IN VIVO CONDITIONAL SIGNALING IN AVIAN DEVELOPMENT

鸟类胚胎是探索脊椎动物发育的组织相互作用和分子机制的强大实验系统。组织移植方法与逆转录病毒介导的异位基因表达或直接重组蛋白应用的结合在过去几年中取得了巨大的进展。然而，这些实验方法受到无法调节引入的基因或蛋白质的活性的限制。在这项提案中描述的实验中，劳弗实验室将改装两个条件信号系统，用于AVEN系统。LBD(激素受体配体结合域)系统利用激素受体的突变配体结合域可逆地失活结构性激活的蛋白：LBD融合。雌激素和孕激素受体LBDS都将被使用。它们分别受4-羟基-三苯氧胺和RU486的调节。FK506结合蛋白(FKBP)系统使用与伙伴蛋白融合的模块化FKBP结合域和二价合成配体(AP20187)来调节蛋白质活性。将使用每个系统开发试剂，并在体外研究它们的性质。将构建表达核GFP：LBD融合的禽类逆转录病毒，并将配体依赖的细胞质到核的易位用作激活的监测。表达截短的成纤维细胞生长因子受体：FKBP融合蛋白的逆转录病毒也将被构建；激活是由配体依赖的二聚引起的。细胞培养中的激活将通过受体磷酸化和DNA合成的诱导来评估。对于这些试剂中的每一种，将确定激活动力学和剂量响应。然后将建立每个融合蛋白-配体对的体内调控参数。鸡胚胎将感染重组逆转录病毒，在允许表达目的蛋白后，将用适当的药物治疗胚胎。NGFP：LBD的激活将通过亚细胞定位来确定。FGFR活性将通过使用非放射性原位杂交组织化学和诱导组织生长的依赖于成纤维细胞生长因子的靶基因表达来评估。不同的药物剂量和应用方案(通过浅血管注射、静脉注射或腔内注射)将决定靶向激活的最有效方法，以及动力学和剂量反应。这些系统共同提供了在禽类发育过程中以空间、时间和数量方式调节蛋白质功能的手段。

The avian embryo is a powerful experimental system for exploring the tissue interactions and molecular mechanisms underlying vertebrate development. The combination of tissue transplantation methodologies and retrovirus-mediated ectopic gene expression or direct recombinant protein application have allowed enormous advances in the past few years. These experimental approaches are limited, however, by an inability to regulate the activity of the introduced gene or protein. In the experiments described in this proposal, the Laufer lab will adapt two conditional signaling systems for use in the avian system. The LBD (hormone receptor ligand binding domain) system uses mutant ligand binding domains of hormone receptors to reversibly inactivate constitutively activate protein: LBD fusions. Both the estrogen and progesterone receptor LBDs will be used. These are regulated by 4- hydroxy-tamoxifen and RU486, respectively. The FK506 binding protein (FKBP) system uses modular FKBP binding domains fused to partner proteins, and a bivalent synthetic ligand (AP20187) to regulate protein activity. Reagents will be developed using each system, and their properties investigated in vitro. Avian retroviruses expressing nuclear GFP:LBD fusions will be constructed, and ligand-dependent cytoplasmic to nuclear translocation used as a monitor of activation. Retroviruses that express a truncated FGF receptor: FKBP fusion protein will also be constructed; activation is caused by ligand-dependent dimerization. Activation in cell culture will be assessed through receptor phosphorylation and induction of DNA synthesis. For each of these reagents, activation kinetics and dose responses will be determined. The in vivo regulatory parameters of each fusion protein-ligand pair will then be established. Chick embryos will be infected with the recombinant retroviruses, and after allowing for expression of the target protein, embryos will be treated with the appropriate drug. nGFP:LBD activation will be determined via subcellular localization. FGFR activity will be assessed by FGF-dependent target gene expression using non-radioactive in situ hybridization histochemistry and induction of tissue growth. Various drug doses and application regimens (via superficial vasculature, intravenous or intralumenal injection) will determine the most efficacious means of target activation, as well as kinetics and dose responses. Together these systems will provide the means to regulate protein function in a spatial, temporal and quantitative fashion during avian development.