SERINE-THREONINE KINASES IN EARLY EMBRYONIC PATTERNING

最近的研究已经确定了几种细胞外因子在 早期脊椎动物胚胎的模式。尽管我们已经学到了很多东西 关于细胞外信号转导的机制 培养的体细胞质膜到细胞核，鲜为人知 早期胚胎发生中的细胞内信号通路。这个 拟议的研究将检验细胞质丝氨酸-苏氨酸的作用。 蛙类早期信号转导中的特异性激酶 发展。我们的注意力将集中在两个激酶上，Raf-1和GSK3 β，它们被认为是两种细胞中的胞浆信号转导 体细胞和早期胚胎细胞的胚胎学作用和 这些激酶的调节和作用的生化机制将是 使用早期雾化胚胎作为检测系统进行了检测。这项研究将 提供了对细胞外细胞外的 负责建立脊椎动物胚胎身体计划的信号 通过其靶细胞的细胞质进行转导。 非洲爪哇GSK3beta将通过与其他GSK3beta和其 在早期胚胎中的表达模式。编码RNA的显微注射 标记GSK3β的表位将用于研究GSK3β的磷酸化和 在正常发育期间和在对特定的 信号分子。GSK3β在胚胎发育中的作用 图案化将通过GSK3β活性在两者中的错误表达来检查 整个胚胎和已定义的早期胚胎组织中。 此外，还将研究Raf-1在早期胚胎发育过程中的调控。 RAF-1的磷酸化，对生理底物的活性，以及 在正常的胚胎发育过程中，将测试与细胞蛋白的关联性， 响应于特定感应信号以及响应于操纵 Raf-1、Raf-1激酶底物上游的特定信号通路 在胚胎中，可能介导Raf-1对图案化影响的基因将被克隆 使用表达文库方法并在体内作为Raf-1底物进行测试 通过将激活的Raf-1显微注射到胚胎中。

Recent work has established the role of several extracellular factors in the patterning of early vertebrate embryos. Although much has been learned concerning the mechanism of transduction of extracellular signals from plasma membrane to nucleus in cultured somatic cells, little is known about the intracellular pathways mediating signals in early embryogenesis. The proposed research will examine the role of cytoplasmic serine-threonine specific kinases in the transduction of signals during early frog development. Attention will be focussed on two kinases, Raf-1 and GSK3 beta, which have been implicated as cytosolic signal transducers in both somatic and early embryonic cells., Both the embryological role and biochemical mechanisms of regulation and action of these kinases will be examined using the early fog embryo as an assay system. This study will provide important insights into the mechanisms by which the extracellular signals responsible for establishing the body plan of a vertebrate embryo are transduced through the cytoplasm of their target cells. Xenopus GSK3beta will be cloned by homology with other GSK3betas and its pattern of expression in the early embryo. Microinjection of RNA encoding epitope tagged GSK3beta will be used to study GSK3beta phosphorylation and activity during both normal development and in response to specific signalling molecules. The functional role of GSK3beta in embryonic patterning will be examined by mis-expression of GSK3beta activity in both whole embryos and in defined early embryonic tissues. The regulation of Raf-1 during early embryogenesis will also be examined. Raf-1 phosphorylation, activity towards a physiological substrate, and association with cellular proteins will tested during normal embryogenesis, in response to specific inductive signals, and in response to manipulation of specific signaling pathways upstream to Raf-1, Raf-1 kinase substrates in the embryo which may mediate Raf-1 effects on patterning will be cloned using an expression library method and tested as Raf-1 substrates in vivo by micro-injection into embryos with activated Raf-1.