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Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

ABSTRACT: Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at successive developmental stages identifies a population displaying a dual muscle and connective tissue signature. BMP signalling is active in this dual population and at the tendon/muscle interface. In vivo and in vitro gain- and loss-of-function experiments show that BMP signalling regulates a fibroblast-to-myoblast conversion. These results suggest a scenario in which BMP signalling converts a subset of lateral plate mesoderm-derived cells to a myogenic fate in order to create a boundary of fibroblast-derived myonuclei at the myotendinous junction that controls limb muscle patterning.

SUBMITTER: Esteves de Lima J

PROVIDER: S-EPMC8219714 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Esteves de Lima Joana J Blavet Cédrine C Bonnin Marie-Ange MA Hirsinger Estelle E Comai Glenda G Yvernogeau Laurent L Delfini Marie-Claire MC Bellenger Léa L Mella Sébastien S Nassari Sonya S Robin Catherine C Schweitzer Ronen R Fournier-Thibault Claire C Jaffredo Thierry T Tajbakhsh Shahragim S Relaix Frédéric F Duprez Delphine D

Nature communications 20210622 1

Positional information driving limb muscle patterning is contained in connective tissue fibroblasts but not in myogenic cells. Limb muscles originate from somites, while connective tissues originate from lateral plate mesoderm. With cell and genetic lineage tracing we challenge this model and identify an unexpected contribution of lateral plate-derived fibroblasts to the myogenic lineage, preferentially at the myotendinous junction. Analysis of single-cell RNA-sequencing data from whole limbs at ...[more]

PMID: 34158501

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Project description:BackgroundThe angiogenic response to ischemia restores perfusion so as to preserve tissue. A role for mesenchymal-to-endothelial transition in the angiogenic response is controversial. This study is to determine if resident fibroblasts contribute to angiogenesis.MethodsWe utilized the murine model of hindlimb ischemia, and in vivo Matrigel plug assay together with lineage tracing studies and single cell RNA-sequencing to examine the transcriptional and functional changes in fibroblasts in response to ischemia.ResultsLineage tracing using Fsp1-Cre: R26R-EYFP mice revealed the emergence within the ischemic hindlimb of a small subset of YFP+ CD144+ CD11b- fibroblasts (E* cells) that expressed endothelial cell (EC) genes. Subcutaneous administration of Matrigel in Fsp1-Cre: R26R-EYFP mice generated a plug that became vascularized within 5 days. Isolation of YFP+ CD11b- cells from the plug revealed a small subset of YFP+ CD144+ CD11b- E* cells which expressed EC genes. Pharmacological or genetic suppression of innate immune signaling reduced vascularity of the Matrigel plug and abrogated the generation of these E* cells. These studies were repeated using human fibroblasts, with fluorescence-activated cell sorting analysis revealing that a small percentage of human fibroblasts that were induced to express EC markers in Matrigel plug assay. Pharmacological suppression or genetic knockout of inflammatory signaling abolished the generation of E* cells, impaired perfusion recovery and increased tissue injury after femoral artery ligation. To further characterize these E* cells, single cell RNA-sequencing studies were performed and revealed 8 discrete clusters of cells expressing characteristic fibroblast genes, of which 2 clusters (C5 and C8) also expressed some EC genes. Ischemia of the hindlimb induced expansion of clusters C5 and C8. The C8 cells did not express CD144, nor did they form networks in Matrigel, but did generate angiogenic cytokines. The C5 fibroblasts most resembled E* cells in their expression of CD144 and their ability to form EC-like networks in Matrigel.ConclusionsTogether, these studies indicate the presence of subsets of tissue fibroblasts which seem poised to contribute to the angiogenic response. The expansion of these subsets with ischemia is dependent on activation of innate immune signaling and contributes to recovery of perfusion and preservation of ischemic tissue.

Project description:Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation. Both X-irradiation and thalidomide-induced phocomelia have been interpreted as patterning defects in the context of the progress zone model, which states that a cell's proximodistal identity is determined by the length of time spent in a distal limb region termed the 'progress zone'. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.

Dataset Information

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

Publications

Unexpected contribution of fibroblasts to muscle lineage as a mechanism for limb muscle patterning.

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