CD201+ fascia progenitors choreograph injury repair.

Optimal tissue recovery and organismal survival are achieved by spatiotemporal tuning of tissue inflammation, contraction and scar formation1. Here we identify a multipotent fibroblast progenitor marked by CD201 expression in the fascia, the deepest connective tissue layer of the skin. Using skin injury models in mice, single-cell transcriptomics and genetic lineage tracing, ablation and gene deletion models, we demonstrate that CD201+ progenitors control the pace of wound healing by generating multiple specialized cell types, from proinflammatory fibroblasts to myofibroblasts, in a spatiotemporally tuned sequence. We identified retinoic acid and hypoxia signalling as the entry checkpoints into proinflammatory and myofibroblast states. Modulating CD201+ progenitor differentiation impaired the spatiotemporal appearances of fibroblasts and chronically delayed wound healing. The discovery of proinflammatory and myofibroblast progenitors and their differentiation pathways provide a new roadmap to understand and clinically treat impaired wound healing.

In vivo fluorescent labeling and tracking of extracellular matrix.

Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N-hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM 'tagging' with fluorophores using N-hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics.

Wound infiltrating adipocytes are not myofibroblasts.

The origins of wound myofibroblasts and scar tissue remains unclear, but it is assumed to involve conversion of adipocytes into myofibroblasts. Here, we directly explore the potential plasticity of adipocytes and fibroblasts after skin injury. Using genetic lineage tracing and live imaging in explants and in wounded animals, we observe that injury induces a transient migratory state in adipocytes with vastly distinct cell migration patterns and behaviours from fibroblasts. Furthermore, migratory adipocytes, do not contribute to scar formation and remain non-fibrogenic in vitro, in vivo and upon transplantation into wounds in animals. Using single-cell and bulk transcriptomics we confirm that wound adipocytes do not convert into fibrogenic myofibroblasts. In summary, the injury-induced migratory adipocytes remain lineage-restricted and do not converge or reprogram into a fibrosing phenotype. These findings broadly impact basic and translational strategies in the regenerative medicine field, including clinical interventions for wound repair, diabetes, and fibrotic pathologies.

Cutting into wound repair.

The skin is home to an assortment of fibroblastic lineages that shape the wound repair response toward scars or regeneration. In this review, we discuss the distinct embryonic origins, anatomic locations, and functions of fibroblastic lineages, and how these distinct lineages of fibroblasts dictate the skin's wound response across injury depths, anatomic locations, and embryonic development to promote either scarring or regeneration. We highlight the supportive role of the fascia in dictating scarring outcomes; we then discuss recent findings that indicate fascia mobilization by its resident fibroblasts supersede the classical de novo deposition program of wound matrix formation. These recent findings reconfigure our traditional view of wound repair and present exciting new therapeutic avenues to treat scarring and fibrosis across a range of medical settings.

Fibroblasts as confederates of the immune system.

Fibroblastic stromal cells are as diverse, in origin and function, as the niches they fashion in the mammalian body. This cellular variety impacts the spectrum of responses elicited by the immune system. Fibroblast influence on the immune system keeps evolving our perspective on fibroblast roles and functions beyond just a passive structural part of organs. This review discusses the foundations of fibroblastic stromal-immune crosstalk, under the scope of stromal heterogeneity as a basis for tissue-specific tutoring of the immune system. Focusing on the skin as a relevant immunological organ, we detail the complex interactions between distinct fibroblast populations and immune cells that occur during homeostasis, injury repair, scarring, and disease. We further review the relevance of fibroblastic stromal cell heterogeneity and how this heterogeneity is central to regulate the immune system from its inception during embryonic development into adulthood.

Connexin43 gap junction drives fascia mobilization and repair of deep skin wounds.

Deep and voluminous skin wounds are repaired with scars, by mobilization of fibroblasts and extracellular matrix from fascia, deep below the skin. The molecular trigger of this novel repair mechanism is incompletely understood. Here we reveal that the gap junction alpha-1 protein (Connexin43, Cx43) is the key to patch repair of deep wounds. By combining full-thickness wound models with fibroblast lineage specific transgenic lines, we show Cx43 expression is substantially upregulated in specialized fibroblasts of the fascia deep beneath the skin that are responsible for scar formation. Using live imaging of fascia fibroblasts and fate tracing of the fascia extracellular matrix we show that Cx43 inhibition disrupts calcium oscillations in cultured fibroblasts and that this inhibits collective migration of fascia EPFs necessary to mobilize fascia matrix into open wounds. Cell-cell communication through Cx43 thus mediates matrix movement and scar formation, and is necessary for patch repair of voluminous wounds. These mechanistic findings have broad clinical implications toward treating fibrosis, aggravated scarring and impaired wound healing.

Injury triggers fascia fibroblast collective cell migration to drive scar formation through N-cadherin.

Scars are more severe when the subcutaneous fascia beneath the dermis is injured upon surgical or traumatic wounding. Here, we present a detailed analysis of fascia cell mobilisation by using deep tissue intravital live imaging of acute surgical wounds, fibroblast lineage-specific transgenic mice, and skin-fascia explants (scar-like tissue in a dish - SCAD). We observe that injury triggers a swarming-like collective cell migration of fascia fibroblasts that progressively contracts the skin and form scars. Swarming is exclusive to fascia fibroblasts, and requires the upregulation of N-cadherin. Both swarming and N-cadherin expression are absent from fibroblasts in the upper skin layers and the oral mucosa, tissues that repair wounds with minimal scar. Impeding N-cadherin binding inhibits swarming and skin contraction, and leads to reduced scarring in SCADs and in animals. Fibroblast swarming and N-cadherin thus provide therapeutic avenues to curtail fascia mobilisation and pathological fibrotic responses across a range of medical settings.

Patch repair of deep wounds by mobilized fascia.

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism1-5. Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.

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

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.