Micromechanical measurements in living embryos

胚胎是一个复杂的系统，其中局部组织的位移和变形是通过复合组织的基本未知的机械性能耦合的局部和远程力产生机制的结果。一个特别的例子是神经形成，这是一个被称为神经外胚层的早期细胞片折叠成三维结构的过程，而三维结构是脊椎动物中枢神经系统生长的框架。在最简单的情况下，比如脊髓中的神经形成，这个过程涉及到将一张纸大致折叠成一个圆柱体，但即使是这样，人们也知之甚少。大脑中的神经形成要复杂得多，但对我们来说是必不可少的；它的形态发生错误是导致虚弱和致命出生缺陷的根本原因。多亏了新的成像和图像处理技术，我们在开发捕捉细胞和组织运动的方法方面取得了长足的进步。使用全细胞跟踪和计算分析的三维时间推移图像显示了丰富的组织重建光谱。然而，尽管这一方案显然很复杂，但我们认为，这些模式可能源于一系列精心策划的刻板印象的力量产生机制，这些机制在空间上形成模式，在影响上重叠。我们已经可以对这些可能的作用做出预测，但为了验证这些模型并取得进一步进展，我们需要更深入地了解组织在发展过程中不断变化的物理属性。在动物胚胎中存在的复杂和不断变化的条件下，生物学家需要工具来解决这些问题。我们的目标是开发这样一种工具，并用它来研究发育中组织的活跃过程和潜在机械特性之间的平衡，这对于形成正确的胚胎形态发生至关重要。在这个项目中，我们计划开发一种能够探测活组织局部机械反应的微创工具。该设备将是相对便携的，可以安装在标准显微镜平台上。它将对位于生物样品中的铁磁珠施加可控的力。要控制的力的方向和大小。我们的初步测试表明，这样的实验可以在斑马鱼胚胎上实现。动物在这些颗粒存在的情况下正常发育，适度的磁场可以用来轻轻地移动这些胚胎中的珠子。这种方法论将使我们能够调查发育生物学中大部分未被探索的领域。首先，我们将描述正常发育的胚胎内活组织的弹性和粘性/塑性特性。这一信息对于确定观察过程所需的力的范围以及区分可能的机制很重要。我们将重点关注发育过程中组织成熟的分析。从囊胚到原肠胚的过渡就是一个很好的例子，细胞被认为逐渐形成了更紧密的连接。我们将跟踪组织力学性质随发育时间的演变，并询问这种时间变化是否对正常发育至关重要。使用许多胚胎的统计数据，我们将能够研究机械性能的空间模式。我们将更具体地描述大脑发育过程中涉及的模式在多大程度上是由于被动属性的变化，以及主动过程和周围组织之间的平衡如何对正常发育至关重要。这些问题和这里为解决这些问题而开发的方法适用于大多数形态发生转变，并预计将在其他地方高度相关。

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.