DEVELOPMENTAL STUDIES OF SEGMENTATION GENE FUNCTION

该提议解决了发育生物学中的一个中心问题： 胚胎不同区域的细胞如何被分配到 不同的发育路径。在果蝇胚胎中， 发展路径由组合的 片段基因的定位表达。几个活体内 提出了几种方法来研究基因如何分割 表达式是本地化的，使用空间作为模型系统 配对规则基因对累加基因的调控。(I)上位性 研究将被用来确定调控途径 积少成多。用于确定调节性的RNA和蛋白质 渐进式的道路。堆积的RNA和蛋白质的表达 将在其调节器的双重突变体中进行测量，使用原位 杂交和免疫标记。(Ii)的表达结构域 渐进式及其监管互动。(Iii) 配对规则基因的异位表达对基因表达的影响 将测量累积性以确定观察到的变化是否 与监管的组合模型一致。基因 将被置于热休克推进器的控制之下 利用胚系转化技术在胚胎中表达。(Iv)A类 累进遗嘱配对规则调节器的功能剖析 在胚胎中使用体内试验进行。嵌合体 不同的配对规则基因将被测试它们的能力 当它们被放置在 控制热休克启动子或其中之一的启动子 对父母有贡献的基因。通过检查嵌合体是否 就像父母的基因一样，应该有可能 确定配对规则基因的哪些部分是 对累进的具体规定。这些实验将提供 对进化的功能重要性的体内测试- 保守的图案，如同源盒子和配对盒子。(V)一个 抑制试验，其中短命的节段调节因子 通过阻止一般蛋白质合成来耗尽基因，将是 用来描述几个配对规则和空间规则 片段极性基因。

The propose addresses a central problem in developmental biology: how cells in different regions of the embryo are assigned to different developmental pathways. In the Drosophila embryo, developmental pathways are specified combinatorially by the localized expression of segmentation genes. Several in vivo approaches are proposed to investigate how segmentation gene expression is localized, using as a model system the spatial regulation of the engrailed gene by pair-rule genes. (i) Epistasis studies will be used to determine the regulatory pathways of engrailed. The RNA and protein used to determine the regulatory pathways of engrailed. The RNA and protein expression of engrailed will be measured in double mutants of its regulators, using in situ hybridization and immunolabeling. (ii) Expression domains of engrailed and its regulatory interactions. (iii) The effects of ectopic expression of pair-rule genes on the expression of engrailed will be measured to determine if the observed changes are consistent with a combinatorial model of regulation. The genes will be placed under the control of a heat-shock promoter and expressed in embryos using germ-line transformation. (iv) A functional dissection of the pair-rule regulators of engrailed will be performed using in vivo assay in the embryo. Chimeras of different pair-rule genes will be tested for their ability to regulate engrailed expression when they are placed under the control of either a heat-shock promoter, or the promoter of one of the contributing parental genes. By examining whether the chimera behaves like one of the parental genes, it should be possible to determine which parts of the pair-rule gene are required for the specific regulation of engrailed. These experiments will provide an in vivo test of the functional importance of evolutionarily- conserved motifs such as the homeobox and paired box. (v) An inhibitor assay, in which short-lived regulators of segmentation genes are depleted by blocking general protein synthesis, will be used to describe the spatial regulation of several pair-rule and segment-polarity genes.