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Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos.

Roddy KA, Prendergast PJ, Murphy P - PLoS ONE (2011)

Bottom Line: Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide.This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates.In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, School of Natural Sciences, Trinity College Dublin, Dublin, Ireland.

ABSTRACT
Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.

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Representation of the altered patterns of regulatory gene expression due to immobilisation.Colour coded expression patterns of markers and regulatory genes of interest in both control and immobilised sections. cc; capsular condensation, cl; chondrogenous layer, il; intermediate layer, IPFp; inter-patella-femoral fat pad, jf; loss of joint line definition, m, meniscus, MFc; medial femoral condyle, p; patella, tc ; tibial crest, tib; tibiotarsus. Note: BMP2 and FGF 2 are expressed in the capsule (not shown in legend).
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pone-0017526-g007: Representation of the altered patterns of regulatory gene expression due to immobilisation.Colour coded expression patterns of markers and regulatory genes of interest in both control and immobilised sections. cc; capsular condensation, cl; chondrogenous layer, il; intermediate layer, IPFp; inter-patella-femoral fat pad, jf; loss of joint line definition, m, meniscus, MFc; medial femoral condyle, p; patella, tc ; tibial crest, tib; tibiotarsus. Note: BMP2 and FGF 2 are expressed in the capsule (not shown in legend).

Mentions: Immobilisation also impacted the process of cell differentiation in the interzone, as indicated by changes in characteristic gene expression patterns and histology. In the absence of normal muscle forces the expression patterns of marker genes Tenascin C and Collagen type II alpha 1 (COL2A1) and regulatory genes PTHLP, BMP2, FGF2, CD44 and HAS2 were altered (summarised in Figure 7). Normal expression of FGF2, BMP2, CD44 and HAS2 was disrupted or lost specifically in the interzone regions of immobilised embryos which acquire cartilage like tissue characteristics as indicated by the inappropriate activation of COL2A1 and PTHLP expression. In addition boundaries of gene expression between cartilage rudiments and the interzone were less distinct suggesting either cell movement and cell mixing between the territories or transdifferentiation of cells in the interzone to a cartilaginous character; the latter interpretation is supported by the findings of Kahn et al [20] of aberrant expression of COL2A1 in lineage labeled interzone cells (descended from Gdf5 expressing cells) in immobile mouse embryo limbs. Finite element models predicted that the patterns of biophysical stimuli created by muscle contractions correspond with the emergence of specific tissues in the joint, suggesting that they could contribute to the patterning of these tissues [30]. For example the chondrogenous layers which ultimately form the articular cartilages are predicted to develop under dynamic elevation of fluid velocity and stress. We propose from these findings that correct differentiation of interzone cells and the maintenance of interzone cell type are dependent on the mechanical environment to which they are exposed. It has been shown that cultured interzone cells initially express the interzone marker Gdf5, however after several days in culture the cells resemble chondrocytes and express markers such as Collagen type II [7]. This strongly supports the conclusion that following initial specification of the interzone, maintenance of the territory and further differentiation toward cell types of particular articular structures is dependent on mechanical stimulation.


Mechanical influences on morphogenesis of the knee joint revealed through morphological, molecular and computational analysis of immobilised embryos.

Roddy KA, Prendergast PJ, Murphy P - PLoS ONE (2011)

Representation of the altered patterns of regulatory gene expression due to immobilisation.Colour coded expression patterns of markers and regulatory genes of interest in both control and immobilised sections. cc; capsular condensation, cl; chondrogenous layer, il; intermediate layer, IPFp; inter-patella-femoral fat pad, jf; loss of joint line definition, m, meniscus, MFc; medial femoral condyle, p; patella, tc ; tibial crest, tib; tibiotarsus. Note: BMP2 and FGF 2 are expressed in the capsule (not shown in legend).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046254&req=5

pone-0017526-g007: Representation of the altered patterns of regulatory gene expression due to immobilisation.Colour coded expression patterns of markers and regulatory genes of interest in both control and immobilised sections. cc; capsular condensation, cl; chondrogenous layer, il; intermediate layer, IPFp; inter-patella-femoral fat pad, jf; loss of joint line definition, m, meniscus, MFc; medial femoral condyle, p; patella, tc ; tibial crest, tib; tibiotarsus. Note: BMP2 and FGF 2 are expressed in the capsule (not shown in legend).
Mentions: Immobilisation also impacted the process of cell differentiation in the interzone, as indicated by changes in characteristic gene expression patterns and histology. In the absence of normal muscle forces the expression patterns of marker genes Tenascin C and Collagen type II alpha 1 (COL2A1) and regulatory genes PTHLP, BMP2, FGF2, CD44 and HAS2 were altered (summarised in Figure 7). Normal expression of FGF2, BMP2, CD44 and HAS2 was disrupted or lost specifically in the interzone regions of immobilised embryos which acquire cartilage like tissue characteristics as indicated by the inappropriate activation of COL2A1 and PTHLP expression. In addition boundaries of gene expression between cartilage rudiments and the interzone were less distinct suggesting either cell movement and cell mixing between the territories or transdifferentiation of cells in the interzone to a cartilaginous character; the latter interpretation is supported by the findings of Kahn et al [20] of aberrant expression of COL2A1 in lineage labeled interzone cells (descended from Gdf5 expressing cells) in immobile mouse embryo limbs. Finite element models predicted that the patterns of biophysical stimuli created by muscle contractions correspond with the emergence of specific tissues in the joint, suggesting that they could contribute to the patterning of these tissues [30]. For example the chondrogenous layers which ultimately form the articular cartilages are predicted to develop under dynamic elevation of fluid velocity and stress. We propose from these findings that correct differentiation of interzone cells and the maintenance of interzone cell type are dependent on the mechanical environment to which they are exposed. It has been shown that cultured interzone cells initially express the interzone marker Gdf5, however after several days in culture the cells resemble chondrocytes and express markers such as Collagen type II [7]. This strongly supports the conclusion that following initial specification of the interzone, maintenance of the territory and further differentiation toward cell types of particular articular structures is dependent on mechanical stimulation.

Bottom Line: Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide.This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates.In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers.

View Article: PubMed Central - PubMed

Affiliation: Department of Zoology, School of Natural Sciences, Trinity College Dublin, Dublin, Ireland.

ABSTRACT
Very little is known about the regulation of morphogenesis in synovial joints. Mechanical forces generated from muscle contractions are required for normal development of several aspects of normal skeletogenesis. Here we show that biophysical stimuli generated by muscle contractions impact multiple events during chick knee joint morphogenesis influencing differential growth of the skeletal rudiment epiphyses and patterning of the emerging tissues in the joint interzone. Immobilisation of chick embryos was achieved through treatment with the neuromuscular blocking agent Decamethonium Bromide. The effects on development of the knee joint were examined using a combination of computational modelling to predict alterations in biophysical stimuli, detailed morphometric analysis of 3D digital representations, cell proliferation assays and in situ hybridisation to examine the expression of a selected panel of genes known to regulate joint development. This work revealed the precise changes to shape, particularly in the distal femur, that occur in an altered mechanical environment, corresponding to predicted changes in the spatial and dynamic patterns of mechanical stimuli and region specific changes in cell proliferation rates. In addition, we show altered patterning of the emerging tissues of the joint interzone with the loss of clearly defined and organised cell territories revealed by loss of characteristic interzone gene expression and abnormal expression of cartilage markers. This work shows that local dynamic patterns of biophysical stimuli generated from muscle contractions in the embryo act as a source of positional information guiding patterning and morphogenesis of the developing knee joint.

Show MeSH
Related in: MedlinePlus