GAP JUNCTION GENE EXPRESSION AND DEVELOPMENT

间隙连接基因表达和发育

• 批准号: 2202000
• 负责人: CECILIA W. LO
• 金额: $ 16.76万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2202000
• 关键词: binding proteins; biological signal transduction; cell cell interaction; developmental genetics; fluorescent dye /probe; gap junctions; gene expression; genetic regulatory element; genetically modified animals; immunocytochemistry; in situ hybridization; laboratory mouse; limbs; mammalian embryology; molecular cloning; neurogenesis; nucleic acid sequence; polymerase chain reaction; regulatory gene; reporter genes; retinoate; second messengers; transfection

Gap junctions are comprised of membrane channels that mediate the diffusion of ions and small molecules such as calcium, cAMP and other second messengers. Given these interesting membrane permeability properties, it has been suggested that gap that play a role in cell signaling. Consistent with gap playing a role in development is the finding that in mouse embryos, gap junctional communication (gjc) and expression of gap junction genes are modulated in a developmentally significant manner. Thus, in the limb bud and central nervous system (CNS), expression of the gap junction gene Cx43 is spatially restricted in a pattern which is coincident with or complementary to those of wnt genes-a family of growth factors that mediate cell signaling in development. Particularly striking is a band of Cx43 and wnt-expression spanning the midbrain/hindbrain junction. That wnt-1 plays an important role in mouse development is indicated by the finding of midbrain/hindbrain ectopically expressing wnt-1 in the limb. Our hypothesis is that gap junctions may mediate the transmission of second messengers derived from signal transduction cascades triggered by wnt-1, and in this manner, play an integral role in wnt-1 mediated events in mammalian development. In Aim 1, we will determine if Cx43 expression and function may be subject to regulation via wnt-1 signaling. Thus, we will examine if the absence or ectopic expression of wnt-1 may perturb gjc or Cx43 expression. In Aim 2, we will examine the developmental effects of inhibiting Cx43 function in the wnt-1 cell signaling, then the loss of Cx43 function may result in developmental defects similar to those found in wnt-1 null mutants. In Aim 3, we will use transgenic approaches to determine if the assembly and gating of Cx43 encoded gap junctions may be subjects to regulation by signal transduction pathways triggered by wnt-1. We will focus these analyses on Cx43 constructs in which mutations have been introduced into the cytoplasmic tail, a region of the protein which undergoes phosphorylation. In Aim 4, we will generate lacZ reporter constructs driven by genomic DNA from the Cx43 gene, and determine if there are regulatory elements that may respond to wnt-1 expression. In addition, using luciferase reporter constructs, we will search for retinoic acid response elements (RARE), as retinoic acid is known to affect gjc, Cx43 expression, as well as patterning in the limb and neural tube.

间隙连接由介导的膜通道组成 离子和小分子的扩散，例如钙，cAMP和其他 第二使者。 鉴于这些有趣的膜渗透性 属性，有人建议在细胞中发挥作用的差距 信号。 与差距一致的发展是 在鼠标胚胎，间隙连接通信（GJC）和 间隙连接基因的表达在发育中调制 重要的方式。 因此，在肢体和中枢神经系统中 （CNS），间隙连接基因CX43的表达在空间上受到限制 以与Wnt相吻合或互补的模式 基因 - 一个介导细胞信号传导的生长因子家族 发展。 特别引人注目的是CX43和WNT表达的乐队 跨越中脑/后脑交界处。 Wnt-1扮演重要 发现在小鼠发育中的作用是通过发现 中脑/后脑异位表达肢体中的Wnt-1。 我们的 假设是间隙连接可能介导第二个的传播 来自Wnt-1触发的信号转导级联的使者， 以这种方式，在Wnt-1中介事件中发挥不可或缺的作用 哺乳动物发育。 在AIM 1中，我们将确定CX43是否表达 功能可能会受到WNT-1信号传导的调节。 因此，我们 将检查Wnt-1的缺失或异位表达可能会扰动 GJC或CX43表达。 在AIM 2中，我们将研究发展的 在Wnt-1细胞信号中抑制CX43功能的影响，然后 CX43功能的丧失可能导致类似的发育缺陷 那些在Wnt-1 null突变体中发现。 在AIM 3中，我们将使用转基因 确定CX43编码间隙的组装和门控的方法 连接可能受信号转导途径的调节 由Wnt-1触发。 我们将把这些分析重点放在CX43构造上 哪些突变已引入细胞质尾部，一个区域 经历磷酸化的蛋白质。 在AIM 4中，我们将 生成来自CX43基因组DNA驱动的LACZ报告基因构造 基因，并确定是否存在可能响应的监管元素 Wnt-1表达。 此外，使用荧光素酶报告基因构建体，我们 将搜索视黄酸反应元件（稀有）作为视黄酸 已知会影响GJC，CX43表达以及在 肢体和神经管。