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Transcriptomic analysis of bone and fibrous tissue morphogenesis during digit tip regeneration in the adult mouse.

ABSTRACT: Humans have limited regenerative potential of musculoskeletal tissues following limb or digit loss. The murine digit has been used to study mammalian regeneration, where stem/progenitor cells (the "blastema") completely regenerate the digit tip after distal, but not proximal, amputation. However, the molecular mechanisms responsible for this response remain to be determined. Here, we evaluated the spatiotemporal formation of bone and fibrous tissues after level-dependent amputation of the murine terminal phalanx and quantified the transcriptome of the repair tissue. Distal (regenerative) and proximal (non-regenerative) amputations showed significant differences in temporal gene expression and tissue regrowth over time. Genes that direct skeletal system development and limb morphogenesis are transiently upregulated during blastema formation and differentiation, including distal Hox genes. Overall, our results suggest that digit tip regeneration is controlled by a gene regulatory network that recapitulates aspects of limb development, and that failure to activate this developmental program results in fibrotic wound healing.

SUBMITTER: Qu F

PROVIDER: S-EPMC7501216 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Transcriptomic analysis of bone and fibrous tissue morphogenesis during digit tip regeneration in the adult mouse.

Qu Feini F Palte Ilan C IC Gontarz Paul M PM Zhang Bo B Guilak Farshid F

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20200607 7

Humans have limited regenerative potential of musculoskeletal tissues following limb or digit loss. The murine digit has been used to study mammalian regeneration, where stem/progenitor cells (the "blastema") completely regenerate the digit tip after distal, but not proximal, amputation. However, the molecular mechanisms responsible for this response remain to be determined. Here, we evaluated the spatiotemporal formation of bone and fibrous tissues after level-dependent amputation of the murine ...[more]

PMID: 32506623

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Project description:Purpose: Repair of distal phalanx amputation is widely studied and thought to occur through a regenerative process. Single-cell RNA sequencing (scRNA) has greatly improved the ability to characterize the cells that are involved in regeneration and abnormal healing of bone and soft tissue. Given the disorganized architecture of digit tip “regenerated” bone and its common progenitor cell to the pathologic process of heterotopic ossification, we hypothesized that digit tip repair after injury follows a program of aberrant wound repair rather than true regeneration of normal bone tissue. Methods: ScRNA data of mouse digit tip amputation (DTA) and embryonic limb development (EMB) was downloaded from NBCI GEO data base ascension number GSE135985. Additionally, scRNA data from heterotopic ossification (HO) was downloaded from GSE126060. HO, DTA, and EMB datasets were clustered individually. HO was merged and aligned with EMB and DT in separate analyses. Cells expressing the mesenchymal progenitor cell (MPC) marker Pdgfra, which we have shown to differentiate into HO and others have shown to differentiate into bone after DTA, were identified and analyzed in the HO-DTA and HO-EMB datasets. Monocle 2, Seurat 3.1.1, and Pearson correlation analysis were used for analysis and visualization. Immunostaining for markers present in DTA (ACAN, FBN-2, LTBP2) was performed on sections collected from a murine model of traumatic HO in Pdgfra-CreER;TdTomato mice induced with tamoxifen one-week prior to HO-forming injury. Results: MPC clusters from DTA and HO and from EMB and HO were aligned and projected using UMAP to compare transcription profiles. Across the timepoints, MPCs in the DTA and HO occupy similar regions of the UMAP plot, while MPCs in EMB and HO occupy largely independent regions, with only overlap at the latest time points. Using Monocle 2, cells were placed on a virtual timeline based on similarities in transcription. MPCs from DTA and HO meet at the same location on the trajectory following injury. In the EMB to HO comparison, cells only occupy similar regions of the trajectory at post-natal day 3 suggesting that bone is formed by different pathways. To understand correlations between time points, transcriptomes were also compared by Pearson analysis. MPCs following HO and DTA injuries occupy similar regions of the plot while MPCs in EMB and HO only occupy similar regions of the correlation plot at P3. Markers previously identified to be upregulated in MPCs in DTA were also upregulated in MPCs in HO by both scRNA and immunofluorescent histology. Conclusions: Cells responsible for bone repair following DTA show similar transcriptional profiles as MPCs in traumatic HO and dissimilar to MPCs in EMB. Furthermore, MPCs in both digit tip repair and HO follow trajectories that do not overlap with embryonic limb development. Thus, we demonstrate for the first time, that the process following DTA is not truly regeneration and follows programs of aberrant repair as seen in HO.

Dataset Information

Transcriptomic analysis of bone and fibrous tissue morphogenesis during digit tip regeneration in the adult mouse.

Publications

Transcriptomic analysis of bone and fibrous tissue morphogenesis during digit tip regeneration in the adult mouse.

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