FUNCTION OF ENDOTHELIN FAMILY IN NEURAL CREST DEVELOPMENT TEM

内皮素家族在神经嵴发育中的作用

• 批准号: 2576552
• 负责人: W PAVAN
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2576552
• 关键词: cell differentiation; developmental genetics; endothelin; gene complementation; gene expression; gene mutation; genetic strain; hormone receptor; hormone regulation /control mechanism; in situ hybridization; laboratory mouse; melanocyte; neural plate /tube; polymerase chain reaction; tissue /cell culture

The mouse mutant lethal spotting (ls) encodes the vasoactive peptide endothelin 3 (EDN3) and the piebald (s) locus encodes its receptor, endothelin receptor B (EDNRB). Mice homozygous for mutations in either of these genes exhibit aganglionic megacolon and a white spotted coat due to the lack of neural-crest derived enteric ganglia and melanocytes in affected portions of the colon and skin. In order to understand the role of these genes in melanocyte development, we are using in situ hybridization to determine the time that these genes are expressed relative to other neural crest markers in mutant and normal embryos. A 1.3 kb cDNA clone of EDN3 was isolated from a mouse embryonic day 10.5 cDNA library and a 950 bp cDNA clone of EDNRB was isolated by RT pcr using gene specific pcr primers. These genes have been used to determine expression patterns in adult and embryonic tissues. We have shown that both genes are expressed very early during mouse embryonic development in neural crest derived cells and expression patterns are altered in various neural crest mutants. In order to determine the function of EDN 3, we are determining how exogenously added EDN3 alters the timing, number, survival and morphology of melanocytes in an in vitro system that we have developed. We have found that EDN3 can successfully replace TPA in this system and results in a 50 fold increase in melanocytes. We have found that of the related family members (EDN1 and EDN2) only EDN1 can substitute for EDN3 in this assay. We have also shown that the effect of EDN3 is dependent upon the presence of its receptor EDNRB on neural crest cells by using chemical inhibitors and piebald mice as donors for the neural crest cultures.

小鼠突变致死斑点（LS）编码血管活性肽 内皮素3（EDN3）和Piebald（S）基因座编码其受体， 内皮素受体B（EDNRB）。两种突变的纯合小鼠 这些基因显示出Aganglionic Megacolon和一件白色斑点外套 由于缺乏神经沟衍生的肠神经节和黑素细胞 影响结肠和皮肤的部分。为了了解角色 在黑素细胞发育中的这些基因中，我们正在原位使用 杂交以确定这些基因表达的时间 相对于突变体和正常胚胎中的其他神经rest标记。一个 从小鼠胚胎第10.5天分离出EDN3的1.3 kb cDNA克隆 通过RT PCR分离EDNRB的cDNA库和950 bp的cDNA克隆 使用基因特异性PCR引物。这些基因已用于确定 成人和胚胎组织中的表达模式。我们已经表明 这两个基因在小鼠胚胎发育过程中都非常早就表达 在神经rest衍生的细胞中，表达模式在 各种神经rest突变体。为了确定EDN的功能 3，我们确定外源添加的EDN3如何改变时间， 黑色素细胞的数量，生存和形态，在体外系统中 我们已经发展了。我们发现EDN3可以成功替换TPA 在该系统中，可导致黑素细胞增加50倍。我们有 发现相关家庭成员（EDN1和EDN2）只有EDN1可以 在此测定中代替EDN3。我们还表明了 EDN3取决于其受体EDNRB在神经波峰上的存在 通过使用化学抑制剂和Piebald小鼠作为供体 神经rest文化。