Request for a Diversity Supplement to GM074619 in Support of Post-Baccalaureate Research Experience and Career Development for Mr. Quentin D. Watson

请求对 GM074619 进行多元化补充，以支持 Quentin D. Watson 先生的学士后研究经验和职业发展

• 批准号: 8845453
• 负责人: DAVID A WEISBLAT
• 金额: $ 5.28万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8845453
• 关键词: Affect; Animals; BMP2 gene; BMP4; BMP5 gene; Basic Amino Acids; Basic Science; Binding; Biological Assay; Biological Models; Bone Morphogenetic Proteins; Caliber; Cell Lineage; Cells; Complement; Complex; Defect; Development; Developmental Process; Disease; Distant; Drosophila genus; Ectoderm; Ectoderm Cell; Embryo; Environment; Epithelium; Epitopes; Extracellular Matrix; Failure; Family; Future; Genetic Markers; Geometry; Goals; Grant; Hair follicle structure; Heterodimerization; Human; Human Development; Individual; Insecta; Label; Leeches; Life; Ligands; Logic; Malignant Neoplasms; Mature Bone; Medical; Membrane; Mesenchyme; Microinjections; Modeling; Molecular; Monitor; N-terminal; Organ; Organogenesis; Pattern; Peptide Hydrolases; Peptides; Physicians; Play; Positioning Attribute; Process; Production; Proteins; Proteolytic Processing; Public Health; Regulation; Request for Proposals; Research; Resistance; Role; Scientist; Signal Transduction; Signaling Molecule; Site; Spatial Distribution; Specimen; Staging; Stem cells; Stereotyping; Subtilisins; Sum; Surveys; System; TGF Beta Signaling Pathway; Techniques; Testing; Textbooks; Tissues; Tooth structure; Training; Transforming Growth Factor beta; Variant; Western Blotting; Work; base; career development; cell type; chordin; clinically relevant; experience; human disease; kexin; member; morphogens; mutant; novel; public health relevance; receptor; research study; signal processing; stem; tumor

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.

描述（由申请人提供）：本项目的目标是研究高度保守的TGF-β信号通路如何发挥作用，使用水蛭胚胎作为方便的模型。TGF-β信号转导的BMP亚家族在整个脊椎动物发育过程中是活跃的，从背腹图案形成和器官发生到牙齿和毛囊的诱导。许多发育缺陷、癌症和其它疾病涉及BMP信号传导的失调，因此理解BMP信号传导的调节是至关重要的。 许多研究集中在分泌的BMP和它们的拮抗剂相互作用以形成形态发生梯度的机制上，所述形态发生梯度将位置信息传递给大的细胞场，例如在脊椎动物和昆虫胚胎的背腹图案化中。但其他重要的BMP信号传导过程涉及短程相互作用，例如癌症中的肿瘤-基质相互作用，或牙齿发育中的上皮-间充质相互作用。 水蛭（Helobdella）胚胎提供了一个互补的模型，脊椎动物和果蝇系统最常用的BMP信号的研究，因为它具有相对简单的细胞几何形状和刻板的细胞谱系，其命运部分由短程信号相互作用。水蛭是经得起精确和可重复的实验操作，基于显微注射的标记和遗传结构到确定的，谱系限制的干细胞（成端细胞）。因此，与Helobdella，它是可能的，进行BMP信号的特殊操作和测定的结果，精确地在完整胚胎的生理相关的上下文中。此外，Helobdella在进化上与标准模型相距甚远;并且理解BMP信号传导如何在不同物种的同源过程中发挥作用可以揭示BMP信号传导可以在个体物种的不同组织和阶段中发挥作用的机制的范围（例如，人类）。水蛭发育的研究对进化发育（Evo-Devo）领域也很重要，因为水蛭属于一种多样的，但研究不足的超级动物门，称为Lophotrochozoa。 拟议项目期间的具体目标是利用Helobdella外胚层系统探索BMP信号在异源细胞类型中的空间调节。待检验的主要假设是，在胚胎中两种不同细胞类型之间观察到的BMP 5 -8信号传导的空间范围的差异反映了通过弗林蛋白酶或蛋白酶的前肽转化酶枯草杆菌蛋白酶/kexin（PCSK）家族的其他成员加工BMP前肽的差异，所述蛋白酶通常将BMP前肽加工成其成熟形式。为了检验这一假设，表达野生型和切割位点突变型水蛭BMP 5 -8的信号传导能力、表位标记变体的构建体将被显微注射以单独或与各种水蛭PCSK一起驱动其在胚胎的特定谱系中的表达。处理表位标记的BMP后进行蛋白质印迹分析，并使用一组既定的分子和形态学标准评估其信号传导作用。在其他实验中，通过可视化活胚胎中有信号传导能力的荧光蛋白标记的BMP 5 -8和/或固定标本中表位标记的BMP 5 - 8变体，直接评估功能性BMP 5 -8的空间分布。最后，将评估可能影响BMP 5 -8信号传导的空间调节的其他因素，包括通过N末端碱性氨基酸的保守簇与细胞外基质的可能相互作用、内化BMP配体-受体复合物以及BMP异二聚体与同二聚体的活性和流动性之间的差异。 这项研究与公共卫生的相关性在于，它将拓宽我们对TGF-β信号传导过程在人类发育或疾病过程中如何调节的理解，在这些过程中，实验是不可能的，并且在复杂的脊椎动物模型系统中，这些系统不适合水蛭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磨练实验技术和严格的分析智力方法，随后应用于更临床相关的问题。