MOLECULAR GENETICS OF DROSOPHILA GASTRULATION

原肠胚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生物体中。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。对这一过程所需要的分子的追求将使我们更好地理解不仅是非常重要的发育程序，而且在神经管形成和癌细胞转移等其他事件中对细胞运动的控制。由于复杂的分子遗传技术的可用性，本应用程序建议使用果蝇（一种常见的果蝇）来研究原肠形成。在果蝇中，蜗牛基因的突变导致腹侧细胞内陷缺陷和中胚层衍生物（如肌肉、心脏、脂肪体和淋巴腺）的丧失。蜗牛蛋白作为一种转录抑制因子，在腹侧推定的中胚层中阻止外侧神经外胚层基因的表达。这使得中胚层细胞的命运得以建立。然而，最近的研究结果表明，神经外胚层基因的抑制虽然会破坏中胚层细胞的命运，但不会干扰内陷。早期的模式如何导致假定的中胚层内陷的问题仍然没有答案。这项提议计划测试蜗牛可以调节另一组腹侧基因的假设，这些基因是原肠形成所必需的。这一假设与蜗牛也参与中枢神经系统和翅盘发育的研究结果相印证。因此，蜗牛可能在多个发育过程中调控多组靶基因。本提案的具体目标包括：1)利用转基因蝇解剖蜗牛蛋白，组织培养，并通过突变等位基因测序来确定原肠胚形成所需的结构域；2)分析腹侧基因，确定可能与蜗牛直接相互作用的启动子元件，并通过酵母dna结合单杂交筛选分离新的靶基因；3)利用第二位点增强子筛选鉴定蝇原肠形成下游基因。

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.