Localization of YAP activity in developing skeletal rudiments is responsive to mechanical stimulation

Abstract

Background Normal skeletal development, in particular ossification, joint formation and shape features of condyles, depends on appropriate mechanical input from embryonic movement but it is unknown how such physical stimuli are transduced to alter gene regulation. Hippo/Yes‐Associated Protein (YAP) signalling has been shown to respond to the physical environment of the cell and here we specifically investigate the YAP effector of the pathway as a potential mechanoresponsive mediator in the developing limb skeleton. Results We show spatial localization of YAP protein and of pathway target gene expression within developing skeletal rudiments where predicted biophysical stimuli patterns and shape are affected in immobilization models, coincident with the period of sensitivity to movement, but not coincident with the expression of the Hippo receptor Fat4. Furthermore, we show that under reduced mechanical stimulation, in immobile, muscle‐less mouse embryos, this spatial localization is lost. In culture blocking YAP reduces chondrogenesis but the effect differs depending on the timing and/or level of YAP reduction. Conclusions These findings implicate YAP signalling, independent of Fat4, in the transduction of mechanical signals during key stages of skeletal patterning in the developing limb, in particular endochondral ossification and shape emergence, as well as patterning of tissues at the developing synovial joint.

Control of skeletal morphogenesis by the Hippo-YAP/TAZ pathway

“…Thus, chondrocytes may also employ the Hippo-YAP/TAZ pathway to sense mechanical forces acting via the ECM and regulate the developmental remodelling response via Ctgf , Cyr61 and several matrix remodelling enzymes. In support of this notion, a recent study has demonstrated that YAP localisation and activity in the embryonic cartilaginous humerus is depended on mechanical stimulation derived from the surrounding muscle ( Shea et al, 2020 ). Our findings also have important medical relevance, further implicating the Hippo-YAP/TAZ pathway in human birth defects including chondrodysplasia and cleft palate ( Williamson et al, 2014 ).…”

Joint development recovery on resumption of embryonic movement following paralysis

“…We have previously hypothesised that local biophysical stimuli generated from movement create a type of positional information that contributes to the correct patterning of emerging tissues in the joint (Roddy et al, 2011b). We have also shown changes in the molecular profiles and signalling pathways active across the territories of the joint (Shea et al, 2019;Rolfe et al, 2018;Singh et al, 2018;Rolfe et al, 2014). In particular, there is partitioning of signalling activity with BMP signalling active within the skeletal rudiment, at a distance from the joint interzone, and the canonical Wnt pathway active at the joint line, but this spatial restriction is lost in immobilised mouse and chick specimens (Rolfe et al, 2018;Singh et al, 2018).…”

“…In particular, there is partitioning of signalling activity with BMP signalling active within the skeletal rudiment, at a distance from the joint interzone, and the canonical Wnt pathway active at the joint line, but this spatial restriction is lost in immobilised mouse and chick specimens (Rolfe et al, 2018;Singh et al, 2018). Cell territories are altered on multiple levels in immobilised specimens, also including localised cell proliferation patterns (Roddy et al, 2011b;Kahn et al, 2009;Germiller and Goldstein, 1997) and nuclear localisation patterns of YAP within skeletal rudiments, related to changes in shape at the joint interface (Shea et al, 2019). Cell migration is also an important feature of the forming joint (Shwartz et al, 2016) which may be another cellular activity affected by biophysical stimuli (Rolfe et al, 2018), of particular interest given the importance of cytoskeletal regulation during cell migration.…”

“…Immobility results in specific effects on synovial joints, including reduction of the interzone region between adjacent skeletal rudiments, with continuity of cartilaginous rudiments across joints (fusion) in many cases; loss of normal cellular organisation with absence of the chondrogenous layers at the ends of rudiments (zones of future articular cartilage marked by increased cell density oriented parallel to the joint line); and failure to commence cavitation (Singh et al, 2018;Nowlan et al, 2014;Roddy et al, 2011b;Nowlan et al, 2010a;Kahn et al, 2009;Osborne et al, 2002). Changes within the rudiment termini also result in abnormal joint shape (Sotiriou et al, 2019;Brunt et al, 2016;Brunt et al, 2015;Roddy et al, 2011b) and all of these changes have been shown to be underpinned by altered gene expression and activation of signalling pathways that guide essential developmental steps including Wnt, BMP and Hippo (Shea et al, 2019;Rolfe et al, 2018;Singh et al, 2018;Brunt et al, 2017;Rolfe et al, 2014;Roddy et al, 2011b;Kahn et al, 2009). Disturbances of the spine due to immobility include curvature abnormalities, posterior and anterior vertebral fusions and altered vertebral shape (Levillain et al, 2019;Rolfe et al, 2017;Hosseini and Hogg, 1991).…”

Joint development recovery on resumption of embryonic movement following paralysis

“…The copyright holder for this preprint this version posted January 8, 2021. ; https://doi.org/10.1101/2021.01.08.425893 doi: bioRxiv preprint Kahn et al, 2009;Osborne et al, 2002). Changes within the rudiment termini also result in abnormal joint shape (Sotiriou et al, 2019;Brunt et al, 2016;Brunt et al, 2015;Roddy et al, 2011b) and all of these changes have been shown to be underpinned by altered gene expression and activation of signalling pathways that guide essential developmental steps including Wnt, BMP and Hippo (Shea et al, 2019;Rolfe et al, 2018;Singh et al, 2018;Brunt et al, 2017;Rolfe et al, 2014;Roddy et al, 2011b;Kahn et al, 2009). Disturbances of the spine due to immobility include curvature abnormalities, posterior and anterior vertebral fusions and altered vertebral shape (Levillain et al, 2019;Rolfe et al, 2017;Hosseini and Hogg, 1991).…”