Novel regulators of BMP signaling in Drosophila

 描述(申请人提供)：BMP途径在多细胞生物体中是普遍重要的，已知在模型系统中调节增殖、构型、细胞命运和其他基本过程。它的成分在进化上是保守的，通路功能障碍会导致人类骨骼、血管和胃肠道系统的疾病，以及结直肠癌的易感性。在果蝇中的工作有助于我们理解信号转导的分子机制及其调控，特别是在体内信号转导途径的研究中。我们发现Lilipod是Lipocalin受体家族中一种尚未鉴定但在进化上保守的跨膜蛋白，在不同的环境中参与BMP信号转导，我们提供了广泛的证据表明Lilipod在卵巢巢中对生殖系干细胞(GSC)的自我更新起到了作用。我们还鉴定了Fly Fabp，一个脂肪酸结合家族的蛋白质，作为Lilipod的一个潜在的配体和调节剂。脊椎动物有两个Lilipod同源基因和至少三个Fabp同源基因。由于氨基酸序列高度保守(类似于其他途径组件)，Lilipod/Fabp在BMP信号转导中的作用可能是保守的。多个Lilipod和Fabp同源基因在脊椎动物中广泛表达，有时可能具有冗余功能，并且由于BMP的多种作用而显示出多效性；因此，体内的研究变得复杂。果蝇是研究Lilipod和Fabp功能的理想模型，因为它只有一个Lilipod和Fabp基因，ii)强大的分子遗传学技术，用于体内功能丧失和功能获得的分析，以及III)丰富的BMP途径(DPP)试剂，既可用于体内模型，也可用于昆虫细胞培养模型。我们发现Lilipod在GSCs中表达；它是GSCs自我更新的必要条件和充分条件；它调节这些细胞中的BMP信号。对于这一提议，我们将首先使用体外系统(S2细胞)，然后在体内测试新出现的模型(在卵巢干细胞利基)，以阐明Lilipod作用的分子机制。此外，我们还将剖析其可能的配体Fabp在体内和体外的功能。根据我们的初步数据，Lilipod 进入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，在S2细胞BMP信号的转导中需要Lilipod。一份详细的生化报告 分析S2细胞中Lilipod的丢失/获得的影响将评估在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)。新出现的分子机制随后将在体内进行测试(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以及它作为Lilipod配体的作用。初步实验表明，在胚系利基中，Fabp是促进干细胞状态所需的且充足的；Fabp亚型直接与Lilipod结合；而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学方法和生化技术(目标3)来剖析FABP在体内(生殖系生态位)和体外(在S2细胞中)的功能。

 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).