Drosophila germ-band extension as a model for understanding the integration of cell intrinsic and extrinsic forces during animal morphogenesis

当受精卵开始发育时，它们面临着巨大的挑战。卵子必须多次分裂才能产生大量的细胞。反过来，这些细胞必须被引导相互分化，同时以刻板的方式重新排列，以塑造身体的组织和器官。这种运动程序被称为形态发生。在所有动物胚胎中发生的最重要的早期变化之一是胚胎的头尾轴从短而宽的组织延伸到细长的窄体。如果这种汇聚和延伸的过程失败，它对胚胎是非常有害的，甚至更通常是致命的。在人类中，神经管缺陷，如脊柱裂和无脑畸形，是异常会聚和伸展的后果的例子。两种类型的信息对于试图理解形态发生是如何发生的至关重要。首先，确定哪些基因被激活来协调它，其次，研究相关基因如何控制驱动胚胎重塑的力量的产生。在过去30年左右的发展生物学中，人们主要关注遗传解释。随着我们对活胚胎发育成像能力的最近革命，以及在自动跟踪发育中的胚胎内的细胞和细胞形状方面的最新进展，这种情况已经开始改变。我们的实验室一直处于这些最新发展的前沿，我们处于非常有利的地位，能够研究发育过程中力量的性质和变化的平衡，以及这些力量是如何由基因协调的。这个项目的目的是用果蝇胚胎作为模型，用来开发方法来理解驱动胚胎发育的力量。果蝇胚胎在胚胎外部有一个相对简单的单层“上皮”，细胞聚集和延伸，它的遗传学是非常清楚的。胚胎中的力是由细胞收缩产生的，其方式类似于肌肉收缩。在会聚和伸展运动中，细胞收缩驱动细胞形状的变化，并导致细胞重新排列。重要的是，活跃的细胞行为产生的力将对邻近的细胞产生影响，这些细胞可以反过来将力传递给邻近的细胞，或者通过改变形状或排列来驱散力量。因此，细胞可以感受到来自远近细胞的各种外力。这个项目的目标之一是将活跃的细胞力从外力(来自其他地方)中分离出来。在同质组织中不会发生会聚和延伸。基因在组织中的表达各不相同，并与活跃的细胞重排行为的强度相关。我们将详细研究行为变异的模式，并将其与基因表达和细胞收缩的模式相关联。我们将应用新的方法来区分内在细胞重排和外力诱导的被动重排。我们将扩展现有的细胞自动跟踪方法，以跟踪细胞在收敛和延伸过程中的完整三维形状。有了这样的数据，我们将能够询问细胞的形状和方向是否存在差异，它们是倾斜的还是楔形的，表明存在局部或远处的力量。我们将使用聚焦激光消融来测试上面产生的关于组织力性质的假设，通过穿孔或切线来测试组织是否以我们的假设预测的方式拉开。在这个简单的模型中，结合开发新的通用方法来分离胚胎力量，将是能够处理更复杂的脊椎动物模型的重要一步，例如斑马鱼和老鼠以及其他与人类出生缺陷和疾病状态相关的模型。

When fertilized eggs start their development they are faced with an enormous challenge. The egg must divide many times to produce huge numbers of cells. In turn, these cells must be directed to become differentiated from one another and simultaneously rearrange in stereotypical ways to shape the tissues and organs of the body. This programme of movements is called morphogenesis. One of the most important early transformations that takes place in all animal embryos is the elongation of the embryonic head-tail axis from a short, wide tissue to an elongated narrow body. If this process of convergence and extension should fail, it is highly detrimental or more usually lethal to embryos. In humans, neural tube defects such as spina bifida and anencephaly are examples of the consequences of abnormal convergence and extension.Two types of information are vital in trying to understand how morphogenesis happens. Firstly, identifying which genes are turned on to orchestrate it and, secondly, working out how the relevant genes control the generation of forces that drive embryonic reshaping. The last 30 years or so of developmental biology have seen a focus primarily on genetic explanations. This has begun to change with the recent revolution in our ability to image live embryonic development and even more recent progress in the automation of the tracking of cells and cell shapes within developing embryos. Our laboratories have been at the forefront d of these recent developments, and we are in an excellent position to be able to investigate the nature and changing balance of forces during development, and how these are orchestrated by the genes. The aim of this project is to use fruit-fly embryos as a model with which to develop methods to understand the forces that drive embryonic development. The fruit-fly embryo has a relatively simple single-layered 'epithelium' of cells on the outside of the embryo that converges and extends, and its genetics is very well understood. Forces in embryos are generated by contractions of cells in ways analogous to how muscles contract. During convergence and extension movements, cell contractions drive changes in cell shape and cause cell to rearrange. Importantly, forces generated by active cell behaviour will exert effects on neighbouring cells, that can transfer force onto their neighbours in turn, or respond by dissipating the force through changing shape or arrangement. Thus cells can experience a variety of extrinsic forces from cells near and far. Disentangling active cell forces from forces imposed extrinsically (from elsewhere) is one of the goals of this project.Convergence and extension does not happen in a homogenous tissue. Gene expression varies across the tissue, and is correlated with patterns of the strength of active cell rearrangement behaviour. We will investigate the patterns of behaviour variation in detail, and correlate these with the patterns of gene expression and of the contraction of the cell. We will apply new methods to distinguish intrinsic cell rearrangement from passive rearrangement induced by extrinsic forces.We will extend our current automated methods for tracking cells to track the full three-dimensional shapes of cells during convergence and extension. With such data we will be able to ask whether there are differences in the shapes and orientations of cells, whether they are tilted or wedge-shaped in ways that indicate the presence of local or distant forces. We will test hypotheses generated above about the nature of tissue forces using focused laser ablation, making punctures or cut lines to test if the tissue pulls apart in ways predicted by our hypotheses. The combination of developing new generic methods to disentangle embryonic forces in this simple model will be a major step in being able to tackle more complicated vertebrate models, such as the zebrafish and mouse and other models relevant to human birth defects and disease states.