Mechanics of development

Embryo movement is essential to the formation of a functional skeleton. Using mouse and chick models, the group has previously shown that mechanical forces influence gene regulation and tissue patterning, particularly at developing joints. However, there remains a lack of knowledge of the molecular mechanisms that underpin the influence of mechanical signals. 

Wnt signalling is required during skeletal development and is altered under reduced mechanical stimulation. To explore Wnt signalling as a mediator of mechanical input, the expression of Wnt ligand and Fzd receptor genes in the developing skeletal rudiments was profiled. Canonical Wnt activity restricted to the developing joint is reduced under immobilization while over-expression of activated b-catenin or the Wnt antagonist Sfrp3 following electroporation of chick embryo limbs, supports the proposed role for Wnt signalling in mechanoresponsive joint patterning. Two key findings advance our understanding of the interplay between Wnt signalling and mechanical stimuli: firstly, the loss of canonical Wnt activity at the joint shows reciprocal, co-ordinated regulation of Wnt and BMP pathways under mechanical influence. Secondly, this occurs simultaneously with increased expression of several Wnt pathway component genes in a territory peripheral to the joint, identifying the importance of mechanical stimulation on a population of potential joint progenitor cells.

Muscle spindles and Golgi tendon organs (GTOs), proprioceptive mechanoreceptors located inside striated muscles and in myotendinous junctions, respectively, are components of the stretch reflex circuitry that control muscle activity. Using genetic mouse models, the group demonstrates the involvement of proprioception in regulating spine alignment and spontaneous realignment of fractured bones, dubbed natural reduction. Failure of the mechanism that maintains posture may result in spinal deformity as in adolescent idiopathic scoliosis. The group shows that null mutants for Runx3 transcription factor, which lack connectivity between proprioceptors and spinal cord, developed peripubertal scoliosis not preceded by vertebral dysplasia or muscle asymmetry. Deletion of Runx3 in the peripheral nervous system or specifically in peripheral sensory neurons, or of enhancer elements driving Runx3 expression in proprioceptive neurons, induced a similar phenotype. Egr3 knockout mice, lacking spindles but not GTOs, displayed a less severe phenotype, suggesting that both receptor types are required for this regulatory mechanism.

Fracture repair involves restoration of bone morphology. Comparison among mice of different ages revealed, surprisingly, that three-month-old mice exhibited more rapid and effective natural reduction than newborns. Fractured bones of Runx3-null mutants failed to realign properly. Blocking Runx3 expression in peripheral nervous system, but not in limb mesenchyme, recapitulated the null phenotype, as did inactivation of muscles flanking the fracture site. Egr3 knockout mice displayed a less severe phenotype, suggesting that both receptor types, as well as muscle contraction, are required for this regulatory mechanism. Overall, these findings uncover physiological roles for proprioception in non-autonomous regulation of skeletal integrity and repair. 

Mechanical stimulation generated by fetal kicking and movements is known to be important for prenatal musculoskeletal development, and there are a number of human conditions that emphasise the link between abnormal fetal movements and delayed or impaired skeletal development. The most common of these is developmental dysplasia of the hip (DDH), a relatively common joint shape abnormality (clinical incidence 1.3 in 1000), the risk of which is strongly associated with restricted fetal movement, such as fetal breech position. The group is using computational modelling approaches (finite element analysis and musculoskeletal modelling), combined with human fetal imaging data to try to understand how the biomechanical stimulation (stresses and strains) caused by fetal movements evolve in the prenatal developing hip joint. Furthermore, the group models a range of intra-uterine conditions and situations that increase the risk of DDH, such as fetal breech position and oligohydramnios (reduced amniotic fluid), in order to be able to understand how a range of factors affecting movement may impact on the developing hip joint.

Tendons transmit muscle-generated forces to bones to enable skeletal movements and stabilize joint structures. Proper development and maintenance of these extracellular matrix-rich tissues is critical to their demanding physical roles throughout the body. Abnormal tendon formation during embryogenesis is associated with frequently occurring musculoskeletal deformities such as congenital tallipes equino varus. Furthermore, tendons injured postnatally fail to recapitulate development during healing, and instead heal with aberrant matrix composition and organization, resulting in reduced functionality and greater susceptibility to re-injury. The group is interested in understanding how tendon mechanical properties elaborate during normal embryonic development to inform the prevention of tendon-related musculoskeletal birth defects and the enhancement of regenerative postnatal healing. This talk will focus specifically on the novel approaches the group has utilized to characterize the mechanical properties of tendons during embryonic development, and crosslinking mechanisms that have been identified to be critical to this process. Furthermore, the talk will discuss the development of engineered hydrogel systems that mimic the embryonic tendon mechanical microenvironment, enabling study of how dynamic changes in tissue stiffness continuously influence cell behaviours (differentiation, extracellular matrix synthesis, etc.) during development. Finally, the talk will discuss the role of physical movements (eg, kicking) in regulating tendon formation during embryonic development. These findings provide new insights into the crucial roles of mechanics and tissue mechanical properties in new tendon formation.

The notochord is a conserved axial structure that serves as a hydrostatic scaffold for embryonic axis elongation and, later on, for proper spine assembly. The vertebrate notochord consists of a core of large vacuolated cells surrounded by an epithelial sheath that is encased by an elaborate extracellular matrix. Each vacuolated cell contains a single fluid filled vacuole that occupies most of the cellular volume. These cells are arranged in a stereotypical staircase pattern within the notochord. The group investigated the origin of this pattern and found that it can be achieved purely by physical principles. Moreover, the group is able to model the arrangement of vacuolated cells within the notochord using a physical model composed of silicone tubes and water absorbing beads, suggesting that the notochord is a self-organizing structure. Furthermore, it was found that the biological structure and the physical model can be accurately described by the theory developed for cylindrical foams. 

In spite of their large size and being under constant mechanical stress, vacuolated cells possess a remarkable structural integrity. This is in part due to the presence of numerous caveolae, omega shaped cell surface invaginations, at the plasma membrane of vacuolated cells, which buffer mechanical tension. In zebrafish that lack caveolae, vacuolated cells collapse under the strain of axial bending during locomotion. Then, release of nucleotides from collapsed vacuolated cell leads to the invasion and trans-differentiation of sheath cells into new vacuolated cells. This regenerative response restores the mechanical properties of the notochord, thus allowing normal spine development.

Recent studies using a variety of animal models support a central role for flow and fluid forces in the development of valves in the heart, blood vessels and lymphatic vessels. An outstanding question in this field is how information sensed by endothelial cells that are directly subject to fluid forces is converted to instructions to underlying cells that participate most directly in formation of valves. To address this question the group has investigated the role of the transcription factor Krupple-like Factor 2 (KLF2) in heart valve development. KLF2 expression is highly upregulated by fluid shear forces in endothelial cells in vitro and its expression in vivo corresponds closely to predicted endothelial fluid shear forces as well. The group finds that loss of endocardial KLF2 in mouse embryos results in mid-gestation death due to an inability to remodel the cardiac cushion to a mature valve as the embryo grows. Loss of KLF2 is accompanied by cell non-autonomous changes in mesenchymal cell proliferation and condensation, two key steps in valve development. Loss of KLF2 results in virtually complete loss of the secreted protein Wnt9b as well as a severe loss of canonical WNT signalling in the sub-endocardial mesenchymal cells that participate in valve formation. Endocardial loss of Wnt9b recapitulates the Klf2 deficient phenotype in mice and studies in zebrafish embryos performed in collaboration with the Vermot lab demonstrate that Wnt9b is strongly regulated by both fluid shear forces and klf2a in vivo. These studies provide one answer to the question of how the information generated by fluid forces at the blood-endothelial interface is transmitted to cells not in contact with blood to orchestrate the creation of heart valves.

The Tzima lab investigates the role of mechanotransduction in regulating cardiovascular function in health and disease. We focus on endothelial mechanosensing and how fluid shear stress affects vessel and cardiac morphogenesis and function. Over the last decade, we identified and systematically characterised one of the most comprehensive models of endothelial mechanotransduction available to date. Using state-of-the-art approaches we showed that PECAM is a part of a junctional mechanosensory complex that senses and transduces mechanical force that ultimately regulates flow-mediated vascular remodelling. PECAM does not act alone; it crosstalks with integrins, another established mechanotransduction site, to collectively determine downstream responses. Recent work from our group, identified the signalling adaptor protein Src homologous and collagen protein (Shc) as a major player in endothelial mechanotransduction. We showed that Shc is tyrosine phosphorylated in response to mechanical force and binds to both the junctional mechanosensory complex and integrins. We now present data showing that Shc tyrosine phosphorylation provides a mechanical coupling checkpoint that allows certain signals to proceed, while blocking others in endothelial cells.

Mechanical forces are fundamental to cardiovascular development and physiology. The interactions between mechanical forces and endothelial cells are mediated by mechanotransduction feedback loops. The group is interested in understanding how hemodynamic forces modulate cardiovascular function and morphogenesis. Overall, their recent work is unravelling the biological links between mechanical forces, mechanotransduction and endothelial cell responses. Here the talk will discuss the recent work aiming at addressing how mechanical forces are spreading into the embryonic heart at each contraction and how these forces are sensed to modulate cardiac valve development.

Actomyosin contractility is a key regulator of tissue dynamics. During development, tissue dynamics, such as cell intercalations and oriented cell divisions, are critical for shaping tissues and organs. However, less is known about how tissues regulate their dynamics during tissue homeostasis and repair, to maintain their shape after development. In this talk, Dr Mao will discuss how tissues respond to mechanical perturbations, such as stretching or wounding, by altering their actomyosin contractile structures, to change tissue dynamics, and thus preserve tissue shape and patterning.

Heart formation involves a complex progression of finely orchestrated biological and biophysical processes. Blood flow dynamics, which is established early during cardiac development, when the heart is a linear tube without valves or chambers, is indispensable for proper heart formation. Cardiac looping, septation and valve formation all happen under blood flow, and, in fact, perturbations in blood flow dynamics during tubular heart stages lead to structural heart malformations. Blood flow mechanics, sensed by cardiac cells, act as a feedback loop regulating heart development. Feedback from blood flow mechanical stimuli is needed so that cardiovascular development addresses the increasing requirements of the embryo, while ensuring proper cardiac function. However, the discovery of the mechanisms by which developing cells sense and respond to blood flow mechanical stimuli is only starting to emerge. Quantification of both mechanical stimuli and biological responses is needed to determine the magnitude of the response to blood flow, affected regulatory processes, and possible implications to congenital heart disease. This talk will discuss these results in quantifying blood flow stimuli and biological responses, as well as implications on heart malformation.