The movements of a baby in the womb (fetal movements) are a critical sign of the baby’s health and development. Such movements are also important for development of the baby’s bones and joints. When a baby doesn’t move enough, or their movements are restricted in some way, the shapes of their joints don’t form correctly, leading to conditions such as developmental dysplasia of the hip (where the hip joint is unstable or dislocated) or arthrogryposis, where multiple joints are angled abnormally. There is a link between the mechanical forces caused by fetal movements and the processes by which the skeleton takes its shape, but the mechanisms underlying this relationship are unknown.
In this paper, we found that a particular ion channel called TRPV4 (transient receptor potential cation channel subfamily V member 4) is involved in the response of the growing skeleton to the mechanical forces caused by fetal movements. We discovered this link by blocking activity of TRPV4 in embryonic mouse limbs, and showing that the normal response of the tissues to mechanical loading was eliminated. We also showed that activation of TRPV4 by mechanical loading affects proliferation of cells and the production of matrix in the cartilage, both of which affect growth of the joint.
A fascinating thing about TRPV4 is that when the gene which codes for the TRPV4 protein is mutated, a range of different severe skeletal conditions can occur including lethal metatropic dysplasia, spondylometaphyseal dysplasia (dwarfism), and autosomal dominant brachyolmia. Our study is the first to demonstrate that TRPV4 activity in the developing skeleton is closely linked to the mechanical loading from fetal movements. Drugs aimed at targeting TRPV4 are being trialled for a range of different conditions including osteoarthritis and heart failure, and we believe that our research indicates that TRPV4 may be a valuable target for future therapeutic disease modifying drugs for abnormalities of paediatric skeletal development, particularly when fetal movements are reduced or restricted.
The paper was featured on the Science Advances homepage! Many congratulations to Nidal and all co-authors.
Vivien’s final paper from her PhD was published in eCM (open access link). What we found was that when embryos develop without any skeletal muscle, surprisingly, the effects on the skeleton are less severe over development.
Work by our group and others have shown that when skeletal muscle is absent, bones and joints are abnormal with missing cavitation, abnormally shaped bones, and decreased mineralisation. BUT how these abnormalities progress over time in utero was unknown. We wanted to look at stages not previously characterised in detail, and chose TS24 (around e15.5) and TS27 (around e18.5). Our previous work showed different bones and joints are differentially affected by the lack of muscle, so we also looked at a range of rudiments.
At TS24, we found similar effects of absent muscle to those reported before; abnormal sizes and shapes of all major joints in the limb. BUT, at TS27, the joint shapes were much more normal, with much of the significant differences eliminated.
We looked at cell-level activities in the joints at TS24 and TS27 and found that cell size is a possible mechanism underlying the recovery in joint shape over gestation.
Interestingly, cavitation did not change between the two stages, so when cavitation was abnormal at TS24, it didn’t improve by TS27. Therefore, improvements in shape occurred *despite* absent cavitation in some joints.
Perhaps most surprising was the finding that mineralisation, as all muscleless long bones had significantly less mineralisation at TS24, which was completely recovered by TS27, and even exceeded in some rudiments like the ulna. The big question is how does this recovery in both cartilage growth/shape and mineralisation occur? It’s one we are still answering, so watch the space for follow up!
Jo‘s first first-author paper entitled “Growth orientations, rather than heterogeneous growth rates, dominate jaw joint morphogenesis in the larval zebrafish” was published in the Journal of Anatomy. Read the paper online here (open access).
In this research, a collaboration with Dr Chrissy Hammond (Bristol, UK), we tackle a long running question: what cell activities determine embryonic joint growth & shape?
We tracked individual cells in 3D in the larval zebrafish jaw joint over a 48-hour window. Using changes between cell centroids, we constructed growth maps of rate and direction of local tissue deformations
Growth maps varied substantially in growth orientation and growth rates both spatially at each developmental time point, and over the duration of development studied.
We synthesised the growth rates in a finite element analysis simulation, which was able to accurately predict joint morphogenesis. What this means is that cell positional information (i.e., orientation and volume) over time is enough to approximate growth and shape change. Then, we were able to use the simulation to test the importance of growth orientation, versus heterogeneous growth rates. We found that growth orientation was much more important for shape than growth rate heterogeneity.
Thank-you to the Anatomical Society for funding Jo’s PhD and congratulations Jo and all the team on a lovely study and paper!
In the paper we investigated how frequency and duration of loading affect cartilage and bone development. We used an in vitro explant culture system to culture embryonic chick limb explants under a range of loading regimes in which the amount of loading and the frequency were systematically varied. We found that increasing the duration (amount) of loading promoted cartilage growth, shape development and mineralisation of the femur and tibiotarsus. However, varying frequency only had significant effects on mineralisation, and not on cartilage growth or shape. Increased glycosaminoglycan deposition and cell proliferation may have contributed to the accelerated cartilage growth and shape change under increasing loading duration. The results demonstrated that frequencies and durations of applied biomechanical stimulation differentially promoted cartilage and bone formation, with implications for developmentally inspired tissue engineering strategies aiming to modulate tissue construct properties.
The work described in this paper was funded by an ERC Starting Grant. Congratulations Nidal and Cristian!
In previous papers by Aurélie, Rebecca and others, we demonstrated that muscle loading is needed for normal development of the spine, including spinal curvature, vertebral shape and vertebral segmentation in the chick embryo. However, the chick embryo does not have the same type of discs as humans (or mammals in general) as it lacks a nucleus pulposus. Therefore, we needed to switch to the mouse to look at the influence of muscles on development of the discs. Our usual mouse line of choice (splotch delayed) wasn’t suitable due to the fact that spine development and vertebral segmentation is known to be abnormal in this line. Therefore we used the “mdg” or muscular dysgenesis line (with grateful thanks to Prof Eli Zelzer, Weizmann Institute) in which skeletal muscles form but do not contract.
In this paper, we investigated how muscle forces affect (1) notochord involution and vertebral segmentation, and (2) intravertebral disc (IVD) development including the mechanical properties and morphology, as well as collagen fibre alignment in the annulus fibrosus. We looked at three different stages of development; Theiler Stage (TS)22 when notochord involution starts, at TS24 when involution is complete, and at TS27 when the IVD is formed. Vertebral and IVD development were characterised using histology, immunofluorescence, and indentation testing. We found that notochord involution and vertebral segmentation occurred independently of muscle contractions between TS22 and TS24. However, in the absence of muscle contractions, we found vertebral fusion in the cervical region at TS27, along with (i) a displacement of the nucleus pulposus towards the dorsal side, (ii) a disruption of the structural arrangement of collagen in the annulus fibrosus, and (iii) an increase in viscous behaviour of the annulus fibrosus. Therefore, mechanical loading due to muscle contractions are important for the later stages of disc development, particularly for annulus fibrosus formation. We believe our results suggest a need for mechanical loading in the creation of fibre-reinforced tissue engineering replacement IVDs as a therapy for IVD degeneration.
The research described in this paper was funded by the Leverhulme Trust and by an ERC Starting Grant.
See @NTAurelie‘s nice twitter thread here! Congratulations Aurélie and all the team!
Congratulations to Devi (together with collaborators Rebecca and Colin Boyle) on publication of her paper entitled “Quantifying the tolerance of chick hip joint development to temporary paralysis and the potential for recovery” in Developmental Dynamics.
In this paper, we wanted to discover when- and for how long- fetal movements are most important for development of the hip joint, in order to better understand conditions affecting the skeletons of babies who may have had restricted or reduced movements in utero, including developmental dysplasia of the hip (where the hip joint is unstable) and arthrogryposis (multiple joint contractures). We also wanted to know if joint development can be “rescued” by external manipulation even when immobilised.
We varied the initiation and duration of drug-induced paralysis in the chick embryo, and found that a three day period between 4 and 7 embryonic days was most important for hip joint shape. In terms of hip joint development, this timeline is equivalent to roughly 10 to 12 gestational weeks in humans, and means that the end of the first trimester would be a useful time for targeted screening for arthrogryposis and hip dysplasia. We found that cavitation was more dependent on duration of paralysis, rather than timing.
When we externally manipulated the immobilised chick limbs, the hip joints had more normal shapes, and more normal progression of cavitation, compared to the contralateral limbs of the same chicks. This implies that the developing limb has the potential to recover from periods of immobility, and external manipulation provides an innovative avenue for prevention and treatment of developmental joint pathologies.
The full paper is available open-access here. The research was funded by ERC Starting Grant #336306.
Saima‘s paper funded by the ERC characterising the collagens in the developing skeletal rudiment was published in eCM. We used immuno-fluorescence to look at the major and minor collagens in sections of the humerus at TS22 (e13.5, before formation of the primary ossification centre), TS25 (e15.5) and TS27 (e17.5).
Several collagens change substantially with the progression of the ossification centre, like for example Col V.
Some develop amazingly complex structures over the period of development studied, like Col II (green) and XI (grey).
Vivien‘s first first-author paper entitled “Effects of Abnormal Muscle Forces on Prenatal Joint Morphogenesis in Mice” has been published in the Journal of Orthopaedic Research. The paper characterises the effects of absent or reduced muscle on prenatal development of the major synovial joints. Vivien used image registration to qualitatively and quantitatively compare joint shapes between muscleless, reduced-muscle and normal mice. Different joints were affected more than others, and what we found most interesting is that a reduction in muscle often led to *more* severe effects than no muscle- like in the humeral distal condyles (blue: control, yellow: reduced-muscle, purple: muscleless)
Congratulations Vivien! You can access the final version of the paper here, or the submitted version here if you don’t have access to JOR.
Aurélie‘s paper (on which Yuming and Rebecca are also authors) entitled “Short-term foetal immobility temporally and progressively affects chick spinal curvature and anatomy and rib development” has been published in eCM (European Cells and Materials). The paper describes how a very short period of paralysis during prenatal development can have severe and lasting effects on development of the spine and ribs, as summarised in the graphical abstract. This work has consequences for understanding congenital scoliosis, for which a change or reduction in fetal movements could be an important factor. The project was funded by the Leverhulme Trust and was a collaboration with Prof James Iatridis in the Icahn School of Medicine at Mount Sinai, New York, USA. View the paper for free here.
Stefaan’s final and Perren Award winning paper from his postdoc in the Developmental Biomechanics has been published online in the Journal of Biomechanics. In the paper, the effects of different risk factors for hip dysplasia (such as fetal breech position, first born babies and oligohydramnios (reduced amniotic fluid)) on fetal kicks and the stresses and strains in their hip joints are quantified. Kick force, stress and strain were found to be significantly lower in cases of breech position and oligohydramnios. Similarly, firstborn fetuses were found to generate significantly lower kick forces than non-firstborns.