Novel regulators of BMP signaling in Drosophila

果蝇 BMP 信号传导的新型调节因子

• 批准号: 9507224
• 负责人: FRANCESCA PIGNONI
• 金额: $ 3.47万
• 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/9507224
• 关键词: Adult; Affect; Animals; BMP2 gene; Binding; Biochemical; Biological Models; Biological Process; Cell Culture Techniques; Cell Maintenance; Cell model; Cells; Cellular biology; Cessation of life; Colorectal Cancer; Communication; Data; Development; Developmental Biology; 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; Stem Cell Research; Stem cells; System; Techniques; Testing; Tissues; Transducers; Vascular System; Vertebrates; Work; base; bone morphogenetic protein receptors; experimental study; 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; public health relevance; receptor; response; self-renewal; skeletal disorder; stem cell division; 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，并且已知在模型系统中调节增殖、模式化、细胞命运和其他基本过程。其组分在进化上是保守的，并且通路功能障碍导致骨骼、血管和胃肠道系统的人类疾病以及结肠直肠癌的易感性。在果蝇中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。我们已经发现，Lilipod，一种未表征但在进化上保守的脂质运载蛋白受体家族跨膜蛋白，在不同的背景下在BMP信号传导中发挥作用，我们提供了大量的证据证明其对生殖系干细胞（GSC）在卵巢生态位中自我更新的贡献。我们还确定了苍蝇Fabp，脂肪酸结合家族的蛋白质，作为一个潜在的配体和调节百合。脊椎动物有两个Lilipod直向同源物和至少三个Fabp直向同源物。考虑到高度的氨基酸序列保守性（类似于其他途径组分），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，并且有时可能冗余地起作用，以及鉴于BMP的许多作用而显示多效性;因此，使体内研究复杂化。果蝇是研究百合足和Fabp功能的理想模型，因为它具有i）仅一个百合足和fabp基因，ii）用于体内功能丧失/获得分析的强大分子遗传技术，以及iii）用于体内和昆虫细胞培养模型的丰富BMP途径（Dpp）试剂。我们表明，百合表达在GSC;这是必要的和足够的自我更新;它调节BMP信号在这些细胞。对于这个提议，我们将首先使用体外系统（S2细胞），然后在体内（在卵巢干细胞生态位中）测试新兴模型，从而阐明Lilipod作用的分子机制。此外，我们还将在体内和体外对其推定的配体Fabp的功能进行剖析。根据我们的初步数据，莉莉波德 进入信号级联的输入发生在受体Tcl 3和活化的R-SMAD pMad之间。初步的实验表明，百合和Tibet在体内的直接相互作用，并在S2细胞中的BMP信号转导的百合的要求。详细的生化分析 在S2细胞中分析失去/获得百合的影响将评估在存在和不存在百合的情况下各种BMP信号传导组分的稳定性、结合和/或活化性质（目的1）。然后将在体内测试新出现的分子机制（目标2）。在目标3中，我们将研究Fabp在生殖系生态位中的功能及其作为推定的Lilipod配体的作用。初步实验表明，fabp是生殖系生态位所必需的，足以促进干细胞状态;一种Fabp同种型直接与Lilipod结合;而Fabp水平的降低（使用缺陷）会损害Lilipod的功能。我们将使用先进的遗传学方法和生物化学技术（目的3）在体内（生殖系生态位）和体外（S2细胞）解剖fabp功能。