Novel regulators of BMP signaling in Drosophila

果蝇 BMP 信号传导的新型调节因子

• 批准号: 9284221
• 负责人: FRANCESCA PIGNONI
• 金额: $ 2.12万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9284221
• 关键词: Adult; Affect; Animals; BMP2 gene; Binding; Biochemical; Biological Models; Biological Process; Cell Culture Techniques; Cell Maintenance; Cell Proliferation; Cell model; Cells; Cellular biology; Cessation of life; Colorectal Cancer; Communication; Data; Development; Developmental Biology; Disease; Drosophila genus; Family; Female; Formulation; Functional disorder; Genes; Genetic; Genetic Techniques; Health; Homeostasis; Human; Human Pathology; Impairment; In Vitro; Insecta; Integral Membrane Protein; Laboratories; Ligands; Malignant Neoplasms; Mammals; Mediating; Modeling; Molecular; Molecular Genetics; Mus; Mutation; Names; Organ; Organism; Orthologous Gene; Ovarian; Ovary; Pathway interactions; Pattern; Physiological; Play; Predisposition; Process; Property; Protein Family; Protein Isoforms; Proteins; Reagent; Regulation; Role; Signal Pathway; Signal Transduction; Skeletal system; Staging; Stem Cell Research; Stem cells; System; Techniques; Testing; Tissues; Transducers; Vascular System; Vertebrates; Work; base; bone morphogenetic protein receptors; fatty acid-binding proteins; fly; gain of function; gastrointestinal system; genetic analysis; genetic approach; germline stem cells; human disease; in vivo; in vivo Model; insight; member; novel; pleiotropism; promoter; receptor; research study; response; self-renewal; stem cell niche

 DESCRIPTION (provided by applicant): The BMP pathway is universally important in multicellular organisms and is known to regulate proliferation, patterning, cell fate and other fundamental processes in model systems. Its components are evolutionarily conserved, and pathway dysfunction leads to human diseases of the skeletal, vascular, and gastrointestinal systems as well as predisposition to colorectal cancer. Work in Drosophila has contributed greatly to our understanding of the molecular mechanisms for signal transduction and its regulation, particularly in the study of the pathway in vivo. We have discovered that Lilipod, an uncharacterized but evolutionarily conserved transmembrane protein of the Lipocalin-receptor family, plays a role in BMP signaling in different contexts and we provide extensive evidence of its contribution to germline stem cell (GSC) self-renewal in the ovarian niche. We have also identified fly Fabp, a protein of the fatty-acid-binding family, as a potential ligand and modulato of Lilipod. Vertebrates have two Lilipod orthologs and at least three Fabp orthologs. Given a high degree of aa sequence conservation (similar to other pathway components), Lilipod/Fabp's role in BMP signaling is likely to be conserved. The multiple Lilipod and Fabp orthologs are broadly expressed in vertebrates and may function sometime redundantly, as well as show pleiotropy given the many roles of BMP; thus, complicating studies in vivo. Drosophila is an ideal model to study Lilipod and Fabp function because it has i) only one lilipod and fabp gene, ii) powerful molecular genetic techniques for loss/gain-of-function analysis in vivo, and iii) a wealth of BMP-pathway (Dpp) reagents for both in vivo and insect cell culture models. We show that lilipod is expressed in GSCs; it is necessary and sufficient for their self-renewal; and it regulates BMP signaling in these cells. For this proposal, we will elucidate the molecular mechanism of Lilipod action by first using an in vitro system (S2 cells) and then testing emerging models in vivo (in the ovarian stem cell niche). In addition, we will also dissect the function of its putative ligand Fabp in vivo and in vitro. Based on our preliminary data, Lilipod's input into the signaling cascade occurs between the receptor Tkv and the activated R-SMAD pMad. Preliminary experiments suggest a direct interaction between Lilipod and Tkv in vivo and a requirement for lilipod in the transduction of the BMP signal in S2 cells. A detailed biochemical analysis of the effect of loss/gain of lilipod in S2 cells will assess the stability, binding and/o activation properties of various BMP signaling components in the presence and absence of Lilipod (Aim 1). The emerging molecular mechanism will then be tested in vivo (Aim 2). In Aim 3, we will investigate the function of Fabp in the germline niche and its role as a putative Lilipod ligand. Preliminary experiments suggest that fabp is required and sufficient in the germline niche to promote the stem cell state; an Fabp isoform directly binds to Lilipod; and a reduction in Fabp level (using a deficiency) impairs Lilipod's function. We will dissect fabp function in vivo (germline niche) and in vitro (in S2 cells) using advanced genetic approaches and biochemical techniques (Aim 3).

 描述（由申请人提供）：BMP 途径在多细胞生物体中普遍重要，并且已知可调节模型系统中的增殖、模式化、细胞命运和其他基本过程。其成分在进化上是保守的，通路功能障碍会导致人类骨骼、血管和胃肠系统疾病以及结直肠癌的易感性。果蝇方面的工作极大地促进了我们对信号转导及其调节的分子机制的理解，特别是在体内途径的研究中。我们发现 Lilipod 是脂质运载蛋白受体家族中一种未表征但进化上保守的跨膜蛋白，在不同背景下的 BMP 信号传导中发挥作用，并且我们提供了其对卵巢微环境中生殖系干细胞 (GSC) 自我更新的贡献的广泛证据。我们还鉴定了果蝇 Fabp（脂肪酸结合家族的一种蛋白质）作为 Lilipod 的潜在配体和调节剂。脊椎动物有两个 Lilipod 直向同源物和至少三个 Fabp 直向同源物。鉴于 aa 序列高度保守（类似于其他途径成分），Lilipod/Fabp 在 BMP 信号传导中的作用可能是保守的。多个 Lilipod 和 Fabp 直向同源物在脊椎动物中广泛表达，有时可能会冗余地发挥作用，并且考虑到 BMP 的多种作用，也显示出多效性；因此，体内研究变得复杂。果蝇是研究 Lilipod 和 Fabp 功能的理想模型，因为它具有 i) 只有一个 lilipod 和 fabp 基因，ii) 用于体内功能丧失/获得分析的强大分子遗传学技术，以及 iii) 丰富的用于体内和昆虫细胞培养模型的 BMP 途径 (Dpp) 试剂。我们发现 Lilipod 在 GSC 中表达；对于他们的自我更新来说这是必要且充分的；它调节这些细胞中的 BMP 信号传导。对于本提案，我们将首先使用体外系统（S2 细胞），然后测试体内新兴模型（在卵巢干细胞生态位中），从而阐明 Lilipod 作用的分子机制。此外，我们还将剖析其假定配体Fabp在体内和体外的功能。根据我们的初步数据，Lilipod 的 信号级联的输入发生在受体 Tkv 和激活的 R-SMAD pMad 之间。初步实验表明 Lilipod 和 Tkv 在体内存在直接相互作用，并且在 S2 细胞中 BMP 信号转导中需要 Lilipod。详细的生化 对 S2 细胞中 Lilipod 损失/增加的影响的分析将评估在 Lilipod 存在和不存在的情况下各种 BMP 信号成分的稳定性、结合和/或激活特性（目标 1）。随后将在体内测试新兴的分子机制（目标 2）。在目标 3 中，我们将研究 Fabp 在种系生态位中的功能及其作为假定的 Lilipod 配体的作用。初步实验表明，在种系生态位中，fabp 是必需的并且足以促进干细胞状态； Fabp 同工型直接与 Lilipod 结合； Fabp 水平的降低（利用缺陷）会损害 Lilipod 的功能。我们将使用先进的遗传方法和生化技术（目标 3）在体内（种系生态位）和体外（S2 细胞中）剖析 fabp 功能。