Systems Analysis of BMP Regulation in Developing Zebrafish Embryos

描述（由申请人提供）：骨形态发生蛋白（BMP）作为细胞外信息的范例在发育模式形成中起作用，其作为形态发生蛋白在空间上以梯度分布，通过形态发生蛋白水平指定不同的细胞类型。在脊椎动物背腹（DV）轴向图案形成的功能，分子伴侣，和每个BMP组件的作用是相当好的理解，但机制的组件的组合功能形成一个强大的BMP梯度是复杂的，仍然知之甚少。该领域处于僵局，无法超越当前的范式，并解决细胞外因子和多个反馈回路相互作用并在空间和时间上调节彼此的活动以产生模式化胚胎的梯度的机制。斑马鱼系统现在已经有了足够好的定义，可以生成有效且可测试的数学模型，从而推动这一领域的发展。此外，我们对BMP调节剂如何调节实际的信号梯度知之甚少。目的是发现和歧视BMP的调节机制，利用定量图像采集和分析，早期斑马鱼胚胎DV图案的几何精确的数学模型，和混合质量约束为基础的优化。在目的1中，BMP信号梯度的时空形成将通过测量野生型和BMP组分突变体斑马鱼胚胎中的磷酸-Smad 1/5水平来定量研究，分割每个胚胎中的所有细胞核，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定量差异和相似性，以了解它们在BMP信号梯度形成中的作用。在目标2中，BMP介导的信号控制网络将通过开发、优化和分析3D时空模型来识别。将开发基于图像的斑马鱼晚期囊胚-原肠胚胚胎BMP模式形成模型，并测试引导模式形成动力学的BMP调节的多种替代机制。在目标3中，我们将使用基于模型的最优实验设计来降低多组分网络综合分析所需的析因设计的复杂性。此外，我们将确定Cvl 2，Tsg 1和Chd调节动态BMP信号传导的机制，以测试模型的预测能力并描述这一重要网络的作用。这个目标的目的是进行同步基因扰动实验，这将提供最大量的有关BMP调控的信息。了解脊椎动物中BMP介导的模式化机制将为在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.