Fibroblasts are an essential cellular and structural component of our organs. Despite several advances, the critical behaviors that fibroblasts utilize to maintain their homeostasis in vivo have remained unclear. Here, by tracking the same skin fibroblasts in live mice, we show that fibroblast position is stable over time and that this stability is maintained despite the loss of neighboring fibroblasts. In contrast, fibroblast membranes are dynamic during homeostasis and extend to fill the space of lost neighboring fibroblasts in a Rac1-dependent manner. Positional stability is sustained during aging despite a progressive accumulation of gaps in fibroblast nuclei organization, while membrane occupancy continues to be maintained. This work defines positional stability and cell occupancy as key principles of skin fibroblast homeostasis in vivo, throughout the lifespan of mice, and identifies membrane extension in the absence of migration as the core cellular mechanism to carry out these principles.

Here, we made use of our established intravital imaging approach to interrogate the principles of adult fibroblast homeostasis in an intact mouse. We show that in unperturbed conditions, fibroblasts are stable in position over the course of weeks to months in non-remodeling skin. Unexpectedly, even following local fibroblast loss, the nuclei of neighboring fibroblasts remain fixed in position. Instead, we find that fibroblasts display dynamic membranes that, following neighbor cell loss, extend into the depleted region, without migration of the main cell body, in a Rac1-dependent manner. Following larger damage, we see that fibroblasts use membrane extension in conjunction with known regenerative behaviors to repair the loss of dermal architecture, or tissue-wide cell depletion. Finally, we demonstrate that the fibroblast loss in density that occurs with aging is not a uniform loss of cells, but an accumulation of localized cell losses, where stably positioned neighboring fibroblasts again compensate via membrane extensions. Collectively, our work demonstrates the remarkable positional stability of fibroblasts in the skin when both unperturbed and following local cell loss while elucidating membrane dynamics as the mechanism to compensate for that loss throughout the lifespan of mice.

Our knowledge of the dynamics of fibroblasts mostly comes from in vitro and ex vivo experiments, where cells can be easily tracked for any period of time. Fibroblast migration has been characterized to involve several types of membrane behaviors, including lamellipodia and filopodia, and affected by the nature of the extracellular environment in both culture and dermal explants (). However, our understanding of the critical homeostatic behaviors in the mammalian skin has been limited by the inability to visualize and track the same individual fibroblast cells over time in vivo.

The easily accessible and anatomically well-defined dermal layer of the mammalian skin has provided an excellent model to interrogate the biology of fibroblasts. The mouse skin dermis contains at least two different and atomically distinguishable layers: the upper dermis nearest the epidermis, and the lower dermis below (). During development, fibroblasts populate the dermis by proliferation, which largely ceases by postnatal day 10 (). However, DNA radiolabeling suggests that proliferation continues to occur at a low rate throughout the lifespan of the animal (). The presence of different and sometimes dynamic appendages specific to the skin complicates our understanding of generalizable fibroblast behaviors in vivo (). Nevertheless, fibroblasts maintain a high potential for proliferation in culture settings and cutaneous wounding ().

Fibroblasts are key cell types that are essential for organ homeostasis through their contributions to both extracellular matrix structure and signaling interactions with neighboring cells (). Current knowledge of the fibroblast is built on understanding their behaviors by in vitro and ex vivo approaches, their cell states by molecular marker analyses, and their unique cell biological features by electron microscopy ().

Finally, to interrogate the membrane distribution within these gaps, we returned to our strain of combined nuclear and maximal membrane labeling (PDGFRα-H2BGFP; PDGFRα-CreER; mTmG). We again observed gaps in the nuclei of mice above 8 months old and found that the gaps contained fibroblast membrane of a similar density to non-gap regions ( Figures 6 E, 6F, and S6 D). We also quantified the membrane occupancy across older mice and found a similar level of occupancy compared to young mice ( Figures 6 G versus 2 G), further supporting that membrane occupancy is maintained even into older tissue. In summary, we identified that, during aging, fibroblast cell loss occurs as an accumulation of localized clusters of loss, rather than a uniform reduction in density. Additionally, as in our laser ablation models, the neighboring fibroblasts to these gaps fail to recover cell number and remain stably positioned but do maintain membrane occupancy of the volume via membrane extensions ( Figure 6 H).

To test whether the increased number of gaps in older individuals resulted from constant cell loss and replacement, or from progressive loss without replacement, we performed 5-month revisits in mice that already contained gaps ( Figures 6 D and S6 E). With this approach, we captured both the appearance of new gaps ( Figure 6 D, top-right inset) as well as the maintenance of existing gaps ( Figure 6 D, bottom-right inset). In both cases, the nuclei surrounding the gaps were stable over the 5-month period, moving approximately as little as the 2-week time period ( Figures 1 A versus 6 D). This uncovered that the fibroblast loss in density that occurs with aging is not a uniform loss of cells, but an accumulation of localized cell losses that are not recovered due to the positional stability of fibroblasts.

The observed positional stability of fibroblasts in non-remodeling skin caused us to interrogate the implications of this behavior during the aging process, as fibroblast density is known to be reduced in older individuals (). To this end, we visualized fibroblast nuclei in the non-hairy paw and non-remodeling hairy skin and compared young mice to two cohorts of older mice: 8–10 months old and 16 months or older ( Figures 6 A–6C). Strikingly, rather than a uniform reduction of cell density in the older mice, we observed highly localized clusters of cell loss. Each cluster was similar in organization to that created with the laser ablations that we employed to perturb fibroblast homeostasis ( Figures 1 3 , and 5 ). This was in contrast to the organization of nuclei in the young (2 months old) mice, which was uniform across the tissue ( Figures 6 A and S6 A). In order to more rigorously quantify the number of gaps, given their heterogeneous size and shape, we used MATLAB scripts to generate Voronoi diagrams based on the nuclei positions determined using Imaris software (see STAR Methods ). In addition to quantification, these provided a helpful visualization of the localized nature of the gaps ( Figure 6 A, right). Quantifications from the three separate age cohorts showed that gap numbers increased with increasing age ( Figure 6 B). These localized clusters of fibroblast loss with age were also observed in non-remodeling hairy skin ( Figures S6 A–S6C). Finally, to explore this further, we counted fibroblast density down to a depth of 40 μm and found that, in this wider range of dermal depth, cell loss occurs earlier than in the upper fibroblasts alone ( Figure 6 C).

(E) Quantification of the number of non-yellow cells across the 5-month period of the same regions in the same mice. Corresponding representative time course shown in Figure 6 D. Red only cells indicate cell loss (left column). Green only cells indicate cell gain (right column). Samples taken from regions varying from 200μmto 2800μmacross three revisited mice. Cell loss far outweighs cell gain. Error bars indicate one standard deviation. n = 3 mice.

(C) Density of cells, sampled from 1000μm x 1000μm x 10μm regions in the upper dermis of non-remodeling hairy skin. n = 4 (2 months old). n = 4 (16 months old or greater). p = 0.0004 (2 months versus ≥ 16 months). Error bars are std. dev.

(B) Number of cell areas in Voronoi analyses exceeding 900μm 2 , sampled from 1000μm x 1000μm x 10μm regions in the upper dermis of non-remodeling hairy skin. n = 4 (2 months old). n = 4 (16 months old or greater). p = 0.0024 (2 months versus ≥ 16 months). Error bars are std. dev.

(A) Representative images of fibroblasts (PDGFRα-H2BGFP) in 2 months (left) and 19 months (right) non-remodeling hairy skin. Hair follicle locations are marked by “HF” and a circle with a diameter approximating the diameter of the hair follicle in this Z section. Density is seen to be reduced in the 19 months old mouse and the reduced density is seen to occur not uniformly through the tissue, but in localized clusters of fibroblast cell loss, similar to the paw skin ( Figure 6 A). Scale bars 50μm. n = 3 mice.

(H) Model of cell loss during aging. Cells are lost in localized clusters that are similar in size and shape to our laser ablation assays. Similar to after laser ablation, the nuclei neighboring physiological cell losses remain stable in position but project new membrane extensions into the region.

(F) Membrane occupancy in 12-month-old mice between normal nuclei and gap regions, computed by thresholding the GFP signal (see STAR Methods ). Compare to Figure 2 G. Error bars are SD. n = 3 mice for each bar.

(D) Revisits of skin across 5 months, beginning in skin already containing gaps. Red-only signal (Merge) is from nuclei that disappeared during the 5-month period. Cells disappeared in localized clusters of cell loss, rather than uniformly throughout the skin. Insets (column 4) highlight examples of localized clusters of cell loss that occurred during (top, 1) and before (bottom, 2) the 5-month period. Quantification is shown in Figure S6 E. Scale bar, 100 μm. Scale bar, 50 μm (insets).

(C) Cell density across randomly sampled 200 × 200 × 40-μm regions of skin dermis from mice in each age cohort. p = 0.0018 (2 months versus 8–10 months). p = 0.0016 (2 months versus ≥16 months). Error bars are SD. n = 3 mice per age group.

(B) Number of cell areas in Voronoi diagrams exceeding 900 μm 2 , sampled from 1,000 × 1,000 × 10-μm regions in the upper dermis (∼2,500 cells). One sample per mouse. n = 5 mice (2 months old). n = 4 mice (8–12 months old). n = 5 mice (≥16 months old). p = 0.0499 (2 months versus 8–10 months). p = 0.0114 (2 months versus ≥ 16 months). Error bars are SD.

(A) Non-hairy paw skin fibroblast nuclei in 2-month-old mice (column 1) and ≥16-month-old mice (column 2). In Voronoi diagrams (top: 2 months old, bottom: ≥16 months old), polygons are color coded by size. n = 5 mice for each age. Scale bars, 100 μm.

Finally, we used laser ablation in the remodeling hairy skin. We observed that the ablated region was repopulated by newly appearing fibroblast nuclei as well as reorganization of the membrane network ( Figures 5 F and S5 D). Altogether, we found that positional stability is not maintained in skin that is actively undergoing physical changes.

Our finding that fibroblasts operate under the principles of stability and membrane occupancy in hairy skin prompted us to interrogate fibroblast behaviors in an actively remodeling dermis ( Figure 5 A, right). During the initiation of the hair growth phase, the follicles extend into the dermis increasing their length several fold (). We captured the nuclei of fibroblasts from the beginning of this initiation of the growth phase and over the subsequent 2-week period ( Figures 5 E and 5G). We observed that in 1 week, a few nuclei remained in the same position, but the majority had moved from their initial position by 2 weeks. Remounts of this region showed much lower image correlation coefficients than remounts in non-remodeling hairy skin, or non-hairy paw skin, suggesting an even greater sensitivity to small differences in tissue mounting ( Figure 5 G). However, the relative movement of nuclei over the 2-week period compared to remounts was much greater than in non-remodeling hairy skin or non-hairy paw skin, indicating that real movement of fibroblasts does occur during tissue remodeling. The degree to which this movement is due to physical deformation of the dermis due to the growing hair follicles ( Figure 5 A, right, day 0 versus +2 weeks) as opposed to differences in signaling pathways that change the behavior of fibroblasts is not clear.

To test whether this stability would be preserved in the face of neighboring fibroblast loss, we performed laser ablations of fibroblasts in the hairy ear skin and observed that positional stability was maintained over 2 weeks ( Figures 5 C and 5G), similar to what we found in the non-hairy paw. In addition, mosaic membrane labeling in combination with laser ablation captured the ability of these fibroblasts to also utilize membrane extensions in order to reoccupy the space ( Figure 5 D). Therefore, the principles of fibroblast positional stability and membrane extension are conserved across distinct skin tissue types.

In order to test the generality of the principles of positional stability and membrane extension following cell loss, we turned to testing these behaviors in a more complex skin tissue. The ear tip is a hairy region that has a stable dermal architecture due to the rarity of actively growing and shrinking hair follicles ( Figure 5 A, left; Video S4 ). Fibroblasts in this region have similar membrane occupancy, but lower nuclear density and larger cell size, compared to the non-hairy paw ( Figures S5 A–S5C). Revisits of the same fibroblast nuclei over 2 weeks showed that these cells maintain stable positions, as was observed in the non-hairy paw skin ( Figure 5 B). To quantify the results, we used the same image correlation technique as in Figures 1 B and 1H ( Figure 5 G). Comparing Figure 1 B remounted tissue with Figure 5 G remounts (positive controls), we saw that the correlation was lower for the ear than the paw, suggesting that that the ear tissue is more sensitive to stretches and bumpiness in the technical mounting process. Comparing the relative correlation coefficients of 2-week revisits to remounts, we found that the movement of nuclei over the 2-week period is the same between non-hairy paw and hairy ear skin ( Figure 5 G versus Figure 1 B).

(D) Representative time course of nuclei and membrane-GFP labeled fibroblasts (PDGFRα-H2BGFP; PDGFRα-CreER; mTmG) in remodeling hairy skin, following laser ablation of nuclei, labeled pink (before ablation). At one day after ablation (+1 day), some new membrane extensions are highlighted by the orange arrowheads. By one week after ablation (+1 week), extensive membrane dynamics are observed (highlighted by orange arrowheads). In this remodeling hairy skin, fibroblast nuclei position is also not stable and nuclei are not in the same positions between +1 day and +1 week and the ablated region appears to be repopulated with nuclei and membrane. Scale bar 50μm.

(C) Quantification of the density of fibroblasts in the ear tip, ear base, and paw. Fibroblasts in the ear (tip and base) are of lower density than in the paw, and these fibroblasts are larger (B). n = 3 mice per region.

(B) Representative images of fibroblasts from ear (left) and paw (right), allowing for a comparison of morphological differences. Fibroblasts in the ear are larger and appear to have fewer fine projections that fibroblasts in the paw. Scale bars = 20μm.

(A) Representative images of fibroblasts labeled by nuclei (PDGFRα-H2BGFP) and membrane-GFP in the paw (column 1), ear tip (column 2), and ear base (column 3). While nuclei density varies between ear and paw, membrane occupancy of the region is similar. Scale bars 50μm.

(G) Image correlation coefficients of data represented in (B) (Non-rem. hairy, +2), (C) (Non-rem. hairy, Abl.), (E) (Rem. hairy, +2), and (F) (Rem. hairy Abl.). Compare to Figures 1 B and 1H. Non-/rem, non-/remodeling. Re, remount. +2, 2 weeks unperturbed. Abl, ablation. Non-rem. hairy positive control correlation coefficient (0.671) is lower than remounts in non-hairy skin ( Figure 1 B, 10 μm, Re.) (0.905), indicating that a lower maximum correlation is expected for this tissue. Rem. hairy remount positive control correlation coefficient is even lower (0.360), nearing the limitations of this analysis approach. Error bars are SD. n = 3 mice for 2-week time course data. n = 3 regions for remounts.

(F) Merge of +1 day and +2 weeks following ablation in remodeling hairy skin. Little overlap of fibroblast nuclei is observed. n = 3 mice. Quantification is shown in (G) (Rem. hairy, Abl.). Scale bar, 20 μm.

(C) Laser ablation (pink dots) and revisits. +1 day, ablated cells are absent. +2 weeks, remaining nuclei are largely stable, and the cell number remains reduced. Quantification is shown in (G) (Non-rem. hairy, Abl.). n = 3 mice. Scale bar, 20 μm.

(B) Revisits in homeostatic non-remodeling hairy skin. A wide field of view is shown with green (PDGFRα-H2BGFP) and red (membrane-tdTomato) (column 1). Day 0, +2 weeks and merge is region highlighted by orange box in column 1. n = 3 mice. Quantification is shown in (G) (Non-rem. hairy, +2). Scale bar, 50 μm (column 1). Scale bar, 20 μm (columns 2–4).

(A) Diagrams are XZ projections, images are XY projections at 40 μm (non-remodeling) and 50 μm (remodeling) depths. Non-remodeling hairy tissue does not change over 2 weeks. In remodeling hairy tissue, hair follicles elongate over 2 weeks. HF, hair follicle. Hair follicles are outlined in orange. Full Z stacks: Videos S4 and S5 . Scale bars, 50 μm.

Second, we specifically targeted fibroblasts by advancing our previous approach of an inducible Cre-dependent diphtheria toxin (PDGFRα-CreER; Rosa26-loxP-eGFP-(stop)-DTA) system to dramatically increase fibroblast loss. This combination resulted in a higher penetrance of cell loss in the upper dermal layer, with minimal cell loss detected in the lower dermal layer ( Figures 4 D and 4F; see STAR Methods ). Following large-scale cell depletion, we observed partial repopulation of the upper dermal region by 2 weeks ( Figures 4 D and 4F). Tracking the tissue out to 6 weeks, we saw little to no further repopulation, leaving the region closest to the epidermis still depleted of cells ( Figures 4 D and 4F). We examined this remaining space in the presence and absence of nuclear signal and observed that, while this sub-epithelial region was devoid of fibroblast nuclei, it contained membrane extensions rising vertically from the large majority of the fibroblasts underneath ( Figures 4 E, 4G, S4 B, and S4C). These vertical membrane extensions were also observed following laser ablation ( Figure S4 D). The ability of fibroblasts to extend membrane vertically, despite their strong polarity on the XY plane ( Figure 2 A), further expanded our understanding of their membrane dynamics. These data show that fibroblast membrane extension works in conjunction with known repair behaviors and provides a mechanism that, whether alone or in cooperation, enables the fibroblast tissue to mediate space reoccupation in vivo.

(D) Representative images showing the presence of vertical membrane projections from below cells after laser ablation within the upper dermis. Z stacks before (row 1) and +1 week following (row 2) laser ablation are projected. Epidermis (0μm depth) is on the far left and images get deeper to the right. No membrane-GFP labeled vertical projections are visible before laser ablation (row 1), but a new vertical projection is visible at +1 week, highlighted by orange arrowheads (row 2). XZ projection is shown in row 3, with epidermis at the top (Epi.) and upper dermis bellow. Before (left image) and at +1 week (right image) of the same area as top and middle rows above, respectively is shown. The same new vertical projection is highlighted by orange arrowheads. Scale bar 10μm.

(C) Representative images from single Z stack at +6 weeks after Tamoxifen induction. Vertical membrane extensions are visible as thin “islands” of membrane in sub-epithelial region. Complete cell bodies are visible at 20μm of depth. Scale bar 20μm.

(B) Single image of vertical membrane extensions in combination with nuclear labeling at +2 weeks after ablation. Depleted space is visible between epidermis and fibroblast nuclei. In this space, two membrane extensions (arrowheads) are faintly seen. As H2BGFP is much brighter than membrane-GFP, and resolution is lower in the Z dimension compared to XY, it is difficult to observe membrane-GFP extensions in combination with H2BGFP. Scale bar 10μm.

(A) Representative cryosection of paw dermis with fibroblast nuclei labeled (PDGFRα-H2BGFP) and collagen visible in blue via second harmonic signal (SHG). Fibroblasts are split into two groups (upper fibroblasts and lower fibroblasts) in order to test if a difference in average brightness of nuclei H2BGFP signal is greater in the upper fibroblasts. Scale bar 30μm. Representative of n = 4 mice.

Our finding that fibroblasts use membrane extensions, rather than proliferation and migration, to reoccupy space following neighboring cell loss prompted us to test the combination of these behaviors in the contexts of larger damage ( Figure 4 A). We made use of two distinct assays. First, we increased the intensity of the ablation to damage collagen (visible by second harmonic signal) in addition to eliminating fibroblasts ( Figure 4 B). As we captured changes in fibroblast position, likely due to proliferation and/or migration events, we additionally noticed the concurrent extension of membranes during the repair process ( Figures 4 B and 4C).

(G) At +3 weeks, membrane-GFP-labeled cells at approximately 30-μm depth were scored for presence of membrane extensions reaching to epidermis (85%). Error bars are SD. n = 28 cells across 3 mice.

(F) Before ablation, fibroblast density varies by depth between ∼13 and 30 cells per (100 μm) 2 . +2 weeks, fibroblast cell density at 30 μm is largely recovered (yellow). Cell density at 10 μm (orange) and 20 μm (light gray) remains unrecovered at +6 weeks. n = 3 mice.

(E) XZ projections in high DTA with revisits highlighting fibroblast membrane. Before ablation (left), cell bodies are visible in the sub-epithelial region. After high-dose tamoxifen (center and right), sub-epithelial region is still empty of cell bodies. At +3 weeks, vertical membrane projections extend from deeper cells and contact epidermis. Quantification is shown in (G). n = 28 cells in 3 mice. Scale bar, 10 μm. Epi, epidermis.

(D) Revisits following widespread genetic ablation of fibroblasts (DTA). Near complete loss of the upper fibroblasts occurs by +1 week following high dose of tamoxifen. By +2 weeks, the upper dermal layer partially recovers in cell number, but sub-epithelial cell number does not recover even at +6 weeks. Quantification is shown in (F). n = 3 mice. Scale bar, 20 μm. Epi, epidermis.

(C) Proliferation and/or migration (8 of 15), and membrane extensions (15 of 15) following collagen-damaging ablations (Abl.) or non-collagen-damaging controls (No Abl.). n = 15 ablations and 4 non-collagen-damaging controls across 4 mice.

(B) Fibroblast elimination + collagen-damaging ablations (dashed circles and second harmonic signal at top) and revisits. Middle and bottom rows are both green channel, but brightness is adjusted to show stronger nuclear GFP, or fainter membrane-GFP. Quantification is shown in (C). n = 15 ablations in 4 mice. Scale bar, 10 μm. Arrowheads, proliferation or migration. Arrows, membrane extension.

This suggested that neighboring fibroblasts could harness membrane dynamics to extend and occupy the space of the eliminated cells without recovering cell number. To capture how this phenomenon occurs, we maintained nuclear labeling for all cells but used sparser membrane labeling. Eliminating non-membrane-labeled fibroblasts allowed us to capture new membrane-labeled extensions into the depleted region. First, we used short revisits following the elimination of fibroblasts and observed that labeled neighboring cells project long membrane extensions into the area of cell loss ( Figure 3 D). Second, taking inspiration from in vitro scratch assays, we identified and ablated non-membrane-labeled cells that were flanked on two sides by the membrane-labeled cells. Through this design, we could clearly capture neighboring fibroblasts project new extensions into the region of cell loss ( Figure 3 E). Finally, to test whether this compensation was mediated by Rac1, we performed laser ablation 2 weeks after inducing Rac1 deletion by tamoxifen (PDGFRα-H2BGFP; PDGFRα-CreER; mTmG; Rac1or Rac1). We observed that Rac1fibroblasts failed to form membrane extensions, while the Rac1mice responded similar to background strains ( Figures 3 F and 3G). These data show that fibroblasts compensate for neighboring fibroblast loss by extending membrane into the depleted regions in a Rac1-dependent mechanism, while maintaining positional stability.

To test the activity of fibroblast membranes during neighboring cell loss, we used the combined nuclear and maximal membrane labeling (PDGFRα-H2BGFP; PDGFRα-CreER; mTmG) and eliminated cells by laser ablation ( Figure 3 A). Upon revisits over 2 weeks, we observed that the neighboring nuclei remained stably positioned, consistent with our previous observations ( Figures 3 B, 1 E, and 1H). Strikingly, we saw that the ablated region contained membranes that occupied the space to a similar extent as before cell elimination ( Figures 3 B and 3C). Cells have been shown to exist and migrate for some time after the loss of the nucleus (). In order to test whether active membrane from dying cells may persist over time in the skin dermis, we performed cell ablation on individually labeled cells and observed that membrane was lost within a single day ( Figure S3 A). This indicated that these membranes must have come from surviving neighboring cells. We wondered whether the new membranes occupied the same positions as the original membranes, but overlaying images from before ablation and 2 weeks after showed that the new membranes were in different positions ( Figure S3 B).

(B) The exact same images used in Figure 3 B, but increased in brightness in order to show the positions of original membrane and newly reoccupied membrane at +2 weeks following cell ablation. While new membrane occupies the region to a similar degree, it does not occupy the exact same positions in that region, as the merge on the right shows several regions of either green only or red only. Scale bar 20μm.

(A) Representative time course following laser cell ablation showing that membrane does not remain following cell ablation. A singly membrane-GFP labeled fibroblast present before and immediately after ablation of the single labeled cell. Revisited at +1 day, neither the nucleus nor cell membrane are any longer present, suggesting that labeled debris does not remain 1 day after laser ablation. Scale bar 10μm.

(G) Membrane occupancy before and after ablation of a 120 × 120-μm region centered on the ablations in Rac1 +/− or Rac1 −/− fibroblast mice. n = 3 mice for each column. p = 0.0049. Error bars are SD.

(F) Revisits of Rac1 +/− (top) and Rac1 −/− (bottom) following laser ablation at 2 weeks after high-dose tamoxifen (maximal recombination of both Rac1 and mTmG). Rac1 +/− reoccupies the ablated region similar to wild-type (E), while Rac1 −/− fails to do so. Insets (far right column) show details of the region highlighted at +3 weeks. n = 3 mice for Rac1 +/− and n = 3 mice for Rac1 −/− . Quantification is shown in (G). Scale bar, 20 μm. Scale bar, 20 μm (insets).

(E) Laser ablation of membrane-unlabeled cells (pink dots) and revisits of labeled neighbors. Membranes from neighboring cells extend into the ablated region up until +5 days. Membranes remain present at +3 weeks. n = 3 mice. Scale bar, 20 μm.

(B) Laser ablation and revisits of high-dose tamoxifen membrane labeling. Laser ablated nuclei (pink dots, before ablation). +2 weeks, ablated area remains depleted of nuclei, but filled with membrane. Membrane occupancy before versus +2 weeks is similar (insets 1 and 2). Quantification is shown in (C). Scale bar, 20 μm. Inset scale bars, 10 μm.

To next understand how the fibroblast network occupies the dermal space, we combined nuclear labeling with high doses of tamoxifen to induce maximal membrane labeling (PDGFRα-H2BGFP; PDGFRα-CreER; mTmG). By these means, we found that 98.2% ±1.0% of the cells were recombined for membrane labeling ( Figure S2 B). With this labeling, we found that upper dermal fibroblasts occupy 77% ±1.5% SEM of this dermal region ( Figures 2 D and 2G). Given the dynamic nature of fibroblast membranes, we wondered whether this membrane occupancy of the dermal space is actively maintained. To this end, we sought to conditionally delete the actin cytoskeletal remodeling GTPase, Rac1, within the fibroblasts (PDGFRα-H2BGFP; PDGFRα-CreER; mTmG; Rac1or Rac1) (). Using a high tamoxifen dosage to both maximally label the membrane and delete Rac1 ( Figure S2 C), we observed that, after 3–4 months, Rac1mice had significantly reduced membrane occupancy as compared to Rac1, or background strain control mice, without a change in fibroblast nuclear density ( Figures 2 E–2G and S2 D). Altogether, these data show that fibroblast membranes actively maintain occupancy of a large portion of the dermal space via a Rac1-dependent mechanism.

(D) The exact same dataset as used in Figures 2 E and 2G, but separated into separate columns for each replicate in order to show homeostatic membrane coverage variation across Rac1and Rac1. Each column, e.g., “g267m1” represents an individual mouse. Each dot represents an individual sample within that mouse. Samples were averaged for each mouse and presented in Figure 2 G.

(C) Representative genotyping gel showing the unrecombined and recombined (+tam) Rac1 mice using DNA primers specific for Rac1(left 8 lanes) and Rac1(right 8 lanes). Alleles are recombined with 3 doses of 60mg/kg body weight Tamoxifen over 6 days. Primers PO33 and PO91 were used to detect both Rac1(115bp) and Rac1(242bp) (right 8 lanes). Primers PO33 and PO45 were used to detect the presence of Rac1(150bp) (left 8 lanes) ().

(B) Quantification of the number of PDGFRα-H2BGFP positive, membrane-GFP negative cells remaining after maximum Tamoxifen induction (3 doses of 60 mg/kg body weight) in PDGFRα-H2BGFP; PDGFRα-CreER; mTmG mice. The y axis is the number of membrane unlabeled fibroblasts per 100 fibroblasts. The average number of recombined fibroblasts is 98.2% +/−1.0% of the total (PDGFRα-H2BGFP). PDGFRα-H2BGFP labels all fibroblasts in the skin. n = 3 mice.

(A) Exactly the same images as Figure 2 A (upper row), but showing both green (membrane-GFP) and red (membrane-tdTomato) channels. PDGFRα-CreER; R26-loxP-membraneTomato-STOP-membraneGFP (mTmG). The membrane-tdTomato labeling is less detectable and is expressed by all cells and so we typically present only the fibroblast specific membrane-GFP labeling of the mTmG construct. Furthermore, we find that a grayscale projection of membrane-GFP enables a better appreciation of the fine detail of the membrane.

Previous work has shown that skin fibroblasts contact each other, thus forming a large network throughout the dermis (). Therefore, to better understand the lack of response when observing fibroblast nuclei, we next sought to investigate the membranes of individually labeled fibroblasts. To this end, we utilized an inducible Cre-dependent fluorescent reporter (PDGFRα-CreER; Rosa26-loxP-membraneTomato-(stop)-membraneGFP (mTmG)) () and modulated the dose of tamoxifen to distinguish individual fibroblasts. We observed that fibroblasts were polarized to have larger area in the XY dimension than the XZ dimension ( Figure 2 A). Tracking the membranes of individual cells over a period of 2 weeks, we observed that cell shape and position were largely stable, similar to our findings with fibroblast nuclei ( Figures 2 A, 1 A, and 1B). However, we noticed membrane changes on a smaller scale and therefore sought to investigate the dynamics of these changes. Time-lapse recordings showed that fibroblast membranes are highly dynamic, with protrusions that rapidly grow and shrink from the more stable cell body ( Figures 2 B and 2C; Videos S2 and S3 ). While some areas of the cell peripheries were more active, all regions showed some level of movement ( Videos S2 and S3 ). Altogether, while stable in overall position, skin fibroblasts have highly dynamic cell membranes when observed in their native environment in an intact mouse.

(F) Density of nuclei between Rac1 +/− and Rac1 −/− . Data are sampled from regions ∼500 × 500 μm. Error bars are SD. n = 3 mice for each bar.

(E) Images of fibroblast nuclei (top), nuclei + membrane (middle) and inset (bottom) in Rac1 +/− (left) and Rac1 −/− (right) fibroblasts 3–4 months after high-dose tamoxifen. n = 3 mice for Rac1 +/− and n = 3 mice for Rac1 −/− . Quantification is shown in (G). Scale bar, 20 μm. Scale bar, 20 μm (insets).

(D) Image of fibroblast nuclei (top), nuclei + membrane (middle) and inset (bottom) in upper dermis. PDGFRα-H2BGFP labels all fibroblast nuclei. Membrane labeling is induced by CreER recombination of mTmG in 98.2% ±1.0% of cells ( Figure S2 B). Images are max projections of 10 μm. n = 3 mice. Quantification is shown in (G). Scale bar, 20 μm. Scale bar, 20 μm (inset).

(B) Frames from time-lapse of membrane-GFP upper dermal fibroblast (full movie: Video S2 ). Minutes (′) within the time-lapse are written in bottom right of orange boxed insets. Scale bar, 20 μm. n = 3 movies of 3 mice.

In order to test the ability of fibroblasts to maintain this stable position, we individually eliminated cells by laser ablation (see STAR Methods ) and observed the response of the remaining neighbors ( Figures 1 C and 1D). Surprisingly, fibroblasts at different depths appeared not to respond to the loss of twenty to thirty neighboring cells and remained in the same positions for at least 2 weeks, demonstrating a similar stability to what we observed in unperturbed conditions ( Figures 1 E, 1F, and 1H). While this approach provided the advantage of selecting the cells we wished to ablate, it may have also led to the unwanted damage of surrounding extracellular structures and/or neighboring unlabeled cells. To this end, we turned to a complementary genetic approach that could more widely control the loss of cells via inducible Cre-dependent diphtheria toxin (DTA) expression (PDGFRα-CreER; Rosa26-loxP-eGFP-(stop)-DTA) (). By eliminating cells with DTA, we again observed that the remaining cells did not appear to respond to the reduction in fibroblast number ( Figures 1 G and 1H). These results highlight an unexpected tolerance of fibroblasts to a loss of neighboring cells and a lack of response to re-establish cell number.

Surprisingly, revisits of the same fibroblast nuclei over days to weeks showed that these cells are remarkably stable in their position in 4- to 8-week adult mice ( Figures 1 A and 1B ). To quantify the results, we compared the pixel data between images across a 2-week time period and measured the degree to which the pixels exactly matched ( Figure 1 A, right column). Image correlation coefficients comparing time points show a strong positive correlation (0.55–0.75) when compared to the coefficients generated by comparing randomly different locations (−0.05 to +0.05) ( Figures 1 B and S1 C). While at greater depths in the lower dermis we noticed some movement of nuclei, the movement was comparable to that obtained at the same depth when remounting and reimaging the paw within a few minutes of the first image ( Figure 1 B). Altogether, these data show that fibroblast position is remarkably stable in unperturbed non-hairy skin.

(H) Image correlation coefficients of data in (E)–(G) between +1 day and +2 weeks (laser), or +2 weeks and +3 weeks (DTA), in the upper and lower dermis. Image correlation coefficients are similar to image correlation coefficients in unperturbed tissue (B). Error bars are SD. n = 3 mice for each bar.

(G) Revisits using genetically inducible ablation. Following low-dose tamoxifen, random cells (pink nuclei) express DTA and die. Before tam, upper dermis before tamoxifen. +2 weeks and +3 weeks, the same area at respective time point. Some recombination occurs without tamoxifen at a basal rate (see STAR Methods ), and some cells continue to be lost between +2 weeks and +3 weeks (red only cells in merge). Quantification is shown in (H) (DTA, upper). n = 3 mice. Scale bar, 20 μm.

(F) Revisits within the lower dermal layer before and after laser ablation. +2 weeks, remaining nuclei remain largely stable in position, similar to equivalent depth in (A) and (B). Quantification is shown in (H) (laser, lower). n = 3 mice. Scale bar, 20 μm.

(E) Revisits within the upper dermal layer before and after laser ablation (pink dots). +1 day, the ablated cells are absent. +2 weeks, the remaining nuclei remain largely stable in position, the depleted region is not filled in, and the fibroblast cell number remains reduced. Quantification is shown in (H) (laser, upper). n = 3 mice. Scale bar, 20 μm.

(D) Revisits of single-cell laser ablation (see STAR Methods ). Damage is visible by photo-bleached square. Three individually ablated nuclei are shown (pink dots) and are absent by 1 day later. Scale bar, 5 μm.

(B) “Identical” (r = 1.0) is the coefficient of an image compared to itself. +2, coefficient of day 0 versus +2 weeks (as in A). Re., coefficient between two remounts of the same location (pos ctrl). Ra., random images at same depth (neg ctrl) (see STAR Methods ). Positive correlation means a high overlap between signals. Zero correlation means a random overlap. Error bars are SD. n = 3 mice for 2-week data. n = 4 regions for remount. n = 3 regions for random.

In order to understand the principles of fibroblast homeostasis in unperturbed skin, we visualized and tracked individual fibroblasts over time by using the intravital imaging approach previously developed in our lab (). We distinguished skin fibroblasts using an established marker, PDGFRα-H2BGFP (histone H2B fused with GFP and knocked into the PDGFRα locus under that promoter) (). As most regions of the skin organ are a complex organization of many sub-structures, we first sought to use a simplified system by focusing on the skin on the base of the paw, which is devoid of hair follicles or other appendages and mature adipocytes ( Figure S1 A) (). We captured dermal fibroblasts by spanning the entire space from just below the epidermis through the upper and lower dermal regions, down to the skeletal muscle ( Video S1 Figure S1 B).

(B) Frames from Video S1 , which is a live imaged Z stack of live mouse paw dermis of PDGFRα-H2BGFP; mTmG. Epidermal cells (column 1) are visibly labeled with membrane-tdTomato. Capillary blood vessels, used as landmarks, are visible in upper dermis (column 2), along with green fibroblast nuclei (PDGFRα-H2BGFP) and collagen (second harmonic signal, described above). Thicker blood vessels are visible in lower dermis (column 3). Skeletal muscle layer is visible in column 4. While mTmG (membrane-tdTomato in the unrecombined form) labels all cells, certain cells are particularly easily detectable, such as epidermis, blood vessels and muscle. Fibroblast membrane-tdTomato is visible, but is difficult to distinguish from neighboring cells (row 2 columns 2 and 3). Therefore, we typically show only fibroblast specific recombined membrane-GFP in subsequent images. Scale bar 50μm. Scale bar 20μm (insets).

(A) A side by side comparison of representative H&E staining of non-hairy underside paw cryosection (left image) and representative Z-projection (∼0.5μm thick) of live imaged mouse paw labeled with alleles PDGFRα-H2BGFP and mTmG and second harmonic signal (SHG). Epidermis, dermis, and underlying skeletal muscle are labeled. Second harmonic signal is light produced at half the incident wavelength from a 2-photon source by certain molecules without any exogenous labeling. Typically, structural molecules, such as collagens emit this light, and it is captured with our imaging conditions in the blue channel. Scale bars 20μm.

Discussion

Organ function relies on the critical cellular behaviors of the different tissue types. Yet, what the principles are that maintain homeostasis in vivo have been elusive. One cell type present in most of our organs is the fibroblast. Despite the abundance and central role of fibroblasts across our bodies, it has been difficult to elucidate how they reside and sustain their tissue, in part, due to the inability to follow the same cells over time in a live uninjured mammal. Here, we overcame this problem by using intravital imaging and directly tracked and manipulated skin fibroblasts in live mice. This study elucidated positional stability and space occupancy as core principles that govern fibroblast homeostasis in vivo. Furthermore, we identified dynamic membrane extension, in the absence of cell migration, as a mechanism that preserves this positional stability while simultaneously enabling space occupancy as a lifelong principle of skin homeostasis.

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Anderson R.R. Fractional photothermolysis: A new concept for cutaneous remodeling using microscopic patterns of thermal injury. The perdurant alteration of tissue architecture resulting from cell loss and membrane extension is particularly insightful in the context of the aging phenotype of the skin. Studies on human skin comparing young versus old individuals noted that older individuals had fewer fibroblasts and with a greater number of longer membrane projections than younger ones (). Our experiments in older mice add important insights to this process by showing that cellular depletion does not occur uniformly, but as an accumulation of clustered cell losses—perhaps a lifetime of localized subtle traumas written permanently into the dermis. However, whether membrane extensions can maintain the macroscopic properties of skin with the same capacity as the uniform organization of fibroblasts is still to be determined, and the correlation of more gaps with the appearance of wrinkles could suggest that membrane occupancy alone is not sufficient. Interestingly, controlled dermal damage that induces fibroblast proliferation and migration is used clinically to alleviate some age associated skin phenotypes such as wrinkles (). Together, this suggests that the tolerance of fibroblasts to reduced cell number contributes to the tendency of the skin to age. Perhaps aging in other non-proliferative cell types may proceed along a similar course.

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Watt F.M. Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin. Establishing the principle of stability of fibroblasts enabled us to recognize that positional stability can be lost in the skin of physiologically remodeling hair follicles, without wounds and inflammation. This provides evidence of new pathways for controlled fibroblast activation without the excessive proliferation and fibrosis of inflammation-activated fibroblasts and may be similar to a previously described pathway triggering neonatal fibroblast behaviors in adults (). Activation of this alternative fibroblast pathway could provide avenues for translational therapies in wound healing and aging that enable the benefits of laser therapy with lower cost and patient inconvenience and more frequent and earlier interventions.

In conclusion, this work elucidates the core principles of positional stability and space occupancy that govern fibroblast homeostasis in vivo and identifies membrane extension without cell migration as a behavioral mechanism that facilitates these principles. These principles can be used to guide the development of future in vitro studies aiming to recreate the conditions of the in vivo environment and also future in vivo work that studies the consequences of these principles in aging, or the differences in fibroblast behaviors in disease states such as fibrosis and cancer.