Novel molecular mechanisms of cell migration in C. elegans

线虫的遗传分析为阐明发育细胞迁移的分子机制提供了有力的途径。在这项建议中，我们重点研究了线虫性腺形成过程中远端尖端细胞(DTC)定向迁移所需的两个新基因。 一个名为UNC-129的基因编码一种转化生长因子-β，它调节轴突引导和DTC迁移所需的UNC-6/Netrin信号的活性。UNC-129在发育中的线虫中形成高背侧-低腹侧梯度，促进腹侧向背侧DTC的迁移。初步研究表明，在低浓度的UNC-6(与UNC-129相反)下，UNC-129通过将信号从不太敏感的UNC-5转移到更敏感的UNC-5+UNC-40信号来增强UNC-6/netrin排斥信号。我们的研究旨在了解UNC-129激活的信号机制，这是新的，因为它不涉及规范的转化生长因子-β受体。我们的方法依赖于UNC-130的已知特性，UNC-130是一种转录因子，当被禁用时，它会抑制腹侧UNC-129基因的表达-扰乱其背腹侧梯度，并导致腹侧向背侧DTC迁移失败。UNC-129功能丧失不会导致DTC缺陷，但会抑制UNC-130突变型DTC缺陷。因此，通过分离抑制UNC-130 DTC迁移缺陷的突变(如UNC-129突变所做的)，我们确定了UNC-129信号所需的3个基因的突变。我们建议通过一步全基因组测序/SNP定位来识别这3个基因，然后对它们的表达和功能进行表征。由于这些基因影响UNC-129信号，将进行进一步的遗传和分子分析，以确定UNC-129信号级联的其他元件。 第二个基因是Ngat-1，编码N-乙酰半乳糖胺转移酶(Ngat-1)，根据序列预测，该酶能将N-乙酰半乳糖胺添加到糖底物上，在糖蛋白或糖脂上产生LacdiNAc修饰。Ngat-1温度敏感突变体通过DTC腹背向迁移的缺陷来鉴定，这表明LacdiNAc的加入是促进DTC在发育中的性腺迁移的信号。温度漂移实验表明，Ngat-1完成了其在胚胎发生中的功能，即在胚胎后期发育期间发生迁移--这是已知ECM分子的一种独特性质。这些发现为一种尚不清楚的胚胎机制提供了证据，这种机制在发育的较晚阶段表现出来。我们建议使用质谱学和免疫印迹来表征NGAT-1诱导的糖链修饰的性质，并确定NGAT-1活性的靶蛋白(S)或糖脂(S)。我们的长期目标是充分了解在动物发育和可能的肿瘤转移过程中指导特定细胞迁移的机制。

Genetic analysis in C. elegans provides a powerful approach for illuminating the molecular mechanisms underlying developmental cell migration. In this proposal we focus on two novel genes required for the directed migration of distal tip cells (DTCs) during the formation of the C. elegans gonad. One gene, unc-129, encodes a TGF-beta that regulates the activity of UNC-6/Netrin signaling required for axon guidance and DTC migration. UNC-129 forms a high dorsal - low ventral gradient in the developing nematode that promotes ventral to dorsal DTC migration. Preliminary studies suggest that UNC-129 enhances UNC-6/netrin repulsive signaling at low concentrations of UNC-6 (graded opposite to UNC-129) by diverting signaling from less sensitive UNC-5 alone to more sensitive UNC-5+UNC-40 signaling. Our studies are directed at understanding the signaling mechanisms activated by UNC-129, which is novel since it does not involve canonical TGF-beta receptors. Our approach relies on known properties of UNC-130, a transcription factor that when disabled de-represses ventral unc-129 gene expression - disrupting its dorsoventral gradient and causing failures of ventral to dorsal DTC migration. unc-129 loss of function does not induce DTC defects, but does suppress unc-130 mutant DTC defects. Thus, by isolating mutations that suppress the unc-130 DTC migration defect (like unc-129 mutations do), we identified mutations in 3 genes required for UNC-129 signaling. We propose to identify these 3 genes using one step whole genome sequencing/SNP mapping then characterize them for expression and function. Because these genes affect UNC-129 signaling, their further genetic and molecular analysis will be carried out to identify additional elements of the UNC-129 signaling cascade. The second gene is ngat-1, encoding an N-acetylgalactosaminyltransferase enzyme (NGAT-1) that, based on sequence, is predicted to add N-acetylgalactosamine to glycan substrates, creating a LacdiNAc modification on glycoproteins or glycolipids. The ngat-1 temperature-sensitive mutant was identified by its defect in ventral to dorsal migration of the DTCs, suggesting that addition of LacdiNAc is a signal that promotes DTC migration in the developing gonad. Temperature shift experiments show ngat-1 completes its function in embryogenesis for a migration that occurs during late post-embryonic development - a unique property of known ECM molecules. These findings provide evidence for an as yet unknown embryonic mechanism that manifests at a much later stage of development. We propose to use mass spectrometry and immunoblotting to characterize the nature of the NGAT-1-induced glycan modifications and to identify the target protein(s) or glycolipid(s) for NGAT-1 activity. Our long term goal is to fully understand the mechanisms that direct specific cell migrations during animal development and possibly tumor metastasis.