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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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Related in: MedlinePlus

Comparison of cartilage shape and cell proliferation in distal femora of control (blue) and immobilised (red) embryos.Outlines of the cartilage anlaga (A–F) were extracted from virtual sections (Fig.2, C, D, F). A minimum of four outlines per treatment were overlaid. Arrow heads indicate region of reduced outgrowth in the immobilised animals. *indicates flattening of rudiment surfaces. Black brackets show extent of growth reductions in the dorsal aspect of the condyles. Coloured brackets compare width of the intercondylar fossa of control (red) and immobilised (blue) embryos (A, D). The distribution of proliferating chondroctyes in the femur across 5 regions, represented in G (boxes 1–5), was compared in control (blue) and immobilised (red) embryos (h). tf; trochlea fibularis, IF; intercondylar fossa.
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pone-0017526-g003: Comparison of cartilage shape and cell proliferation in distal femora of control (blue) and immobilised (red) embryos.Outlines of the cartilage anlaga (A–F) were extracted from virtual sections (Fig.2, C, D, F). A minimum of four outlines per treatment were overlaid. Arrow heads indicate region of reduced outgrowth in the immobilised animals. *indicates flattening of rudiment surfaces. Black brackets show extent of growth reductions in the dorsal aspect of the condyles. Coloured brackets compare width of the intercondylar fossa of control (red) and immobilised (blue) embryos (A, D). The distribution of proliferating chondroctyes in the femur across 5 regions, represented in G (boxes 1–5), was compared in control (blue) and immobilised (red) embryos (h). tf; trochlea fibularis, IF; intercondylar fossa.

Mentions: This and other detailed aspects of local shape changes in the distal femur were visualised by outlining cartilage (alcian blue stained) in comparable sections of 3D reconstructions. Outlines were generated using the sections shown in Figure 2 C, D and F and physically overlaid so that the medial and lateral sides of the femora in the sections were parallel and the midpoints of the intercondylar fossa were overlapping. Overlaying outlines of this characteristic view of control and immobilised specimens highlighted the effect of rigid paralysis on the emergence of shape in the femoral condyles (Figure 3A–F). Without muscle contraction the general shape of the knee joint is much simpler, joint surfaces are flattened (e.g. flattening of the lateral condyle shown in Figure 3C and F) and functional outgrowths are lost. After 4 days of immobilisation the reduction in width of the intercondylar fossa was very obvious, particularly ventrally (Figure 3A). The surface of the lateral condyle also appeared to be flattened by immobilisation (Figure 3C). Five days of immobilisation caused greater simplification. In particular note the reduction in height of the medial and lateral condyles in the dorsal aspect (Figure 3D, brackets) and the reduced outgrowths on the ventral aspect of the condyles (arrowheads Figure 3D) in the region of the trochlea fibularis grove where the femur interfaces with the fibula enabling smooth movement in later life. A flattening of the articular surfaces was now apparent in both condyles (Figure 3E,F).


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)

Comparison of cartilage shape and cell proliferation in distal femora of control (blue) and immobilised (red) embryos.Outlines of the cartilage anlaga (A–F) were extracted from virtual sections (Fig.2, C, D, F). A minimum of four outlines per treatment were overlaid. Arrow heads indicate region of reduced outgrowth in the immobilised animals. *indicates flattening of rudiment surfaces. Black brackets show extent of growth reductions in the dorsal aspect of the condyles. Coloured brackets compare width of the intercondylar fossa of control (red) and immobilised (blue) embryos (A, D). The distribution of proliferating chondroctyes in the femur across 5 regions, represented in G (boxes 1–5), was compared in control (blue) and immobilised (red) embryos (h). tf; trochlea fibularis, IF; intercondylar fossa.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017526-g003: Comparison of cartilage shape and cell proliferation in distal femora of control (blue) and immobilised (red) embryos.Outlines of the cartilage anlaga (A–F) were extracted from virtual sections (Fig.2, C, D, F). A minimum of four outlines per treatment were overlaid. Arrow heads indicate region of reduced outgrowth in the immobilised animals. *indicates flattening of rudiment surfaces. Black brackets show extent of growth reductions in the dorsal aspect of the condyles. Coloured brackets compare width of the intercondylar fossa of control (red) and immobilised (blue) embryos (A, D). The distribution of proliferating chondroctyes in the femur across 5 regions, represented in G (boxes 1–5), was compared in control (blue) and immobilised (red) embryos (h). tf; trochlea fibularis, IF; intercondylar fossa.
Mentions: This and other detailed aspects of local shape changes in the distal femur were visualised by outlining cartilage (alcian blue stained) in comparable sections of 3D reconstructions. Outlines were generated using the sections shown in Figure 2 C, D and F and physically overlaid so that the medial and lateral sides of the femora in the sections were parallel and the midpoints of the intercondylar fossa were overlapping. Overlaying outlines of this characteristic view of control and immobilised specimens highlighted the effect of rigid paralysis on the emergence of shape in the femoral condyles (Figure 3A–F). Without muscle contraction the general shape of the knee joint is much simpler, joint surfaces are flattened (e.g. flattening of the lateral condyle shown in Figure 3C and F) and functional outgrowths are lost. After 4 days of immobilisation the reduction in width of the intercondylar fossa was very obvious, particularly ventrally (Figure 3A). The surface of the lateral condyle also appeared to be flattened by immobilisation (Figure 3C). Five days of immobilisation caused greater simplification. In particular note the reduction in height of the medial and lateral condyles in the dorsal aspect (Figure 3D, brackets) and the reduced outgrowths on the ventral aspect of the condyles (arrowheads Figure 3D) in the region of the trochlea fibularis grove where the femur interfaces with the fibula enabling smooth movement in later life. A flattening of the articular surfaces was now apparent in both condyles (Figure 3E,F).

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