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Synovial joint cavitation during mouse embryo limb skeletogenesis entails Na/K-ATPase pump expression and osmoregulatory activity

ABSTRACT: The developmental biology of limb synovial joints and their water/lubricant-filled cavity remains unclear. Joint formation is known to start with emergence at each prescribed anatomical site of a compacted Gdf5-expressing mesenchymal interzone flanked by cartilaginous skeletal primordia. We and others previously showed that the interzone cells serve as progenitors and generate most joint tissues over time, including articular cartilage and ligaments. But how is the cavity formed within the compacted interzone? Because water accrual would be needed to create and inflate the cavity and in turn distance the flanking skeletal elements, cavity formation may require energy-consuming osmoregulatory mechanisms able to draw water and exert mechanical force. Our data now reveal that interzone cells in cavitating knee and digit mouse embryo joints strongly express the Na/K-ATPase pumps Atp1a1, Atp1b1 and Atp1b3, members of a key cell surface osmoregulatory protein family that regulates fluid tonicity and drawing of water. There was also local and specific expression of the water channel Aquaporin 1 (Aqp1). Single cell RNA seq (scRNAseq) showed that Gdf5+ interzone cells expressing those genes culminated in number at cavitation and were characterized by other osmoregulatory and mechano-sensing genes also. Indeed, when pregnant mice were administered ouabain -a physiological glycoside that normally limits Na/K-ATPase pump activity and osmoregulatory processes-, limb joint cavitation in the embryos was inhibited as was lubricant gene expression. Joint development is known to depend on signals from Indian hedgehog-expressing growth plate chondrocytes in flanking skeletal primordia. Interference with hedgehog signaling did coordinately inhibit expression of pump, mechano-sensing and lubricant genes as well as cavitation. Together, our data suggest a novel understanding of joint cavitation as an active energy-requiring osmoregulatory process able to accrue a water-based fluid, distance the flanking articulating surfaces and establish a synovial cavity in close coordination with long bone development and growth.

ORGANISM(S): Mus musculus

PROVIDER: GSE276551 | GEO | 2025/07/29

REPOSITORIES: GEO

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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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Synovial joint cavitation during mouse embryo limb skeletogenesis entails Na/K-ATPase pump expression and osmoregulatory activity

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