Systems Analysis of BMP Regulation in Developing Zebrafish Embryos

描述(申请人提供)：骨形态发生蛋白(BMPs)在发育模式的形成中发挥作用，是一种细胞外信息的范例，作为形态生成物在空间上以梯度分布，通过形态生成物水平指定不同的细胞类型。在脊椎动物背腹(DV)轴型的形成中，每个BMP组分的功能、分子伙伴和作用都已被很好地理解，但这些组分的组合功能形成强大的BMP梯度的机制是复杂的，仍然知之甚少。该领域正处于僵局，无法超越当前的范式，解决细胞外因子和多个反馈环在空间和时间上相互作用和调节彼此活动的机制，以产生形成胚胎图案的梯度。斑马鱼系统现在已经定义得足够好，可以产生有效的和可测试的数学模型，从而推动这一领域的发展。此外，我们对BMP调节器如何调节实际的信号梯度知之甚少。其目的是利用定量图像采集和分析、早期斑马鱼胚胎DV构型的几何精确数学模型和基于混合质量约束的优化来发现和识别BMP的调节机制。在目标1中，将通过测量野生型和BMP组分突变斑马鱼胚胎中磷酸化Smad1/5的水平来定量研究BMP信号梯度的时空形成，分割每个胚胎中的所有核，并将数据注册到标准胚胎。这些研究将提供首次的(半)定量数据，可用于区分单个BMP细胞外调节剂的空间和数量的差异和相似之处，以了解它们在BMP信号梯度形成中的作用。在AIM中，将通过开发、优化和分析3D时空模型来识别用于BMP介导的信号控制的网络。将开发一个基于图像的斑马鱼晚期囊胚-原肠胚BMP模式形成模型，并对引导模式形成动力学的BMP调节的多种替代机制进行测试。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，我们还将确定Cvl2、Tsg1和CHD对动态BMP信号的调节机制，以检验模型的预测能力，并描绘这一重要网络的作用。这一目标的目的是同时进行基因扰动实验，以提供与BMP调控有关的最大信息量。了解脊椎动物BMP介导的模式形成机制将为在组织再生和其他人类疾病的预期治疗中严格控制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.