing a single or a group of live hair follicles in growth phase (Anagen II), respectively. The movie captures cell divisions within the stem cell population in the bulge which occur in various orientations. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes.
Two time-lapse movies of a single optical sections showing a single or a group of live hair follicles in growth phase (Anagen II), respectively. The movie captures cell divisions within the stem cell population in the bulge which occur in various orientations. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes.
A time-lapse movie showing a group of live hair follicles in growth phase. The movie captures the rapid downward expansion of the progeny compartment. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2a and S5.
A time-lapse movie showing a group of live hair follicles in growth phase. The movie captures the rapid downward expansion of the progeny compartment. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2a and S5.
A time-lapse movie of a single optical section showing a group of live hair follicles in growth phase. The movie shows epithelial cells in the progeny compartment as they transition from a disorganized pattern to a single row aligned around the mesenchyme (arrow). Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2b.
A time-lapse movie showing two different focal planes (1 and 2, respectively) of the lower part of a hair follicle in growth phase. The movie captures the downward movement of nuclei in the outermost (Outer Root Sheath – ORS) epithelial layer of the hair follicle. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2c.
A time-lapse movie showing the bulge compartment of a hair follicle in growth phase. The movie captures the upward movement of nuclei in the bulge. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. S7.
Acknowledgements
We are grateful to A. Horwich, V. Reinke and A. Giraldez for critical discussion of the work. We thank S. King for feedback on the manuscript, and E. Fuchs for the K14H2BGFP, Lef1RFP and pTREH2BGFP mice and A. Glick for the K5tta mice. P.R. is supported by the James Hudson Brown – Alexander Brown Coxe postdoctoral fellowship. G.Z. is a New York Stem Cell Foundation–Druckenmiller Fellow. E.D. is supported by the Cell and Molecular Biology Training Grant of the National Institute of General Medical Sciences, US National Institutes of Health, Public Health Service. This work was supported in part by the American Skin Association, the American Cancer Society and the Yale Rheumatologic Disease Research Core Center grant NIH P30AR053495.
Footnotes
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
Author Contributions P.R. and V.G. designed experiments and wrote the manuscript, and P.R. performed the experiments and analysed the data. E.D. performed two-photon laser-scanning timelapses. G.Z. performed immunostainings. I.S. set up the mouse colonies and staining protocols. D.G. and A.H. assisted on initial intravital imaging set-up. D.G. performed data analysis.
The authors declare no competing financial interests.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
A time-lapse movie of serial optical sections (2 µm apart), from skin imaging in live mice by means of two-photon microscopy. The sequence (top to bottom) shows the different layers of the epidermis as well as the hair follicles located underneath; several micrometers below the surface of the skin of a transgenic live mouse expressing the GFP-Histone H2B fusion protein under the control of the Keratin 14 promoter (K14H2BGFP). Also see Fig. S2.
A time-lapse movie of a single optical section showing a group of live hair follicles in growth phase (Anagen II). The movie captures several mitotic events (arrows) which appear to be coordinated in both follicles. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes.
Two time-lapse movies of a single optical sections showing a single or a group of live hair follicles in growth phase (Anagen II), respectively. The movie captures cell divisions within the stem cell population in the bulge which occur in various orientations. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes.
Two time-lapse movies of a single optical sections showing a single or a group of live hair follicles in growth phase (Anagen II), respectively. The movie captures cell divisions within the stem cell population in the bulge which occur in various orientations. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes.
A time-lapse movie showing a group of live hair follicles in growth phase. The movie captures the rapid downward expansion of the progeny compartment. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2a and S5.
A time-lapse movie showing a group of live hair follicles in growth phase. The movie captures the rapid downward expansion of the progeny compartment. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2a and S5.
A time-lapse movie of a single optical section showing a group of live hair follicles in growth phase. The movie shows epithelial cells in the progeny compartment as they transition from a disorganized pattern to a single row aligned around the mesenchyme (arrow). Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2b.
A time-lapse movie showing two different focal planes (1 and 2, respectively) of the lower part of a hair follicle in growth phase. The movie captures the downward movement of nuclei in the outermost (Outer Root Sheath – ORS) epithelial layer of the hair follicle. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. 2c.
A time-lapse movie showing the bulge compartment of a hair follicle in growth phase. The movie captures the upward movement of nuclei in the bulge. Epithelial nuclei are marked with K14H2BGFP and individual frames were acquired every five minutes. Also see Fig. S7.