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)编码血管活性多肽 内皮素3(EDN3)和花椒(S)基因座编码其受体， 内皮素受体B(EDNRB)。任何一种基因突变的纯合子小鼠 这些基因中有无神经节细胞的巨结肠和白色斑点被毛 神经脊源性肠神经节和黑素细胞的缺乏 受影响的部分结肠和皮肤。为了理解这一角色 在黑素细胞发育的这些基因中，我们使用了原位 杂交以确定这些基因表达的时间 相对于突变胚胎和正常胚胎中的其他神经脊标记。一个 从小鼠胚胎第10.5天分离到1.3kb的EDN3基因克隆 利用逆转录聚合酶链式反应(RT-PCR)技术获得了EDNRB基因的cDNA文库和950bp的cDNA克隆 使用基因特异的聚合酶链式反应。这些基因已经被用来确定 成体和胚胎组织中的表达模式。我们已经证明了 这两个基因在小鼠胚胎发育的早期就有表达。 在神经沟衍生的细胞中，表达模式在 各种神经脊突变体。为了确定EDN的功能 3，我们正在确定外源添加的EDN3如何改变时间， 黑素细胞在体外系统中的数量、存活和形态 我们已经开发了。我们发现EDN3可以成功地取代TPA 在这个系统中，并导致黑素细胞增加50倍。我们有 发现在相关的家族成员(EDN1和EDN2)中，只有EDN1可以 可替代本试验中的EDN3。我们还表明， Edn3依赖于其受体ednrb在神经脊上的存在。 细胞通过使用化学抑制剂和花椒鼠作为供体 神经脊培养。