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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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Anatomical changes in the knee joints of immobilised embryos.Longitudinal sections through the chick knee joint of control and immobilised embryos (4.5 days of immobilisation) at low (A, B, E, F, I, J) and high magnification (C, D, G, H, K, L). Histological sections (E–D) were stained using alcian blue and counter stained with haematoxlyin and eosin. Other sections show the expression of COL2A1 and TNC mRNA in control and immobilised knee joints. c; cavity, cl; chondrogenous layers, jc; joint capsule, lg ; ligament, m; meniscus, p; patella, pc ; perichondrium, t; tendon. Scale bar 0.5 mm (A, B, E, F, I, J) and 0.1 mm (C, D, G, H, K, L).
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pone-0017526-g001: Anatomical changes in the knee joints of immobilised embryos.Longitudinal sections through the chick knee joint of control and immobilised embryos (4.5 days of immobilisation) at low (A, B, E, F, I, J) and high magnification (C, D, G, H, K, L). Histological sections (E–D) were stained using alcian blue and counter stained with haematoxlyin and eosin. Other sections show the expression of COL2A1 and TNC mRNA in control and immobilised knee joints. c; cavity, cl; chondrogenous layers, jc; joint capsule, lg ; ligament, m; meniscus, p; patella, pc ; perichondrium, t; tendon. Scale bar 0.5 mm (A, B, E, F, I, J) and 0.1 mm (C, D, G, H, K, L).

Mentions: Comparison of embryos immobilised with 0.5% DMB for 4–5 days, commencing on day 4.5 of incubation, and control specimens, revealed a number of consistent abnormalities. Staging of the embryos, using the Hamburger and Hamilton criteria [56], insured that only stage matched specimens were compared. Drug treated embryos showed previously reported effects of immobilisation including spinal curvature and joint contracture (not shown) [10], [21]. Specifically in the knee joint, histological sections showed a general reduction in the separation of the rudiments, altered cellular organisation in the interzone with no clear definition of chondrogenous layers and no sign of cavitation in the altered mechanical environment of immobilised specimens (Figure 1A–D and K,L). Additional alterations to knee joint associated tissues were revealed through marker gene expression analysis. Collagen type II alpha1 (COL2A1) gene expression marks the joint capsule, developing ligaments and tendons and initial appearance of the patella, in addition to the cartilaginous rudiments in control specimens at this stage (Figure 1E and G). Tenascin C (TNC) expression also marks the joint capsule and patella and reveals the chondrogenous layers, the perichondrium and the appearance of the menisci in the joint interzone (Figure 1I and K). In immobilised knee joints, expression analysis of these tissue markers revealed absence of the inter-articular ligaments (Figure. 1F and H), the chondrogenous layers and menisci (Figure 1J and L). The expression of both markers also appeared to be reduced or absent in the joint capsule and patella region (Figure 1F, J) in immobilised joints.


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)

Anatomical changes in the knee joints of immobilised embryos.Longitudinal sections through the chick knee joint of control and immobilised embryos (4.5 days of immobilisation) at low (A, B, E, F, I, J) and high magnification (C, D, G, H, K, L). Histological sections (E–D) were stained using alcian blue and counter stained with haematoxlyin and eosin. Other sections show the expression of COL2A1 and TNC mRNA in control and immobilised knee joints. c; cavity, cl; chondrogenous layers, jc; joint capsule, lg ; ligament, m; meniscus, p; patella, pc ; perichondrium, t; tendon. Scale bar 0.5 mm (A, B, E, F, I, J) and 0.1 mm (C, D, G, H, K, L).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017526-g001: Anatomical changes in the knee joints of immobilised embryos.Longitudinal sections through the chick knee joint of control and immobilised embryos (4.5 days of immobilisation) at low (A, B, E, F, I, J) and high magnification (C, D, G, H, K, L). Histological sections (E–D) were stained using alcian blue and counter stained with haematoxlyin and eosin. Other sections show the expression of COL2A1 and TNC mRNA in control and immobilised knee joints. c; cavity, cl; chondrogenous layers, jc; joint capsule, lg ; ligament, m; meniscus, p; patella, pc ; perichondrium, t; tendon. Scale bar 0.5 mm (A, B, E, F, I, J) and 0.1 mm (C, D, G, H, K, L).
Mentions: Comparison of embryos immobilised with 0.5% DMB for 4–5 days, commencing on day 4.5 of incubation, and control specimens, revealed a number of consistent abnormalities. Staging of the embryos, using the Hamburger and Hamilton criteria [56], insured that only stage matched specimens were compared. Drug treated embryos showed previously reported effects of immobilisation including spinal curvature and joint contracture (not shown) [10], [21]. Specifically in the knee joint, histological sections showed a general reduction in the separation of the rudiments, altered cellular organisation in the interzone with no clear definition of chondrogenous layers and no sign of cavitation in the altered mechanical environment of immobilised specimens (Figure 1A–D and K,L). Additional alterations to knee joint associated tissues were revealed through marker gene expression analysis. Collagen type II alpha1 (COL2A1) gene expression marks the joint capsule, developing ligaments and tendons and initial appearance of the patella, in addition to the cartilaginous rudiments in control specimens at this stage (Figure 1E and G). Tenascin C (TNC) expression also marks the joint capsule and patella and reveals the chondrogenous layers, the perichondrium and the appearance of the menisci in the joint interzone (Figure 1I and K). In immobilised knee joints, expression analysis of these tissue markers revealed absence of the inter-articular ligaments (Figure. 1F and H), the chondrogenous layers and menisci (Figure 1J and L). The expression of both markers also appeared to be reduced or absent in the joint capsule and patella region (Figure 1F, J) in immobilised joints.

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