Micromechanical measurements in living embryos

The embryo is a complex system wherein local tissue displacement and deformation is the result of local and distant force-generating mechanisms coupled through the largely-unknown mechanical properties of the composite tissues. One particular case in point is that of neurulation, the process by which the early sheet of cells, called the neural ectoderm, folds itself into the three dimensional structure that is the framework upon which the vertebrate central nervous system grows. At its simplest, such as neurulation in the spinal cord, the process involves the folding of a sheet roughly into a cylinder but even that is poorly understood. Neurulation in the brain is far more complex but essential for us to understand; errors in its morphogenesis are the root cause of debilitating and fatal birth defects. Thanks to novel imaging and image processing technologies, we have made great strides in developing methods to capture the movements of cells and tissues. Three-dimensional time-lapse images, analysed using in toto cell tracking and computational analyses show a rich spectrum of tissue remodelling. However, despite this apparently complex scheme, we believe that these patterns could originate from a well-orchestrated series of stereotypical force-generating mechanisms that are patterned in space and overlapping in their influence. We can already make predictions of how these may act but to verify these models and progress further we need far greater insight into the changing physical properties of tissues as they develop. Biologists are in need of tools to address such problems in the context of the complex and changing conditions that exist within the animal embryo. Our aim is to develop such a tool and use it to study the balance between active processes and the underlying mechanical properties of developing tissues that is essential in shaping correct morphogenesis of the embryo.

We plan in this project to develop a minimally-invasive tool able to probe the local mechanical response of living tissues. The device will be relatively portable, mountable on a standard microscope stage. It will impose a controlled force upon a ferromagnetic bead located in the biological sample. The direction and magnitude of force to be controlled. Our preliminary tests have demonstrated that such an experiment is achievable in zebrafish embryos. Animals develop normally with these particles in place and modest magnetic fields can be used to gently displace beads within these embryos. This methodology will enable us to investigate largely unexplored areas of developmental biology. First, we will characterise for the the elastic and viscous/plastic properties of living tissues within a normally developing embryo. This information is important to establish the range of forces required to observed processes, and to discriminates between possible mechanisms. We will focus our attention to the analysis of tissue maturation during development. The transition from blastula to gastrula is a good example where cells thought to progressively form tighter junctions. We will follow the evolution of the tissue mechanical properties with developmental time and ask if this temporal variation is key to normal development. Using statistics on many embryos, we will be able to study spatial patterns of mechanical properties. We will more specifically characterise how much of the patterning involved in brain development is due to variations in passive properties, and how the balance between active processes and the surrounding tissue is critical for normal development. Such questions and the methodology developed here to address them apply to most morphogenetic transformations in and are expected to be highly relevant elsewhere.

Our challenge is to understand how tissue mechanical properties are patterned to ensure proper morphogenesis. To address this question requires tools to probe in vivo the local stiffness and plasticity of tissues as development proceeds. We propose to develop a portable device, mountable on a standard microscope stage, that can impose a controlled force upon a ferromagnetic bead located in a living zebrafish embryo. The device will be designed with multiple poles to permit the direction and magnitude of force to be controlled. We will be able to characterize for the first time, using our own established methods of confocal imaging and image processing, the deformation field of the tissue around the magnetic bead for a known force, and from this estimate the local stiffness and visco-elastic properties of the surrounding tissue. Each experiment will use a single bead placed at a precise location in the embryo. We will use induced displacement of the bead to follow local mechanical properties as that tissue develops. Such longitudinal studies are impossible with more invasive techniques such as laser ablation or explant characterisation. Further, by accumulating statistics on these quantities over many embryos, we will generate mechanical maps aligned with morphogenetic strain maps of those same embryos. We will apply this approach to problems of increasing complexity.

Our first study will be of the visco-elastic properties of blastula cells. We will then investigate the mechanical coupling between yolk flow and cell spreading during the fish epiboly. Third, we will monitor the maturation of blastula cells towards the onset of gastrulation. Finally, we will explore the spatial patterning of strains and tissue properties during zebrafish brain and spinal cord neurulation. In all cases, we will show how the patterning of mechanical properties direct tissue morphogenesis.

This proposal will benefit the public by increasing the stock of useful knowledge, by contributing to the creation of new scientific methodologies, by stimulating the development of scientific networks and thus increasing social interaction, by increasing the supply of skilled graduates and researchers, and by enhancing the problem-solving capacity through undergraduate, graduate and postdoctoral teaching.

Increasing the stock of knowledge on morphogenesis is very important as this will impact many other fields and will support progress in medicine. Gaining a deeper understanding of morphogenetic mechanisms is essential to improve human health in three areas. First, to better understand birth defects, which are now the leading cause of infant mortality in developing countries, with neural tube closure malformations alone affecting 0.5-2/1000 pregnancies. Second, to understand cancer metastasis: there is evidence that many cancers invade healthy tissues through collective cell movements that are very reminiscent of embryonic morphogenetic movements. Finally, to develop regenerative medicine: tissue and organ engineering will require in-depth knowledge of morphogenetic mechanisms to be able to build three-dimensional structures following stem cell manipulation. Creation of new scientific methodologies will complement the increase in knowledge about morphogenesis and support progress in the above areas as well. In addition, software tools and protocols developed through the proposed work will increase the stock of scientific methodologies that can be applied to a variety of other problems.

The proposed research aims to reduce particular aspects of a complex biological system in to a simpler but quantitative description, using engineering skills, physical concepts and state of the art imaging. The general approach and some of the underlying techniques are directly applicable to a wide range of problems in science and society. We are actively training students at undergraduate, graduate and postdoctoral level, who will benefit from gaining numerical and problem-solving skills. The knowledge and methodologies gained from the proposed research will also be disseminated through the creation of specific forums such as a wiki site, the Cambridge Advanced Imaging Centre and the QuanTissue Research Network Programme. This will develop scientific networks at national and international levels and thus increasing social interaction within the UK and beyond.