FUNCTION OF ENDOTHELIN FAMILY IN NEURAL CREST DEVELOPMENT TEM

小鼠突变致死斑点（ls）编码血管活性肽 内皮素3（EDN 3）和花斑基因座编码其受体， 内皮素受体B（EDNRB）。突变纯合子小鼠， 这些基因表现出无神经节巨结肠和白色斑点的外套， 缺乏神经嵴来源的肠神经节和黑素细胞， 结肠和皮肤的受影响部分。为了理解 这些基因在黑素细胞发育中的作用， 杂交以确定这些基因表达的时间 相对于突变和正常胚胎中的其他神经嵴标记物。一 1.3从10.5天的小鼠胚胎中分离EDN 3的kb cDNA克隆 用RT PCR方法扩增了EDNRB的cDNA文库，并克隆了一个950 bp的cDNA克隆。 使用基因特异性PCR引物。这些基因被用来决定 在成体和胚胎组织中的表达模式。我们已经证明 这两种基因在小鼠胚胎发育的早期就表达， 在神经嵴衍生的细胞中， 各种神经嵴突变体为了确定EDN的功能， 3，我们正在确定外源性添加的EDN3如何改变时间， 体外系统中黑素细胞的数量、存活和形态， 我们已经开发了。我们已经发现EDN3可以成功地取代TPA 并导致黑素细胞增加50倍。我们有 发现在相关家族成员（EDN 1和EDN 2）中，只有EDN 1可以 在该测定中取代EDN3。我们还表明， EDN3依赖于神经嵴上其受体EDNRB的存在 通过使用化学抑制剂和花斑小鼠作为供体， 神经嵴培养

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.