MOLECULAR GENETICS OF DROSOPHILA GASTRULATION

原肠形成是胚胎发生过程中最早的形态发生过程，发生在包括人类在内的所有经历胚胎发育的生物体中。这一过程涉及高度协调的细胞运动，并导致三个胚层的形成。对这一过程所需分子的追求将使我们不仅更好地理解非常重要的发育程序，而且还可以更好地控制其他事件中的细胞运动，如神经管形成和癌细胞转移。由于先进的分子遗传学技术的应用，本申请建议使用常见的果蝇--黑腹果蝇来研究原肠形成。在果蝇中，蜗牛基因的突变会导致腹侧细胞内陷的缺陷，以及肌肉、心脏、脂肪体和淋巴腺等中胚层衍生物的丢失。蜗牛蛋白作为转录抑制因子，在腹侧假定中胚层阻止侧向神经外胚层基因的表达。这允许建立中胚层细胞的命运。然而，最近的结果显示，神经外胚层基因的去抑制，虽然破坏了中胚层细胞的命运，但不干扰内陷。早期的图案化是如何导致假定的中胚层内陷的问题仍然没有答案。这项提议计划测试这样一个假设，即Snail可以调节另一组原肠形成所需的腹侧基因。这一假设得到了Snail也参与中枢神经系统和翼盘发育的研究结果的证实。因此，Snail可能在多个发育过程中调节多组靶基因。这一建议的具体目的包括：1)利用转基因果蝇分离蜗牛蛋白，组织培养，并通过对突变的等位基因进行测序来确定原肠形成所需的结构域；2)腹侧基因的分析以确定可能与蜗牛直接相互作用的启动子元件，并通过DNA结合的单杂交筛选在酵母中分离新的靶基因；3)利用第二位点增强子基因筛选在果蝇中鉴定下游的原肠形成基因。

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.