Construction of a novel Digital Scanning Lightsheet Microscope and its application in measuring 3D cell behaviour and movement in embryos

了解胚胎发育是生命科学研究的重大挑战之一。所有高等生物胚胎发育的第一个阶段是精子与卵细胞融合形成受精卵，随后通常是一系列快速的细胞分裂，产生许多细胞。由于卵细胞细胞质的不对称或母体提供的外部不对称，不同的细胞启动不同的基因表达程序，从而使细胞增殖，经历程序性细胞死亡，分化和细胞移动。为了正常发育，这些过程必须在三维空间和时间中精确协调，这是通过广泛的细胞间通讯来实现的。细胞间的交流包括通过直接的细胞间接触（短距离）或通过分泌信号分子来传递信号，这些信号分子可以在细胞之间的空间中扩散并传播相对较远的距离。这些过程只能产生有限数量的形式和形状，并且在许多情况下，细胞需要从它们产生的地方移动到需要它们的地方。这在原肠胚形成过程以及神经系统的形成和连接过程中尤为重要。原肠胚形成是胚胎发生的关键阶段，在此阶段胚胎的主体平面被确定，对称轴出现。在此过程中，三个胚层（外胚层、中胚层和内胚层）的细胞在胚胎中占据正确的拓扑位置。内胚层位于胚胎和成体的最内层，内胚层细胞形成消化道和相关腺体（肝脏、胰腺等）的内层。内胚层被中胚层包围，中胚层形成肌肉和骨骼。中胚层由最外层覆盖，外胚层将形成表皮和神经系统。在原肠胚形成过程中，不适当的细胞运动导致严重的发育流产，在不太严重的情况下，形成动物和人类许多先天性缺陷的基础。在各种实验模型系统中研究了原肠胚形成的信号机制和细胞过程。高等脊椎动物如羊膜动物（鸟类、爬行动物和哺乳动物）的原肠胚形成研究主要集中在小鸡和老鼠胚胎的发育上。鸡胚的优点是发育发生在母体外，因此很容易进行实验操作。鸡胚扁平且半透明，这使得观察原肠形成过程中的细胞运动成为可能。为了理解复杂的过程，如原肠胚形成，有必要能够跟踪胚胎中所有细胞的运动。这需要非常强大的显微镜技术，而这项研究的目标之一就是建立和开发一种显微镜，有了它，这将成为可能。这需要物理学家、计算机科学家和生命科学家之间的密切合作，我们已经组建了这样一个联盟。一旦仪器建成，我们将用它来绘制小鸡早期发育过程中的细胞分裂和运动，并生成这一过程的蓝图。在第二阶段，我们将开始研究通过实验扰动控制这些运动的信号系统，我们将上下调节关键信号分子并研究它们对早期发育的定量影响。由此，我们将建立一个控制原肠胚形成的最关键过程的图片，这将有助于理解许多先天性缺陷和以后生活中的疾病，在这些研究中获得的知识将是必不可少的，能够在未来预防和治疗这些病例。

Understanding embryonic development is one of the big challenges of Life Science Research The first stage in the embryonic development of all higher organisms is the formation of the zygote from the fusion of a sperm with an egg cell, which is typically followed by a series of rapid cell divisions in which many cells are generated. Due to asymmetries present in the cytoplasm of the egg, or external asymmetries provided by the mother, different cells initiate distinct gene expression programs, which allow the cells to proliferate, undergo programmed cell death, differentiate and cell move. For proper development to occur these processes have to be coordinated precisely in 3 dimensional space and time, which is achieved by extensive cell-cell communication. Cell-cell communication can involve signaling through direct cell-cell contacts (short-range) or through secretion of signaling molecules that can diffuse in the space in between the cells and travel a relatively long distance. There are only a limited number of forms and shapes that can be generated by these processes and in many cases cells will need to move from the place where they produced to the site where they are required. This is particularly important in the process of gastrulation and during the formation and wiring of the nervous system. Gastrulation is a critical stage in embryogenesis where the main body plan of the embryo is laid down and the axes of symmetry emerge. It involves large-scale long-range cell movements during which cells of the three germlayers (ectoderm, mesoderm, and endoderm) take up the correct topological positions in the embryo. The endoderm is located innermost in the embryo and adult and endoderm cells form the lining of the digestive tract and associated glands (liver pancreas etc). The endoderm is surrounded by the mesoderm that will give rise to the muscles and the skeleton. The mesoderm is covered by the outmost layer, the ectoderm that will form the epidermis and the nervous system. Improper cell movements during gastrulation results in severe cases in abortive development and in less severe cases form the basis of many congenital defects in animals and humans. The signaling mechanisms and the cellular processes underlying gastrulation have been studied in a variety of experimental model systems. The study of gastrulation in higher vertebrates such as amniotes (birds reptiles and mammals) has focused on the development of the chick and mouse embryo. The chick embryo has the advantage that development takes place outside the mother and is therefore easily accessible to experimental manipulation. The chick embryo is flat and translucent which makes observation of cell movements during gastrulation possible. To understand complex processes such as gastrulation it is essential to be able to follow the movements of all cells in the embryo. This requires very powerful microscopic techniques and one of the aims of this research is to build and develop a microscope with which this will be possible. This will require close collaboration between physicists, computer scientist and life scientists and we have assembled such a consortium. Once the instrument is build we will use it to map out cell division and movement during early chick development and generate a blueprint of this process. In a second phase we will start to investigate the signaling systems that control these movements by experimental perturbation, we will up and down regulate critical signaling molecules and study their quantitative effects on early development. From this we will build up a picture of the most critical processes that control gastrulation which will help in understanding many congenital defects and diseases in later life and knowledge obtained in these studies will be essential to be able to prevent and cure some of these cases in the future.