MOLECULAR GENETICS OF DROSOPHILA GASTRULATION

原肠胚形成是胚胎发育过程中最早的形态发生过程，发生在所有生物体中，包括人类，经历胚胎发育。 这个过程涉及高度协调的细胞运动，并导致三个胚层的形成。 对这一过程所需的分子的追求将导致更好地理解不仅是非常重要的发育程序，而且还控制其他事件中的细胞运动，如神经管形成和癌细胞转移。 本申请提出使用果蝇（Drosophilamelanogaster）（常见的果蝇）来研究原肠胚形成，因为可利用复杂的分子遗传技术。 在果蝇中，蜗牛基因的突变会导致腹侧细胞内陷缺陷以及肌肉、心脏、脂肪体和淋巴腺等中胚层衍生物的丧失。 Snail蛋白作为转录抑制因子阻止腹侧假定中胚层中外侧神经外胚层基因的表达。 这允许建立中胚层细胞命运。然而，最近的研究结果表明，神经外胚层基因的去抑制，虽然破坏中胚层细胞的命运，不干扰内陷。 早期的模式化是如何导致假定的中胚层内陷的，这个问题仍然没有答案。这项提案计划测试蜗牛可以调节原肠胚形成所需的另一组腹侧基因的假设。 这一假设与蜗牛也参与中枢神经系统和翅盘发育的发现相印证。因此，Snail可能在多个发育过程中调节多组靶基因。 本研究的具体目标包括：1）利用转基因果蝇、组织培养和突变体等位基因测序技术对Snail蛋白进行解剖，以确定原肠胚形成所需的结构域; 2）分析腹侧基因，以确定可能与Snail直接相互作用的启动子元件，并通过酵母中DNA结合的单杂交筛选分离新的靶基因; 3）利用第二位点增强子基因筛选技术对果蝇原肠形成下游基因进行鉴定。

Gastrulation is the earliest morphogenetic process during embryogenesis and takes place in all organisms, including humans, that go through embryonic development. The process involves highly coordinated cell movements and results in the formation of the three germ layers. The pursuit of molecules required for this process will lead to a better understanding of not only the very important developmental program, but also the control of cell movements in other events such as neural tube formation and cancer cell metastasis. This application proposes to use Drosophila melanogaster, the common fruit fly, to study gastrulation because of the availability of sophisticated molecular genetic techniques. In Drosophila, mutations in the snail gene cause defects in ventral cell invagination and the loss of mesodermal derivatives such as muscles, heart, fat bodies, and lymph gland. Snail protein functions as a transcriptional repressor to prevent the expression of lateral neuroectodermal genes in the ventral presumptive mesoderm. This allows the establishment of mesodermal cell fate. However, recent results reveal that the derepression of neuroectodermal genes, although disrupts mesodermal cell fate, does not interfere with invagination. The question of how the early patterning leads to the invagination of the presumptive mesoderm remains unanswered. This proposal plans to test the hypothesis that Snail can regulate another set of ventral genes that are required for gastrulation. Such postulation is corroborated with the findings that Snail also participates in CNS and wing disc development. Therefore, Snail may regulate multiple sets of target genes in multiple developmental processes. The specific aims of this proposal include: 1) dissection of the Snail protein using transgenic flies, tissue culture, and by sequencing mutant alleles to identify the domain required for gastrulation; 2) analysis of ventral genes to define the promoter elements that may interact directly with Snail, and isolation of novel target genes through DNA-binding one-hybrid screen in yeast; 3) the identification of downstream gastrulation genes using second site enhancer genetic screens in flies.