Spatial Regulation of BMP Signaling in Dorsoventral Patterning

描述(申请人提供)：这个项目的目标是研究高度保守的转化生长因子-β信号通路是如何发挥作用的，使用水蚤的胚胎作为一个方便的模型。BMP亚家族的转化生长因子-β信号在脊椎动物的整个发育过程中都是活跃的，从背腹模式和器官发生到牙齿和毛囊的诱导。许多发育缺陷、癌症和其他疾病都涉及BMP信号的异常调节，因此了解BMP信号的调节具有重要意义。许多研究集中在分泌的BMPs和它们的拮抗剂相互作用形成形态梯度的机制，这些梯度将位置信息传递给大范围的细胞，例如在脊椎动物和昆虫胚胎的背腹模式中。但其他重要的BMP信号过程涉及短程相互作用，例如癌症中肿瘤-间质的相互作用，或牙齿发育中的上皮-间充质相互作用。水蚤(Helobdella)胚胎为脊椎动物和果蝇系统提供了一个补充模型，最常用于BMP信号研究，因为它具有相对简单的细胞几何结构和典型的细胞谱系，其命运部分由短程信号相互作用决定。这种水蚤可以进行精确和可重复性的实验操作，其基础是将标记和基因结构显微注射到已识别的、谱系受限的干细胞(成体细胞)中。因此，利用Helobdella可以对BMP信号进行特殊操作，并在完整胚胎的生理相关背景下准确地分析结果。此外，Helobdella在进化上与标准模型相距甚远；了解BMP信号如何在不同物种的同源过程中发挥作用，可能会揭示BMP信号可能在单个物种(例如人类)的不同组织和阶段中发挥作用的一系列机制。对水蚤发育的研究对进化发展(Evo-Devo)领域也很重要，因为水蚤属于一个多样性的、但研究不足的动物超级门，即Lophotrochozoa。项目期间的具体目标是利用Helobdella外胚层系统探索BMP信号在异种细胞类型中的空间调节。需要检验的主要假设是，在胚胎中观察到的两种不同细胞类型之间BMP5-8信号空间范围的差异反映了Furin或其他前肽转换酶枯草杆菌/可信(PCSK)家族成员处理BMP前肽的不同，这些酶通常将BMP前肽加工成成熟形式。为了验证这一假设，我们将显微注射表达野生型和裂解位点突变的水蛭BMP5-8信号活性表位标记变体的构建物，以驱动其在特定胚胎谱系中的表达，无论是单独的还是与各种水蚤PCSK一起表达。对标记了表位的BMP进行处理后，将进行蛋白质印迹分析，并将使用一套已建立的分子和形态标准来评估它们的信号效应。在其他实验中，功能BMP5-8的空间分布将通过在活胚胎和/或固定标本中的表位标记的BMP5-8变体中可视化具有信号活性的荧光蛋白标记的BMP5-8来直接评估。最后，可能影响BMP5-8信号空间调控的其他因素将被评估，包括通过保守的N末端碱性氨基酸簇与细胞外基质的可能相互作用，内化BMP配体-受体复合体，以及BMP异二聚体和同源二聚体的活性和迁移率的差异。这项研究与公共卫生的相关性在于，它将拓宽我们对转化生长因子-β信号传递过程在人类发育或疾病期间如何调控的理解，在这些疾病中，实验是不可能的，以及在复杂的脊椎动物模型系统中，不能服从在水蚤胚胎中可能进行的详细分析。此外，这项研究提供了一个宽容和相对便宜的培训环境，未来的医生和医学科学家可以在其中磨练实验技术和严格的分析智力方法，以便随后应用于更多临床相关问题。

DESCRIPTION (provided by applicant): The goal of this project is to investigate how the highly conserved TGF-beta signaling pathway functions, using the embryo of the leech as a convenient model. The BMP sub-family of TGF-beta signaling is active throughout vertebrate development, ranging from dorsoventral patterning and organogenesis to the induction of teeth and hair follicles. Many developmental defects, cancers and other diseases involve dis- regulation of BMP signaling, so understanding the regulation of BMP signaling is of fundamental importance. Many studies have focused on the mechanisms by which secreted BMPs and their antagonists interact to form morphogen gradients that impart position information to large fields of cells, for example in dorsoventral patterning of vertebrate and insect embryos. But other important BMP signaling processes involve short-range interactions, for example tumor-stroma interactions in cancer, or epithelia-mesenchyme interactions in tooth development. The leech (Helobdella) embryo provides a complementary model to vertebrate and Drosophila systems most commonly used for studies of BMP signaling, because it features a relatively simple cellular geometry and stereotypical cell lineages whose fates are determined in part by short-range signaling interactions. The leech is amenable to precise and reproducible experimental manipulations, based on the microinjection of markers and genetic constructs into identified, lineage-restricted stem cells (teloblasts). Thus, with Helobdella it is possible to carry out speciic manipulations of BMP signaling and assay the results precisely in the physiologically relevant context of the intact embryo. Moreover, Helobdella is evolutionarily distant from the standard models; and understanding how BMP signaling functions in homologous processes across diverse species may reveal the range of mechanisms with which BMP signaling may operate across diverse tissues and stages in individual species (e.g., human). Studies of leech development are also of importance to the field of evolutionary development (Evo-Devo) because leeches belong to a diverse, yet under-studied super-phylum of animals known as Lophotrochozoa. The specific aims for the proposed project period are to explore the spatial regulation of BMP signaling across heterologous cell types, using the Helobdella ectoderm system. The main hypothesis to be tested is that the observed differences in spatial extent of BMP5-8 signaling between two different cell types in the embryo reflects differences in the processing of BMP pro-peptides by Furin or other members of the pro- peptide convertase subtilisin/kexin (PCSK) family of proteases, which normally process BMP pro-peptides to their mature forms. To test this hypothesis, constructs expressing signaling competent, epitope-tagged variants of wild-type and cleavage site-mutant leech BMP5-8 will be microinjected to drive its expression in specific lineages of the embryo, either alone or with various leech PCSKs. Processing of the epitope-tagged BMPs will be followed by western blot analysis, and their signaling effects will be assessed using an established set of molecular and morphological criteria. In other experiments, the spatial distribution of functional BMP5-8 will be assessed directly by visualizing signaling competent, Fluorescent Protein-labeled BMP5-8 in living embryos and/or the epitope-tagged BMP5-8 variants in fixed specimens. Finally, other factors that may affect the spatial regulation of BMP5-8 signaling will be evaluated, including possible interactions with extracellular matrix via a conserved cluster of N terminal basic amino acids, internalization BMP ligand-receptor complexes, and differences between the activity and mobility of BMP heterodimers versus homodimers. The relevance of this research to public health is that it will broaden our understanding of how TGF- beta signaling processes might be regulated during human development or disease where experimentation is not possible, and in complex vertebrate model systems that are not amenable to the type of detailed analysis that is possible in the leech embryo. In addition, this research provides a forgiving and relatively inexpensive training environment in which future physicians and medical scientists can hone experimental techniques and rigorous analytical intellectual approaches for subsequent application to more clinically relevant problems.