Systems Analysis of BMP Regulation in Developing Zebrafish Embryos

描述（由申请人提供）：骨形态发生蛋白（BMPs）作为细胞外信息的范例在发育模式形成中起作用，这些信息作为形态因子在空间上以梯度分布，通过形态因子水平指定不同的细胞类型。在脊椎动物背-腹（DV）轴向模式形成中，BMP各组分的功能、分子伙伴和作用已被很好地理解，但各组分的组合功能形成强大BMP梯度的机制是复杂的，仍然知之甚少。细胞外因子和多个反馈回路在空间和时间上相互作用和调节彼此的活动，从而产生一个胚胎模式的梯度，这一领域处于僵局。现在，斑马鱼系统已经得到了充分的定义，可以生成有效的、可测试的数学模型，从而推动这一领域的发展。此外，我们对BMP调节因子如何调节实际的信号梯度知之甚少。目的是通过定量图像采集和分析，早期斑马鱼胚胎DV模式的几何精确数学模型，以及基于混合质量约束的优化，发现和区分BMP调控机制。在Aim 1中，将通过测量野生型和BMP成分突变斑马鱼胚胎中TOTO中磷酸化- smad 1/5水平来定量研究BMP信号梯度的时空形成，分割每个胚胎的所有细胞核，并将数据记录到标准胚胎中。这些研究将提供首次（半）定量数据，可用于辨别单个BMP细胞外调节剂的空间和数量差异和相似性，以了解它们在BMP信号梯度形成中的作用。在Aim 2中，将通过开发、优化和分析3D时空模型来确定bmp介导的信号控制网络。基于图像的斑马鱼囊胚晚期-原胚胚胎BMP模式形成模型将被开发和测试多种BMP调节机制，指导模式形成动力学。在目标3中，我们将使用基于模型的实验优化设计来降低综合分析多组分网络所需的析因设计的复杂性。此外，我们将确定Cvl2、Tsg1和Chd调控动态BMP信号的机制，以测试该模型的预测能力，并描述这一重要网络的作用。这个目的的目标是同时进行基因扰动实验，将提供有关BMP调控的最大量的信息。了解脊椎动物BMP介导的模式机制将为组织再生和其他人类疾病的前瞻性治疗中严格控制BMP信号提供基础。

DESCRIPTION (provided by applicant): Bone Morphogenetic Proteins (BMPs) act in developmental pattern formation as a paradigm of extracellular information that is spatially distributed in a gradient as a morphogen, specifying distinct cell types via morphogen levels. In vertebrate dorsal-ventral (DV) axial pattern formation the function, molecular partners, and role of each BMP component are fairly well understood, but the mechanism by which the combined function of the components form a robust BMP gradient is complex and still poorly understood. The field is at an impasse to go beyond the current paradigms and solve the mechanism by which the extracellular factors and multiple feedback loops interact and regulate each other's' activity spatially and temporally to generate a gradient that patterns the embryo. The zebrafish system is sufficiently well defined now to allow effective and testable mathematical models to be generated that could move this field forward. Moreover, we know very little about how BMP regulators modulate the actual signaling gradient. The objective is to discover and discriminate mechanisms of BMP regulation by utilizing quantitative image acquisition and analysis, geometrically accurate mathematical models of early zebrafish embryo DV patterning, and mixed-quality constraint based optimization. In Aim 1 the spatiotemporal formation of the BMP signaling gradient will be quantitatively investigated by measuring phospho-Smad 1/5 levels IN TOTO in wild-type and BMP component mutant zebrafish embryos, segment all nuclei in each embryo, and register the data to a standard embryo. These studies will provide the first-ever (semi)-quantitative data that can be used to discern the spatial and quantitative differences and similarities of individual BMP extracellular modulators to understand their roles in BMP signaling gradient formation. In Aim 2 networks for BMP-mediated signaling control will be identified by developing, optimizing, and analyzing 3D spatiotemporal models. An image-based zebrafish late blastula- gastrula embryo BMP pattern formation model will be developed and tested for multiple alternative mechanisms of BMP regulation that guide pattern formation dynamics. In aim 3 we will use Model-Based Optimal Design of Experiments to reduce the complexity of factorial design required for comprehensive analysis of multiple-component networks. Additionally, we will determine the mechanism of Cvl2, Tsg1, and Chd regulation of dynamic BMP signaling to test the model's predictive ability and delineate the action of this important network. The goal of this aim is to carry out simultaneous gene perturbation experiments that will provide the greatest amount of information pertaining to BMP regulation. Understanding the mechanism of BMP-mediated patterning in vertebrates will provide the basis for tightly controlling BMP signaling in tissue regeneration and other prospective treatments of human disease.