Col4a2-eGFP mouse model reveals the molecular and functional dynamics of basement membrane remodelling in hair follicle morphogenesis

  1. Duligengaowa Wuergezhen
  2. Eleonore Gindroz
  3. Ritsuko Morita
  4. Kei Hashimoto
  5. Takaya Abe
  6. Hiroshi Kiyonari
  7. Hironobu Fujiwara

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Abstract

The precisely controlled remodelling of the basement membrane (BM) is considered vital for morphogenesis. However, the molecular and tissue-level dynamics of the BM during morphogenesis and their functional significance remain largely unknown, especially in mammals, due to limited visualization tools. We developed knock-in mouse lines in which the endogenous collagen IV gene ( Col4a2 ) was fused with a fluorescent tag. Through live imaging of developing hair follicles, we revealed a spatial gradient in the turnover rate of COL4A2 that is closely coupled with the BM expansion rate. The proliferation of epithelial progenitors coincided with the increased expansion of their underlying BM. Epithelial progenitors displaced with directionally expanding BM, but did not actively migrate on stable BM. The addition of a matrix metalloproteinase inhibitor delayed the turnover of COL4A2, restrained the expansion of the BM, and induced a directional shift in the division angle of epithelial progenitors, altering the hair follicle morphology. Our findings revealed spatially distinct BM dynamics within the continuous epithelial BM and affirmed their significance in orchestrating the proliferation, movement and fate of progenitor cells, as well as the macro-level shape of organs during development.

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    Referee #3

    Evidence, reproducibility and clarity

    Abe et al. generated multiple novel mouse model that follows and labels Col4a2 by fusing it with GFP. The creation of this mouse model allowed for the analysis of the basement membrane in real-time using live imaging of embryonic explant cultures. The combination of the mouse model with live imaging revealed new insights into how the basement membrane turnover occurs during early hair follicle development. After setting the stage the authors establish its utility with embryonic tissue, which revealed membrane buds that could be visualized with eGFP at different time intervals for 19h. The growth of the hair follicle bud during that time could be divided three distinct areas, the upper, lower stocks, and the tip. Where the tip and lower stalk increased in BM length, while the upper stalk decreased. BM length correlated with increased cellular replication. Using a double transgenic model system, they evaluated the differences between 'cell displacement', 'BM expansion', and 'cell autonomous movement'. Diving deeper with the Col4a2-mKiGR mouse model investigated Col4a2 turnover to reveal similar results mentioned above. Lastly MMP inhibitors were used to investigate how hair follicle growth is affected, which revealed inhibition of cellular replication and inhibition of Col4a2 turnover. These results inhibited hair follicle elongation which made the forming hair follicles thicker.

    Overall, this is an interesting paper for hair follicle biologists since it investigates the early moments of the budding follicle in real time using a ex vivo culture model. In addition, there is additional appeal because the authors use live imaging the hair follicle neogenesis as a basement membrane model, which is interesting and novel. The manuscript is clearly written, and the data support the overall conclusions. My comments below are to help in clarity which may be used to develop clearer figures and additional text.

    Major:

    In Figure 2 for the cell displacement and expansion part. I found the figure and text confusing, particularly in regard to how the data shows the bleached edge of the BM moving alongside the cells. Could the authors make this clearer in the figure and in the writing of the text?

    The authors created a Col4a2-mKikGR mouse line that is supposed to follow Col4a2 turnover. It is difficult to understand how the authors can claim that it was turnover with the explanation in the text of how the mouse model works. Could the authors write a better description of the mouse model in the text?

    Significance

    The strengths of the paper are the use of a novel mouse model that can track collagen (Col4a2) with a tagged GFP and the live imaging model system. This has led to a novel and important knowledge on the interplay between the BM and cells. The limitation of the manuscript is that the entire manuscript utilizes a single protocol (live imaging) to investigate BM dynamics. This might be due to the sophisticated nature of the model systems, but it is a limitation.

    To my knowledge the study is novel because of the mouse model to track Col4a2 dynamics with GFP. This model led to some interesting findings about hair follicle development in that different regions expand differently with the BM. This is good foundational knowledge using the latest state of the art techniques in live imaging of embryonic tissue culture systems.

    The audience that would be interested in this manuscript is broad, which would span cell and developmental biology but also specialists in membrane and protein biology that can finally see in real time the dynamics of Col4a2 building skin and hair.

    My expertise in in hair follicle development and repair and stem cell biology.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors successfully generated a knock-in mouse line expressing fluorescence-tagged endogenous collagen IV. Homozygous mice exhibited normal survival with no apparent developmental abnormalities. Through imaging of the back skin of COL4A2-eGFP mice in ex vivo culture, the authors observed a faster expansion rate of the basement membrane (BM) at the growing tips of developing hair follicles, while the upper stalk region showed a slower BM expansion rate. Real-time imaging also revealed that the BM and basal epithelial cells moved in the same direction. Fluorescence recovery after photobleaching (FRAP) and the photoconvertible protein mKikGR experiments demonstrated a faster turnover rate of COL4A2 at the tips of the hair follicles. The authors used MMP inhibitors to suppress the recovery of COL4A2-eGFP, BM expansion, and observed abnormalities in the developmental process of hair follicle morphology. The findings are novel and interesting, some aspects of how the experiments and quantifications were conducted should be clarified to allow the readers to fully appreciate the results.

    Major Comments:

    1. BM Expansion Statistics: Regarding the statistical analysis of BM expansion, is the length of the BM affected by the z-axis plane at the time of imaging? How is it ensured that the BM length measured at different time points corresponds to the same z-axis?
    2. Cell Movement Statistics (Figure 2): In the statistical analysis of cell movement in Figure 2, the lack of other references raises questions about ensuring that the cells measured at different time points are the same cells.
    3. The authors concluded that the inhibition of MMP delayed the recovery of COL4A2-eGFP after photobleaching, indicating the crucial role of MMP activity in the incorporation of COL4A2 into the BM. Does MMP inhibition lead to a reduction in the synthesis of the COL4A2 protein, thereby delaying the recovery of COL4A2 in the BM?

    Minor Comments:

    In Figure 1D, the representative image and the statistical graph are inconsistent. For example, in the upper stalk region, all basal epithelial cells have Ki67 signals according to the representative image.

    Significance

    The authors successfully generated knock-in mice expressing fluorescence-tagged endogenous collagen IV. With this novel mouse model, researchers can directly observe the dynamic changes in the extracellular matrix of different organs in mammals during development and disease, revealing the potential roles of the extracellular matrix.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    Wuergezhen et al. generated two fluorescently tagged Col4a2 mouse models, including EGFP-Col4a2 and the photoconvertible mKikGR-Col4a2. They showed that tagged collagen IV get incorporated in the basement membranes of various tissues in the developing and adult mice, and fully support embryonic development, fertility, and physiological functions. The authors followed up investigating Col4a2 dynamics during embryonic development of the hair follicle. Using FRAP, they showed that the basement membrane expands with a faster rate near the tip region. This coincided with faster turnover rates of Col4a2 at the tip relative to the lower and upper stalk. In addition, the authors demonstrated that Col4a2 turnover depends on MMPs.

    Major comments:

    • Are the key conclusions convincing?

    The major conclusions of the manuscript are convincing. These include that tagging collagen IV does not compromise its function, differential expansion of the basement membrane, differential turnover of the Col4a2, the MMP dependence for normal basement membrane expansion and turnover. However, some claims detailed below need to be clarified or addressed to improve the manuscript.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    In Figure 2B-C, the authors conclude that basement membrane expands at different rates, depending on the region with higher expansion rates near the hair follicle tip. From their methods, they conducted repetitive photobleaching cycles every 2-3 hours to maintain the bleached status of ROIs and tracked changes in basement membrane length. However, it sounds challenging to repetitively photobleach the same ROI in a dynamically expanding tissue, which may compromise the accuracy of length measurements. It would be helpful if the authors could provide movies corresponding to these experiments for clarity. In addition, this repetitive bleaching should be highlighted in the figure and figure legend, as readers could get confused about why there is no recovery here when comparing to the FRAP experiment.

    In Lines 180-198 and Figure 2F-H, the authors interpreted cell movement as contributed by cell-autonomous vs basement membrane expansion, which is speculative. Another possibility is that cell movement was all autonomous, while basement membrane expansion was caused by mechanical stretching that was in turn caused by cell proliferation. This conclusion should be rephrased.

    Following MMPi treatment, the authors found that basement membrane expansion was halted (Figure 4C-D). Yet, they later showed that hair follicles widened under MMPi treatment (Figure 4E-J). Under these conditions, I would have expected such widening to be accompanied by basement membrane expansion at the lower or upper stalk, which is not the case, at least for the first 7 hours (Figure 4C-D). How do the authors interpret this?

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    Considering the rapid expansion of BM near the tips, I wonder whether the authors have explored possible structural differences of the basement membrane in different regions? Is the basement membrane near the tips thinner and/or does it have microperforations as reported in other systems? Looking into this may further support their observations, not only in control conditions but also in MMPi treated follicles.

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

    Yes, for possible structural changes, 3D rendering of fluorescence imaging as shown in Figure 1J may be sufficient.

    Increasing sample number using existing mouse strains should also be feasible. It would take 2-3 weeks from setting up timed pregnant mice to imaging the embryonic skin explants. Adding image analysis and figure preparation, it should be doable within 1 month.

    • Are the data and the methods presented in such a way that they can be reproduced?

    In Figure 2D/ Figure 5C, it is unclear how ROIs corresponding to tip/lower/upper stalk are being drawn for quantification of Ki67+ cells.

    The images in 2B and 4C, 3A and 4E, are identical. Images should not be re-used in figures. This raised the concern that probably not sufficient samples were imaged to have different representative images. It should also be clarified where the data was re-used, for example, the control data in 2C and 4D, 3B and 4B.

    • Are the experiments adequately replicated and statistical analysis adequate?

    A few experiments have low sample numbers while the data was quite variable. These include Figure 2G-H (n = 3 cells), Figure 3C (unknown sample number), Figure 4B (n = 3 hair follicles), Figure 5B (n = 3 hair follicles). More sample numbers (~10 total) should be included to solidify the findings.

    For the mKikGR experiment in Figure 3C, quantification should be included. A ratiometric measurement of green/red fluorescence over time should be a good complementary way of demonstrating region-specific recovery.

    In Figure 5B, the authors claim that the percentage of dividing cells in control follicles being different from that of MMPi-treated follicles. How are they extracting these percentages? From the plots, control and MMPi-treated columns do not appear to be normalized as 100% to make such comparisons. Moreover, having two mean+/-SD in each column makes these data confusing to interpret. The authors should consider replotting their data either by combining their data into a unique population per conditions and reporting the percentages, or alternatively, they may consider splitting each of these columns into two (i.e., dividing vs. non-dividing cells), and comparing both conditions as ratios of dividing versus/non-dividing cells.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Already addressed above.

    • Are prior studies referenced appropriately?

    I believe so.

    • Are the text and figures clear and accurate?

    All plots measuring basement membrane length are labeled as 'increase in BM length', even when in some cases the BM length is reduced. The authors should consider relabeling these as 'BM length change' or something similar.

    The second and third paragraphs of the discussion are too long and should be condensed into a single paragraph.

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Use better color combination for visualization of multi-channel images. For instance, magenta/green is a better combination than red/green for the color blind. Color combinations are not consistent across figures in the manuscript.

    Include movies for all data derived from live imaging.

    Include statistical tests used for all plots, some are missing.

    The authors should consider fitting their FRAP data in each condition and report percentages corresponding to the mobile and immobile fractions.

    Examples of horizontal vs perpendicular cell division appear to be mislabeled in Video5.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
    • The large molecular weight of extracellular proteins makes it challenging to generate genetically engineered versions of such proteins to investigate their function and dynamics in vivo. The present study has addressed such issues for Col4a2, a major component of the basement membrane. This study further provides insights towards the understanding of BM dynamics during embryonic organ development.
    • Place the work in the context of the existing literature (provide references, where appropriate).
    • Addressed above
    • State what audience might be interested in and influenced by the reported findings.
    • Cell and developmental biology, extracellular matrix biology, organ development and regeneration.
    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.
    • Tissue morphogenesis, extracellular matrix
  5. Version published to 10.1101/2023.10.31.564866v1 on bioRxiv