Fibronectin meshwork controls epithelial stem cell fate

  1. Soline Estrach
  2. Lionel Tosello
  3. Floriane S. Tissot
  4. Laurence Cailleteau
  5. Ludovic Cervera
  6. Kim B Jensen
  7. Chloé C. Féral

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Abstract

Adult stem cell fate is tightly balanced by the local microenvironment called niche and sustain tissue regeneration 1–4 . How niche signals are integrated and regulate regeneration remains largely unexplored. The extracellular matrix and integrin ligand fibronectin is a crucial and well-characterized wound healing actor that has never been involved in skin regeneration. Here, we show that fibronectin displays a highly specific enrichment in hair follicle stem cells (HFSC) at the onset of regeneration. Conditional deletion of fibronectin in HFSC compartment (Lrig1, K19) leads to hair regeneration blockade, impaired stem cell location and fate. Dermal injection of exogenous fibronectin rescues these phenotypes. To elucidate molecular mechanism underlying fibronectin function, we used conditional deletion models of SLC3A2, the main integrin coreceptor. We show that, via its role in integrin-dependent assembly of fibronectin matrix, SLC3A2 acts as molecular relay of niche signals. Thus, fibronectin-integrin-SLC3A2 cascade finely tunes HFSC fate and tissue regenerative power.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Estrach and colleagues seek to identify the ECM components that are key to regulating hair follicle stem cell (HFSC) activation using the highly-characterized mouse hair follicle as a model. They first use a targeted approach to examine key ECM components expressed by HFSC and find that Fibronectin (FN) is highly expressed. Further, wholemount analysis of the hair follicle reveals a meshwork of FN enveloping the hair follicle. They hypothesize that FN is a fundamental regulator of hair follicle (HF) cycling and then proceed to carry out longterm studies required to examine hair follicle cycling and knockout FN with two different HFSC Cre lines (Lrig1 and Krt19), as well as integrin coreceptor SLC3A2. They clearly show that absence of Fibronectin (FN) and SLC3A2 is detrimental to hair follicle stem cell activation and cycling (FN) and hair follicle identity (SLC3A2).

    Overall comments:

    The authors use the tail hair follicles as a model similarly to the highly-characterized, synchronous back skin hair follicles. However, the tail hair follicles are asynchronous (Braun et al. 2003, PMID: 12954714), thus reporting the age of the mouse from which the tail whole mounts came from is not sufficient to claim a HF cycle disorder - HF should be imaged in an unbiased manner and subsequently quantified for phase. The manuscript would greatly benefit from including more information in the figure legends, such as age of mice, number of mice and HF quantified, as well as what the error bars represent. Further, in samples where many HF were counted per mouse, these should be averaged and then the average per mouse displayed; super plots would be great to use here.

    Major comments:

    1. In Figure 1, the use of tail whole mount images indeed provides striking display of the fibronectin meshwork that envelops the hair follicle. However, addition of a marker of the regenerative phase (e.g. proliferation) and resting phase would provide more convincing evidence that this is the particular phase of the hair cycle that you have captured, especially given my overall comment regarding the asynchronous nature of the tail HF cycle.
    2. The authors show that FN is expressed in early-mid anagen and conclude that FN is a regenerative signal. This claim should be substantiated with FN staining on more time points across the HF cycle to substantiate the argument that it is a regeneration-specific signal, found only in the telogen-anagen transition.
    3. Lrig1-cre and K19-cre-mediated FN knockout result in HF that are thinner at D158 - this is not immediately apparent from histological sections. Can you use your thick sections to give better perspective?
    4. The authors measure the width of the infundibulum from lightsheet microscope images. It is a bit difficult to position whole tissues using this technique, and the images that are shown are not from the same perspective, and thus measurement of the width is not accurate from these images. I suggest either removing this analysis or using more comparable images. Further, if this is a true phenotype, can you speculate on what the thickened infundibulum might mean?
    5. The authors then show mislocalization of Lrig1+ cells to the infundibulum in absence of FN. Are other stem markers localized to the infundibulum or outside of the bulge? Further, what might the mislocalization of Lrig1+ cells might mean?
    6. Please explain your conclusion after Figure 3i and at the end of the manuscript that states that FN is required for stem cell anchorage. I think that a very plausible explanation is that FN is required for stem cell function and identity, but anchorage of the SC lacks sufficient evidence. Further, your only evidence to support the anchoring theory only comes from expression of Lrig1 in FN knockout and no other markers. Are they also mislocalized? Please either tone down this conclusion on SC anchorage or provide stainings for more SC markers to show mislocalization in absence of FN.
    7. In Figure 3l-o, you examine proliferation on the control vs the conditional deletion of FN in D30 and D158. However, in D30, these tissues are not at all directly comparable since one is obviously in anagen and the knockout in telogen. You must compare the anagen knockout sample, although this occurs a bit later than the control. Further, how was the infundibulum distinguished from the bulb in these control images?
    8. In Figure 3P, you carry out RT-qPCR on whole skin to detect HFSC markers. This should have been carried out on sorted epithelial cells as isolation of whole back skin introduces bias to the system in that the number of stem cells may artificially look different in skin that is in anagen vs skin that is in telogen as the anagen skin has a different proportion of SC to progenitor cells to dermal cells. This concern is also similar to point 9 - the control and FN knockout at D30 are not comparable given that they are in different phases of the hair cycle.
    9. Figure 4a these images need to be of the whole mouse - it is not possible to determine what we are looking at or where - there is not even a scale bar.
    10. After Figure 4, you argue that because fibronectin expression resolves from healing dermis is the reason that hair follicles do not form, and site Dekonick and Blanpain (PMID: 30602767) - however this review makes no mention of the dynamics of fibronectin in wound healing. Further, evidence from Driskell et al (2013, PMID: 30602767) would suggest that it is the fibroblast population that responds to the wound that determines whether HF regenerate. And further, very large wounds do regenerate HF (Ito et al PMID: 30602767). In addition, this would all be fibroblast-derived FN, as opposed to the current study which examines keratinocyte-derived FN. Please reconsider this argument.
    11. The authors knockout SLC3A2, an integrin coreceptor that is localized to the plasma membrane. They show a very similar, yet more severe phenotype to the Lrig1- and K19- mediated knockout of FN. Given the bidirectional communication that SLC3A2 is responsible for, can you reconcile whether the defects in the HF cycle and the HFSC are a result of outside-in or inside-out signaling? Further, is it possible that integrin function regulated by SLC3A2 is necessary for more than FN assembly? This could be especially relevant given that your targeted screen also identified Col17A1, which is well known to be required for HFSC function (Matsumura et al., PMID: 26912707)
    12. It is intriguing that in the absence of HFSC-derived SLC3A2 that no FN network forms. Is FN expressed or is the assembly perturbed in the absence of properly functioning integrins? The authors conclude that the signaling cascade flows from fibronectin to integrin to SLC3A2, but do not test where the FN phenotype arises in the SLC3A2 knockout - is it due to aberrant assembly of the FN meshwork or a change in transcriptional or translational levels?
    13. In the grafting assay in Supplemental Figure 3, keratinocytes undergo a de novo hair follicle morphogenesis - is Lrig1 expression maintained in order to carry out cre-mediated deletion? Further, the fibroblasts in this assay may adopt a wound-like phenotype, expressing FN, which you earlier claim to be required for hair follicle production in wounds. Yet in the absence of epithelial FN, no HF form. Can the authors reconcile this?

    Minor comments:

    1. In Figure 1a, the two populations are Lgr5+ and basal; please define what the basal population is in this experiment.
    2. Significative is not a word.
    3. In Figure 4 figure legend, there is reference to a grafting experiment but no experiment shown.
    4. The authors delete FN in Lrig1+ or K19+ cells starting D19 and harvest at D30, and conclude that the hair follicles do not enter anagen after the second telogen, can you please include the data supporting the statement that mutant HF did not reenter the hair cycle after D65.

    Significance

    The authors show for the first time that fibronectin is expressed during cutaneous homeostasis and that it is required for normal function of the hair follicle stem cells. This is significant conceptual advance for the field of skin biology because fibronectin is thought to only be present in wounds: derived first from infiltrating serum and second from fibroblasts to act as provisional dermal ECM to support epithelialization during wound-response, which is ultimately resolved upon the conclusion of wound healing (reviewed in: Singer and Clark, PMID: 10471461). Further, FN has also been characterized as an EMT marker during cancerous progression (Lamouille et al, PMID: 24556840). Estrach and colleagues show that fibronectin is actually expressed by hair follicle stem cell keratinocytes and then is assembled into a meshwork that envelops the hair follicle and is in fact necessary for hair follicle stem cell homeostasis. This work would be broadly interesting to the field of stem cell biology as well as those working on extra cellular matrix signaling. My field is epithelial stem cells and more specifically hair follicle development and cycling.

    Referee Cross-commenting

    I have no disagreement with any of the points raised by the other reviewers. In fact, we seem to agree on the majority of the concerns. This includes the use of the tail wholemount model, the use of Lrig1-cre, selection of timepoint vs phase of the hair cycle, the appropriateness of the link between Fibronectin and SLC3A2, and further significant issues related to display of data and their reproducibility. Further, all of the major comments raised need to be addressed in order to properly evaluate the conclusions that the authors make. In my opinion, none of the comments raised here are unreasonable.

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    Referee #2

    Evidence, reproducibility and clarity

    In this manuscript, Estrach et al., investigated whether extracellular matrix component fibronectin function in hair follicle regeneration, using a range of approaches including FACS, RT-qPCR, immunofluorescent staining, and mouse genetics. They proposed that fibronectin in Lrig1+ cells was necessary for hair follicle stem cell maintenance and activation, and the fibronectin expression and assembly relayed on the integrin co-receptor SLC3A2.

    Significance

    Major points:

    1. In Figure 1a, the author used Lrig1+GFP and a6 to isolate Lrig1+ cells in the infundibulum junction zone above the sebaceous gland at Day 28. However, in Figure 1f-h, the GFP expression was not only in infundibulum above SG, but also in some inner root sheath cells. Since the Lrig1+ cells do not include the hair follicle stem cells (CD34+ bulge cells), result in Figure 1a does not support fibronectin expression in HFSCs.
    2. In Figure 1b-e, the author detected fibronectin expression by IF staining with tail skin whole mount and back skin section. The fibronectin is mainly detected in differentiated cells in the inner root sheath (IRS) in anagen (Figure 1b and 1d), upper IRS in catagen (Figure 1c), hair germ in telogen (Figure 1e), but not in the bulge region (Figure 2i-l). Again, these results do not support fibronectin enrichment in HFSCs either.
    3. In Figure 2a-c, the author knocked out fibronectin in Lrig1+ cells with Lrig1-CreERT2, FN fl/fl mice, and then validated the knockout efficiency by IF staining. However, result shows the fibronectin expression was not only depleted in the GFP+ Lrig1+ cells, but also depleted in GFP- inner root sheath and matrix. Similarly in Figure 4n, fibronectin was only knocked out in Lrig1+ cells, however, result showed the fibronectin cannot be detected in any cell types in skin. The author should explain why fibronectin depletion in Lrig1+ cell lead to completely fibronectin depletion.
    4. In Figure 2r, by Flow cytometry, the author found a significative reduction of a6+CD34+ SC population when fibronectin is conditional knocked out in Lrig1+ cells. As the Lrig1+ cells and a6+CD34+ HFSCs are two distinct cell populations, the author needs to explain how fibronectin depletion in Lrig1+ cells affect the number and activation of HFSCs population.
    5. In Figure 3b-c and 2g-h, the author reported the HF thinning in Lrig1-CreERT cKO mice by back skin HE staining. However, by tail skin wholemount staining, the HF thinning was not observed in those cKO mice (Figure 3f-g; Figure 2k-l). The author needs to explain the discrepency. In addition to this, the low quality of HE staining and poor orientation of HF (Figure 2h and 3b), coupled with lack of quantification, made these results and conclusion unconvincing.

    Mini Points:

    1. Color information in each IF staining panel is only completely presented in Figure 4. It is incomplete or lost in other 4 Figures.
    2. In Figure 1a, FACS profile and representative figures are needed to demonstrate the author isolated the correct population at correct hair follicle stage.
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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    In the manuscript, Estrach et al addressed the role of skin epithelial stem cell-derived extracellular matrix in hair follicle regeneration. They found that Lrig1+ epithelial stem cells highly express fibronectin gene compared to other basal epithelial cells. Conditional deletion of fibronectin gene using Lrig1CreERt2-GFP or K19CreER (the latter is expressed in the bulge stem cells) resulted in hair follicle regeneration blockade, change in the expression pattern of Lrig1-GFP. Injection of fibronectin protein into the dermis of the conditional fibronectin mutants (Lrig1CreERt2-GFP) rescued the hair regeneration blockade phenotype. The authors also conditionally deleted SLC3A2, an integrin coreceptor, using the same CreER lines and found a decrease in fibronectin deposition and CD34+ bulge stem cell number. With the results from these mouse genetics and phenotype analysis, they conclude that fibronectin-SLC3A2 cascade finely tunes hair follicle stem cell fate and their tissue regenerative capacity.

    Major comments

    1 Immunostaining results of fibronectin in the tail epidermal wholemounts are not convincing enough and would require improvements. First, the tail epidermal wholemounts lack the mesenchymal matrix and the basement membrane (Fig. 1b, c, f-h; Fig. 2b, c; Fig. 5d-j; Fig. S1b, c) (Braun et al., 2003 Development (PMID: 12954714)). Fibronectin is localized mainly in the mesenchymal matrix and the basement membrane in the skin and other organs (Stenman and Vaheri, 1978 JEM (PMID: 650151); Couchman et al., 1979 Archives of Dermatological Research (PMID: 393184); Jahoda et al., 1992 J. Anat (PMID: 1294570)), thus this sample preparation method is not appropriate to assess fibronectin tissue distribution. The authors use thick back skin sections, which contain entire skin tissues, thus I would recommend this method. Furthermore, fibronectin antibody signals in the tail epidermal wholemounts are detected in the inner part of the hair follicle epithelium, where there is no expected ECM structure (see Couchman et al. and Jahoda et al. above). Consistently, fibronectin signals are localized inside the Lrig1-GFP+ epithelial basal cells (Fig. 1f-h). Thus the specificity of the fibronectin staining needs to be confirmed. The reviewer understands that the authors provide an image showing the great reduction of fibronectin staining in a D30 tail epidermal wholemount of 4-OHT-treated Lrig1CreERt2GFP,FNfl/fl mice (Fig. 2c). However, as the D65 tail epidermal wholemount from wildtype mice also show many hair follicles without fibronectin signals (Fig. 1c), rigorous assessments would be required.

    2 Lrig1+ stem cells have been reported to maintain the upper pilosebaceuos unit, containing the infundibulum and sebaceous gland, but contribute to neither the hair follicle nor the interfollicular epidermis under normal homeostatic condition (Page et al., 2013 Cell Stem Cell (PMID: 23954751)). However, only 11 days after the first 4-OHT treatment on Lrig1CreERt2GFP;FNfl/fl mice, Estrach et al found the defects in hair cycle blockade, reduced cell proliferation in the hair bulb, and significant reduction in fibronectin deposition in entire hair follicle structure. Please explain how the deletion of fibronectin gene in Lrig1+ stem cells, which do not contribute to hair follicle lineages, lead to significant hair regeneration defects in a short period of time. Current data do not well explain a causal relationship between the genetic perturbation and the observed phenotypes.

    3 In some experiments (listed below), description about the methods, replication and statistics is not adequate, raising concerns about reproducibility. 3.1 Fig. 1a: data variation for basal cells should be presented. Biological replicate number should also be indicated in the figure legend. 3.2 Fig. 2g, h: hair follicle thinning is described here, but only one HE staining image with only one hair follicle is not enough to support this important claim. 3.3 Fig. 2r, 3i: flow cytometric data should be presented. 3.4 Fig. 4: No biological replicate and reproducibility information are provided. 3.5 Fig. 5j: how many biological replicates and hair follicles were analysed? The authors should also perform statistical tests. 3.6 Fig. S3g, h: information for biological replicates should be described. Statistical tests should be applied to Fig. S3h. 3.7 Fig. 5k-n: only one HE staining panel from each mouse line cannot provide rigorous evidence of the defects, which are not obvious from the HE staining.

    4 In Fig. 3j, k, n, hair follicles in the control and 4-OHT treated skin are in different hair cycle phases. Therefore there is a possibility that the difference in their PCNA pattern simply reflects the difference in the cell proliferative activity between different hair cycle phases, but not indicates direct effects from the deletion of fibronectin gene in Lrig1+ cells.

    5 To assess the expression levels of signaling-related genes (Fig. 3p, S2), the authors used mRNA extracted from whole skin tissues, which contain all epithelial and mesenchymal cell populations in different hair cycle phases. Thus, the time and spatial resolution of the analysis is low and it also cannot eliminate confounding factors derived from the difference in hair cycle phases between control and cKO.

    6 In order to provide the characteristics and purity of the FACS isolated cell populations at D28 (Fig. 1a), their flow cytometry data and some marker gene expression data should be presented (see Page et al., 2013 Cell Stem Cell). This assessment is particularly important for the skin compared to other static organs, as it exhibits dynamic gene expression and tissue structural changes during the hair cycle. It is also important to check whether fibronectin protein accumulates around Lrig1+ stem cells in D28 dorsal skin, where upregulation of fibronectin gene expression was detected. The authors should not use tail epidermal wholemounts for the reason described above.

    Minor comments

    7 The increase in stem cell marker expressions shown in Fig. 3p contradicts to the reduction in the number of bulge stem cells shown in Fig. 2r and 3i. Please provide an explanation for this apparent discrepancy.

    8 Although Fig. 5s-v show reduction of a6+CD34+ bulge cell population, the bulge tissue structure can be observed in Fig. 5p. Please explain how to interpret this apparent discrepancy. They just lost the expression of CD34?

    9 Connectivity of the data in the fibronectin cKO with that of SLC3A2 cKO is weak. For example, it could be strengthened if the authors show colocalization of fibronectin and SLC3A2 in vivo.

    10 Although the format of the manuscript is free in Review Commons, the Introduction and Discussion of this manuscript are too brief for us to understand the background and significance of this study. So I would recommend the authors to provide more detailed background information and discussion.

    11 The authors use the term 'HFSC', but it is unclear which stem cell populations they mention; bulge, Lrig1+ or other stem cell populations?

    12 Please provide details of fibronectin protein and antibody used in this study.

    13 Due to the short for experimental information for Fig S3a, b, d, e, I cannot evaluate the data, thus several questions are raised. As the SLC3A2 level was significantly reduced in most cells in the plot, I assumed that Lrig1-GFP+ cells were gated before examining the expression level of SLC3A2. However, no information on the procedures for 4OHT treatment, isolation of cells and flow gating strategy is described. In the case of K19CreER mice (Fig. S3d, e), if the authors gated GFP+ cells before analysis, what GFP means in this case?

    14 The manuscript wants to be checked for copyediting.

    Significance

    These findings might provide a conceptual advance into the role of epithelial stem cell-derived extracellular matrix in regulating stem cell behaviour and tissue regeneration. As fibronectin is upregulated in development, wound healing and cancers in many other organs, their findings may point to the importance of fibronectin in activating tissue progenitors and stem cells in these processes. Thus, this manuscript is likely to be of interest to a wide range of readers, not only in skin biology, but also in stem cell, regenerative and matrix biology. The contributions of this paper could be enhanced if the documentation were to be made stronger and more rigorous in a revised manuscript.

    Referee Cross-commenting

    I totally agree with Reviewer #3's comments in this consultation session. I have no disagreement with any of the points raised by other reviewers.

  5. Version published to 10.1101/2021.05.26.445735v2 on bioRxiv
  6. Version published to 10.1101/2021.05.26.445735v1 on bioRxiv