Investigation of the mechanics of gastrulation in the chick embryo using new transgenic chicken lines

研究包括人类在内的高等动物的发育的一个重要目标是了解数十万到数百万的细胞如何组装成组织，形成器官和结构，构成一个有机体，以及哪些机制控制和整合这些行为。这种细胞行为的协调在胚胎发育的关键早期阶段，即原肠胚形成中尤为重要。在原肠胚形成过程中，胚胎的主体平面形成，主体轴出现。原肠胚形成过程涉及大范围、长时间的细胞运动和组织变形，在此过程中，三个身体层（外胚层、中胚层和内胚层）的细胞在胚胎中占据正确的位置。内胚层位于胚胎的最内侧，形成消化道和相关腺体的内层。它被一层中间层——中胚层包围，中胚层形成肌肉和骨骼。中胚层被最外层外胚层包围，外胚层将形成表皮（皮肤）、大脑和神经系统。原肠胚形成过程中细胞运动协调的缺陷会导致胚胎死亡，或者在不太严重的情况下导致许多重大出生缺陷，如各种形式的脊柱裂和各种心脏缺陷。我们研究鸡胚原肠胚形成的机制，其优点是发育发生在母体动物外的鸡蛋中，因此易于实验观察和操作。胚胎也是扁平和半透明的，这有助于观察原肠胚形成过程中的细胞运动。鸡胚胎的原肠胚形成与人类的原肠胚形成非常相似，这意味着研究结果可以外推到人类的发育过程中。我们研究了不同的细胞行为，如细胞分裂、形状变化、分化和运动是如何通过特定的化学和机械细胞-细胞信号传导机制协调产生这三种胚胎组织的。由于这些过程是高度动态的，我们使用延时显微镜来观察构成这一发育阶段胚胎的大约50万个细胞的这些行为。为了做到这一点，我们开发并制造了一种新型显微镜，一种光片荧光显微镜，它使我们能够看到一种特殊鸡系胚胎中的几乎所有细胞，这种鸡系的细胞膜上有绿色荧光蛋白的标记。我们还开发了广泛的计算方法来提取在正常和实验扰动条件下驱动原肠胚形成的相关细胞和组织行为的定量信息。在这个项目中，我们专门测试了细胞行为产生的力量，如细胞生长和运动，在发育中的胚胎中许多细胞的复杂细胞行为的协调中起着重要作用的假设。此外，我们期望这些力量对控制基因表达的反馈，控制胚胎中各种不同类型细胞的位置和数量。为了验证这一假设，我们将生成新的鸡系，使我们能够监测和干扰肌动蛋白-肌球蛋白细胞骨架的关键元素的活动，这些细胞骨架负责产生这些力，并使用上述实时成像和数据分析方法将这些力与不同的细胞和组织行为联系起来。

An important goal of the study of development of higher animals including humans is to understand how hundreds of thousands to millions of cells assemble into tissues that make organs and structures that make up an organism and which mechanisms control and integrate these behaviours. This coordination of cell behaviours is especially important at a critical early stage in embryonic development, gastrulation. During gastrulation the main body plan of the embryo is laid down and the main body axes emerge. Gastrulation involves large-scale, long-range cell movements and tissue deformations during which cells of the three body layers, the ectoderm, the mesoderm and endoderm take up their correct positions in the embryo. The endoderm is located inner most in the embryo forming the lining of the digestive tract and associated glands. It is surrounded by a middle layer the mesoderm that will make the muscles and the skeleton. The mesoderm is surrounded by the outmost layer, the ectoderm, which will form the epidermis (skin) and the brain and nervous system. Defects in the coordination of cell movements during gastrulation results in death of the embryo or in less severe cases in many major birth defects, such as the various forms of spina bifida and diverse heart defects.We study the mechanism of gastrulation in the chick embryo, that has the advantage that development takes place in the egg outside the mother animal and is therefore easily accessible to experimental observation and manipulation. The embryo is also flat and translucent which helps observation of cell movement during gastrulation. Gastrulation in chick embryos greatly resembles gastrulation in humans, which means that findings can be extrapolated to human development. We study how different cell behaviours such as cell division, shape changes, differentiation and motion are coordinated to generate these three embryonic tissues through specific chemical and mechanical cell-cell signalling mechanisms. Since these processes are highly dynamic we use time lapse microscopy to visualise these behaviours of the around 500.000 cells that make up the embryo of this stage of development. To do this we have developed and built a novel type of microscope, a light-sheet fluorescence microscope, that allows us to see almost all the cells in the embryo of a special chicken line in which the membranes of cells are marked with a green fluorescent protein. We have also developed extensive computational methods to extract quantitative information about relevant cell and tissue behaviours that drive gastrulation under normal and experimentally perturbed conditions. In this project we specifically test the hypotheses that forces produced by cell behaviours such as cell growth and movement are instrumental in the coordination of the complex cell behaviours of many cells in the developing embryo. Furthermore we expect that these forces feedback on the control of gene expression controlling the location and quantity of the various different types of cells in the embryo. To test this hypothesis we will generate new chicken lines that allow us to monitor and perturb the activity of critical elements of the actin-myosin cytoskeleton of cells that is responsible for generating these forces and correlating these with distinct cell and tissue behaviours using the above described live imaging and data analysis methodology.