DEVELOPMENTAL STUDIES OF SEGMENTATION GENE FUNCTION

该提案解决了发育生物学的一个核心问题： 胚胎不同区域的细胞是如何分配给 不同的发展路径。 在果蝇胚胎中， 发育途径是由 分节基因的局部表达。 几项体内 提出了研究分割基因 表达是本地化的，使用空间模型系统， 通过配对规则基因调节所述的错切基因。 (i)上位性 研究将用于确定调节途径， 有刺的 用于确定调节蛋白的RNA和蛋白质 的路径。 RNA和蛋白质表达 将在其调节剂的双突变体中进行测量，使用原位 杂交和免疫标记。 (ii)表达域 和它的监管互动。 (iii)的影响 异位表达对规则基因的表达， 将进行测量，以确定观察到的变化是否 符合监管的组合模型。 的基因 将置于热激促进剂的控制下， 使用种系转化在胚胎中表达。 (iv)一 对规则调节器的功能解剖 在胚胎中使用体内测定进行。 的嵌合体 不同的配对规则基因将被测试它们的能力， 当它们被置于 控制热激启动子或以下之一的启动子 父母的基因。 通过检查嵌合体是否 表现得像父母的基因之一，它应该是可能的， 确定配对规则基因的哪些部分是需要的， 具体的监管制度。 这些实验将提供 一个在体内测试的功能重要性的进化- 保守的基序，如同源框和配对框。 (v)一个 抑制剂测定，其中短寿命的分段调节剂 基因通过阻断一般蛋白质合成而耗尽， 用于描述多对规则的空间调节， 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.