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Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring

Abstract

During fetal development, mammalian back-skin undergoes a natural transition in response to injury, from scarless regeneration to skin scarring. Here, we characterize dermal morphogenesis and follow two distinct embryonic fibroblast lineages, based on their history of expression of the engrailed 1 gene. We use single-cell fate-mapping, live three dimensional confocal imaging and in silico analysis coupled with immunolabelling to reveal unanticipated structural and regional complexity and dynamics within the dermis. We show that dermal development and regeneration are driven by engrailed 1-history-naive fibroblasts, whose numbers subsequently decline. Conversely, engrailed 1-history-positive fibroblasts possess scarring abilities at this early stage and their expansion later on drives scar emergence. The transition can be reversed, locally, by transplanting engrailed 1-naive cells. Thus, fibroblastic lineage replacement couples the decline of regeneration with the emergence of scarring and creates potential clinical avenues to reduce scarring.

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Fig. 1: EPFs replace ENFs during mouse back-skin development.
Fig. 2: EPFs direct dermis development.
Fig. 3: EPFs exhibit three migration behaviours in vivo.
Fig. 4: Dermal lattice is actively changing during development.
Fig. 5: Regeneration-to-scar transition is coupled to EPFs development.
Fig. 6: EPFs are a mature scar-matrix-depositing population.
Fig. 7: ENF and ECM transplantations improve scar outcomes.

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Acknowledgements

Y.R. was supported by the Human Frontier Science Program Career Development Award (CDA00017/2016), the German Research Foundation (RI 2787/1-1 AOBJ: 628819) and the Fritz–Thyssen–Stiftung (2016-01277). J.L. was supported by the China Scholarship Council. D.C.-G. was supported by the Consejo Nacional de Ciencia y Tecnología (CONACYT) and the Deutscher Akademischer Austauschdienst (DAAD).

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Contributions

Y.R. outlined and supervised the research narrative and designed all the experiments. D.J. performed the wound-healing studies, the flow cytometric analysis and sorting of EPFs and ENFs, the cell transplantations and decellularized skin transplantations. D.C.-G. and A.S. performed and analysed the clonal analysis studies and generated the confocal images. D.C.-G. performed the fractal analysis. S.C. and P.R. performed live-imaging and cell-tracking analysis. J.L. and V.R. assisted with clonal analysis, confocal imaging and histological sectioning. M.M.D.S. and D.E.W. generated the decellularized dermis. Y.R. wrote the manuscript.

Corresponding author

Correspondence to Yuval Rinkevich.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Flow cytometric analysis of mesenchymal markers on ENFs and EPFs.

a, single cell suspension was prepared from the pooled back-skin from three E12.5, three E15.5 and one E19.5 En1Cre;R26mTmG embryos, and subjected to flow cytometric analysis. b, Sytox blue was used to exclude dead cells, and APC-conjugated lineage markers (CD45, CD31, Ter119, EpCAM, Tie-2, Lyve-1) were used to exclude non-mesenchymal cells. c–e, representative analysis plots of fibro-adipogenic progenitors (FAPs) (Lin-ITGA7-Sca1+PDGFRa+) (c), pericytes (Lin-CD146+) (d), and mesenchymal stem cells (Lin-CD29+CD105+) (e), and their distributions in ENFs and EPFs. f-g, the percentages of ENFs and EPFs (f) and the ratio of ENFs:EPFs (g) within Lin- cells (initial), FAPs, pericytes and MSCs.

Supplementary Figure 2 Expression of mesoderm markers on ENFs and EPFs by immunofluorescence staining.

Cryosections were prepared from En1Cre;R26VT2/GK3 neonates (P1), and stained with primary antibodies against mouse FABP4 (a), Dlk1 (b), Sca1 (c), aSMA (d), TNC (e), and CD26 (f), and AF647-conjugated respective secondary antibodies. Depicted are representative high-power images of AF647 channel for antibodies (left), GFP-RFP channels for ENFs-EPFs (middle), and merged channels (right). Dotted lines indicate the border of epidermis and dermis, and the border of upper dermis (UD) and lower dermis (LD). Squares indicate the locations of enlarged inserts in merged images. Scales: 50?µm. The depicted are representative images of three individual embryos.

Supplementary Figure 3 EPFs associate with Collagen I fibers.

ad, Immunofluorescence for Collagen I of E11.5 (a), E12.5 (b), E14.5 (c), or E16.5 (d) En1Cre;R26VT2/GK3 embryos. e-h, EPF- or ENF-signal-derived ROI mean grey value (mgv) ± SEM of the Collagen I signal from anterior or posterior regions of E11.5 (e), E12.5 (f), E14.5 (g), or E16.5 (h) En1Cre;R26VT2/GK3 embryos. n?=?3 optical fields of anterior and posterior regions at each development stage. Two-tailed unpaired t-test. * p<0.05. Scale: 50?µm. Images represent 1 out of three optical fields. The exact p values are listed in Supplementary Table 1.

Supplementary Figure 4 EPFs actively displace provisional ENF dermis.

3D representations of 24 h time-lapse images of EPF-positive anterior region in E12.5 En1Cre;R26mTmG embryo (up). Max projection of whole live embryo showing the area analyzed (down). Scales: 100?µm and 500?µm, respectively. Images are taken from the one representative video used for cell tracking analysis showed in Fig. 3.

Supplementary Figure 5 Fractal analysis of ENFs organizations during development. and subsampled fractal analysis.

a, Graphical representation of the quantitative assessment of complexity and porosity in 2D images by Fractal analysis. b, Representative confocal images of clumped posterior E11.5 EPFs (up), and stretched anterior E12.5 (down) from En1Cre;R26VT2/GK3 embryos. c, Fractal dimension (up) and lacunarity (down) values derived from ENFs of different regions and developmental stages. Mean ± SEM. n?=?9 optical fields of anterior or posterior regions at each developmental stage. RM ANOVA, p?<?0.05, Newman-Keuls test. d, Subsampled (local) fractal analysis (right) of low-power magnification confocal images of E16.5 En1Cre;R26VT2/GK3 embryos (left). Scale: 50?µm. e, Fractal dimension (up) and lacunarity (down) values derived from ENFs of different regions and developmental stages, n?>?60 subsampled values pooled from three confocal images. ANOVA, Tukey test, *p?<?0.05. Box and whiskers plots with minimum, lower quartile, median, upper quartile, and maximum. The exact p values are listed in Supplementary Table 1.

Supplementary Figure 6 Fibronectin matrix stiffens concurrent with EPF expansion.

ad, Immunofluorescence for Fibronectin in E11.5 (a), E12.5 (b), E14.5 (c), or E16.5 (d) En1Cre;R26VT2/GK3 embryos. eh, Representative linear profiles of diagonal lines on the Fibronectin signal from E11.5 (e), E12.5 (f), E14.5 (g), or E16.5 (h) images. Dashed lines above the plots represent the calculated distances between fibers. Scale: 50?µm. Images are representatives of 3 optical fields.

Supplementary Figure 7 The fibroblastic lineage replacement drives the transition of dermal response to injury from regeneration to scarring.

ENFs are the primary sculptors that drive dermal lattice development and regeneration during early development, and that their cell lineage declines during development. EPFs that produce mature matrix fibres expand concurrently, and predispose back-skin to scarring.

Supplementary information

Supplementary Information

Supplementary Figures 1–7, Supplementary Table and Videos legends.

Reporting Summary

Supplementary Table 1

Statistics source data.

Supplementary Video 1

EPFs arch in the developing mouse embryo.

Supplementary Video 2

EPFs actively displace ENFs.

Supplementary Video 3

EPFs actively displace ENFs (only ENFs).

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Jiang, D., Correa-Gallegos, D., Christ, S. et al. Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring. Nat Cell Biol 20, 422–431 (2018). https://doi.org/10.1038/s41556-018-0073-8

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