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Chromatin architecture and cis-regulatory landscape of the DACT2-SMOC2 locus in the developing synovial joint.

ABSTRACT: We report the 3D chromatin organization of the 3.4Mb DACT2-SMOC2 locus generated for interphalangeal interzone and phalange, tissues collected during synovial joint formation. The interzone and phalange samples have been dissected from developing hindlimb digits of the chicken embryo at Hamilton Hamburger stage 32 (HH32). Collected material has been processed using the Targeted Chromatin Capture protocol optimized for the 1mln cell. Generation of the T2C-interaction maps has revealed the 3D chromatin architecture of the DACT2-SMOC2 locus in the interzone and phalange tissues as well as identified potential candidate enhancers located in spatial proximity of DACT2 and SMOC2 gene promoters. We report the ChIPseq derived Candidate Enhancer (CE) atlas generated for interphalangeal interzone and phalange, tissues collected during synovial joint formation. Three replicates for interzone and three replicated for phalange were immunoprecipitated (IP) with the H3K27ac antibodies. Also, two replicates for interzone and two replicates for phalange were immunoprecipitated with H3K4me1 antibodies. The IP samples for both histone marks were sequenced and used for annotation of CEs. Further, the CEs were functionally annotated. In parallel, the total RNA for three interzone and three phalange replicates was isolated and mRNA sequencing libraries were constructed. Next the identification of differentially expressed genes (DEGs) and integrative analysis of DEGs and CEs was performed.

ORGANISM(S): Gallus gallus

PROVIDER: GSE210108 | GEO | 2024/07/01

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.

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Chromatin architecture and cis-regulatory landscape of the DACT2-SMOC2 locus in the developing synovial joint.

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