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Versican knockdown reduces interzone area during early stages of chick synovial joint development.

ABSTRACT: Much has been learned regarding factors that specify joint placement, but less is known regarding how these molecular instructions are translated into functional joint tissues. Previous studies have shown that the matrix chondroitin sulfate proteoglycan, versican, exhibits a similar pattern of expression in the embryonic joint rudiment of chick and mouse suggesting conserved function during joint development. In this study, versican's importance in developing joints was investigated by specific inhibition of its expression in the early joint interzone, tissue that gives rise to articular cartilages and joint cavity. In ovo microinjection of adenoviral shRNA constructs into the HH25 chick wing was employed to silence endogenous versican protein in developing appendicular joints. Results showed statistically significant (12-14%) reduction of nonchondrogenic elbow joint interzone area in whole-mount specimens at HH36 in response to versican knockdown. Attenuated expression of key versican-associated molecules including hyaluronan, tenascin, CD44, and link protein was also noted by histochemical and immunohistochemical analysis. Versican knockdown also lowered collagen II expression in presumptive articular chondrocytes indicating possible delay in chondrogenesis. Results suggest that versican functions interactively with other matrix/cell surface molecules to facilitate establishment or maintenance of early joint interzone structure.

SUBMITTER: Nagchowdhuri PS

PROVIDER: S-EPMC3339409 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Versican knockdown reduces interzone area during early stages of chick synovial joint development.

Nagchowdhuri Partha S PS Andrews Kristen N KN Robart Savannah S Capehart Anthony A AA

Anatomical record (Hoboken, N.J. : 2007) 20111220 3

Much has been learned regarding factors that specify joint placement, but less is known regarding how these molecular instructions are translated into functional joint tissues. Previous studies have shown that the matrix chondroitin sulfate proteoglycan, versican, exhibits a similar pattern of expression in the embryonic joint rudiment of chick and mouse suggesting conserved function during joint development. In this study, versican's importance in developing joints was investigated by specific ...[more]

PMID: 22190409

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Project description:Synovial joints are among the most important structures that give us complex motor abilities as humans. Degenerative joint diseases, such as arthritis, cause loss of normal joint functioning and affect over 40 million people in the USA and approximately 350 million people worldwide. Therapies based on regenerative medicine hold the promise of effectively repairing or replacing damaged joints permanently. Here, for the first time, we introduce a model for synovial joint regeneration utilizing the chick embryo. In this model, a block of tissue that contains the prospective elbow is excised, leaving a window with strips of anterior and posterior tissue intact (window excision, WE). In contrast, we also slice out the same area containing the elbow and the distal piece of the limb is pinned back onto the stump (slice excision, SE). Interestingly, when the elbow is removed via WE, regeneration of the joint takes place, whereas the elbow joint does not regenerate following SE. In order to investigate whether the regeneration response recapitulates the developmental program of forming joints, we used GDF-5 and Autotaxin (Atx) as joint tissue specific markers, and Sox-9 and Col-9 as cartilage markers for in situ hybridization on sections at different time points after WE and SE surgeries. Re-expression of GDF-5 and Atx is observed in the WE samples by 60h after surgery. In contrast, the majority of the samples that underwent SE surgery did not express GDF-5 and Atx. Also, in SE fusion of cartilage elements takes place and the joint interzone does not form. This is indicated by continuous Col-9 expression in SE limbs, whereas Col-9 is downregulated at the joint interzone in the regenerating WE samples. This order and pattern of gene expression observed in regenerates is similar to the development of a joint suggesting that regeneration recapitulates development at the molecular level. This model defines some of the conditions required for inducing joint regeneration in an otherwise nonregenerating environment. This knowledge can be useful for designing new therapeutic approaches for joint loss or for conditions affecting joint integrity in humans.

Project description:Limb development requires the coordinated growth of several tissues and structures including long bones, joints and tendons, but the underlying mechanisms are not wholly clear. Recently, we identified a small drug-like molecule - we named Kartogenin (KGN) - that greatly stimulates chondrogenesis in marrow-derived mesenchymal stem cells (MSCs) and enhances cartilage repair in mouse osteoarthritis (OA) models. To determine whether limb developmental processes are regulated by KGN, we tested its activity on committed preskeletal mesenchymal cells from mouse embryo limb buds and whole limb explants. KGN did stimulate cartilage nodule formation and more strikingly, boosted digit cartilaginous anlaga elongation, synovial joint formation and interzone compaction, tendon maturation as monitored by ScxGFP, and interdigit invagination. To identify mechanisms, we carried out gene expression analyses and found that several genes, including those encoding key signaling proteins, were up-regulated by KGN. Amongst highly up-regulated genes were those encoding hedgehog and TGF? superfamily members, particularly TFG?1. The former response was verified by increases in Gli1-LacZ activity and Gli1 mRNA expression. Exogenous TGF?1 stimulated cartilage nodule formation to levels similar to KGN, and KGN and TGF?1 both greatly enhanced expression of lubricin/Prg4 in articular superficial zone cells. KGN also strongly increased the cellular levels of phospho-Smads that mediate canonical TGF? and BMP signaling. Thus, limb development is potently and harmoniously stimulated by KGN. The growth effects of KGN appear to result from its ability to boost several key signaling pathways and in particular TGF? signaling, working in addition to and/or in concert with the filamin A/CBF?/RUNX1 pathway we identified previously to orchestrate overall limb development. KGN may thus represent a very powerful tool not only for OA therapy, but also limb regeneration and tissue repair strategies.

Dataset Information

Versican knockdown reduces interzone area during early stages of chick synovial joint development.

Publications

Versican knockdown reduces interzone area during early stages of chick synovial joint development.

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