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PDGFRα signaling regulates cartilage and fibrous tissue differentiation during synovial joint development [scRNA-seq]

ABSTRACT: Limb development begins with limb buds that develop into long appendicular bones separated by joint-forming structures called interzones. Mesenchymal progenitor cells of the interzone differentiate into cartilaginous and fibrous tissues of the synovial joint, including articular cartilage, menisci, and ligaments. Platelet-derived growth factor receptor-α (PDGFRα) is expressed by mesenchymal cells of the limb bud, but it is not known to have a role in limb or joint development. By analyzing PDGFRα expression in single-cell RNA sequencing data, immunofluorescence, and lineage tracing, we observed dynamic PDGFRα expression in the mouse limb bud, interzone, and perichondral mesenchyme. To investigate PDGFRα function, we use Prrx1-Cre to cause mouse limb bud mesenchyme to express mutant PDGFRα with a kinase domain point mutation (D842V) that increases receptor signaling. Overall limb development was unaffected. However, the hind limbs were immobile with knee joints fused by cartilage and lacking ligaments and menisci. The mutant perichondrium exhibited ectopic cartilage matrix and the enthesis of the patellar tendon was enlarged. The interzone marker Gdf5 was initially expressed at E12.5 but was downregulated at E13.5 and thereafter, suggesting a defect in maintenance of the interzone. Spatial transcriptomics of the mutant joint identified co-expression of genes for both cartilage (Sox9, Col2a1) and fibrotic tissue (Postn, Col1a1, Col1a2), suggesting fibrocartilage. These data suggest that elevated PDGFRα signaling corrupts joint tissue differentiation by downregulating Gdf5 and redirecting interzone progenitors into a fibrocartilage fate. These results highlight the need to balance chondrogenic and fibrogenic cell fates during joint development, with tight regulation of PDGFRα signaling as a necessity for the development of the mouse knee joint.

ORGANISM(S): Mus musculus

PROVIDER: GSE289874 | GEO | 2025/04/01

REPOSITORIES: GEO

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Project description:Objectives. Fibroblasts in synovium include fibroblast-like synoviocytes (FLS) in the lining and Thy1+ connective-tissue fibroblasts in the sub-lining. We aimed to investigate their developmental origin and relationship with adult progenitors. Methods. To discriminate between Gdf5-lineage cells deriving from the embryonic joint interzone and other Pdgfrα-expressing fibroblasts and progenitors, adult Gdf5-Cre;Tom;Pdgfrα-H2BGFP mice were used and cartilage injury was induced to activate progenitors. Cells were isolated from knees, sorted by fluorescence-activated cell-sorting based on developmental origin, and analysed by single-cell RNA-sequencing. Flow cytometry and immunohistochemistry were used for validation. Clonal-lineage mapping was performed using Gdf5-Cre;Confetti mice. Results. In steady state, Thy1+ sub-lining fibroblasts were of mixed ontogeny, while Thy1-Prg4+ lining fibroblasts predominantly derived from the embryonic joint interzone. The latter included Prg4-expressing progenitors distinct from molecularly defined FLS. Clonal-lineage tracing revealed compartmentalisation of Gdf5-lineage fibroblasts between lining and sub-lining. Following injury, lining hyperplasia resulted from proliferation and differentiation of Prg4-expressing progenitors, with additional recruitment of non-Gdf5-lineage cells, into FLS. Consistent with this, a second population of proliferating cells, enriched near blood vessels in the sub-lining, supplied activated multipotent cells predicted to give rise to Thy1+ fibroblasts, and to feed into the FLS differentiation trajectory. Transcriptional programmes regulating fibroblast differentiation trajectories were uncovered, identifying Sox5 and Foxo1 as key FLS transcription factors in mice and humans. Conclusions. Our data blueprint a cell atlas of mouse synovial fibroblasts and progenitors in healthy and injured knees, and provide novel insights into the cellular and molecular principles governing the organisation and maintenance of adult synovial joints.

Project description:Menisci play a vital role in load transmission, shock absorption and joint stability. The current dogma is that the menisci simply protects the cartilage and play no role in osteoarthritis (OA) unless they are injured. However, there is increasing evidence suggesting that OA menisci may not merely be bystanders in the disease process of OA. This study sought: 1) to determine the prevalence of meniscal degeneration in OA patients, 2) to examine gene expression in OA meniscal cells compared to normal control meniscal cells, and 3) to test the hypothesis that OA meniscal cells are different from normal meniscal cells. The grades of meniscal degeneration correlated in a positive fashion with the grades of articular cartilage degeneration (r = 0.672; P < 0.0001). Many genes classified in the biological processes of immune response, inflammatory response, biomineral formation and cell proliferation, including major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1), integrin, beta 2 (ITGB2), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), ankylosis, progressive homolog (ANKH) and fibroblast growth factor 7 (FGF7), were expressed at higher levels compared to normal control meniscal cells. In addition, many genes that were previously implicated in OA were also expressed at higher levels in OA meniscal cells, including ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). Our findings suggest that OA is a whole joint disease. Meniscal cells may play an active role in the development of OA. Investigation of the gene expression profiles of OA meniscal cells may reveal new therapeutic targets for OA therapy and also may uncover novel disease markers for early diagnosis of OA. Studies were approved by our human subjects Institutional Review Board. Menisci and articular cartilage were collected during joint replacement surgery for OA patients (OA specimens) and lower limb amputation surgery for osteosarcoma patients (normal control specimens), and graded. Meniscal cells were prepared from the meniscal tissues and expanded in monolayer culture. Differential gene expression in OA meniscal cells and normal control meniscal cells was examined using Affymetrix microarray and real time RT-PCR.

Project description:Objective: Joint formation begins with the establishment of an interzone within the cartilaginous anlagen of the future skeleton and both Gdf5 and Erg are proposed as regulators of chondrocyte differentiation during and post interzone formation. The aim of this study was to examine the relationship between Gdf5 and Erg expression and downstream effects on chondrocyte gene expression. Design: Erg expression was identified in mouse knee joints at E13.5. Expression and microarray analyses were performed using micromass cultures of murine C3H10T1/2 mesenchymal cells undergoing induced chondrogenesis in the presence of absence of Gdf5 and Erg. Results: At E13.5, Erg expression was found to surround epiphyseal chondrocytes and span the interzone up to the intermediate zone. Erg splice forms were expressed in micromass cultures, and their expression profile was altered by the addition of recombinant Gdf5 depending on the stage of differentiation. Overexpression of Erg-010 resulted in a downregulation of Col2a1 and Col10a1. Microarray analysis following Erg-010 overexpression identified two potential downstream targets, Ube2b and Osr2, which were also differentially regulated by Gdf5. Conclusion: Erg regulation by Gdf5 in mesenchymal cells in vitro is dependent on the stage of chondrogenesis, and its expression in vivo demarcates chondrocytes that are not destined to be consumed by endochondral ossification. Functionally, Erg expression causes downregulation of Col2a1 and Col10a1 expression and this effect is potentially mediated by Osr2, which is a known regulator of chondrocyte differentiation. The identification of Ube2b as a putative downstream target of Erg-010 suggests that it may contribute to the regulation of the ubiquitination pathway and thereby BMP2 signaling, which is essential for normal knee joint development. RNA from overexpression experiments was extracted as described above. Total RNA quality was confirmed using a Bioanalyzer 2100 (Agilent). Labelled targets were generated from total RNA and hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymatrix) (Genomics Centre, University of Manchester). The raw fluorescence intensity values were normalized using a Robust Multi-array Average approach using dChip (V2005)

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PDGFRα signaling regulates cartilage and fibrous tissue differentiation during synovial joint development [scRNA-seq]

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