Endothelin 3 對雞隻胚胎黑色素母細胞分佈之影響

Abstract

色素過度沈澱（hyperpigmentation）為絲羽烏骨雞（Silky）外貌重要特徵之一，原因是由於成年烏骨雞之腸繫膜、腹膜等結締組織有大量黑色素細胞（melanocytes）存在。黑色素細胞源自於具分化多能性（pluripotent）之神經嵴細胞（neural crest cells），於雞胚胎發育第15-16期，神經嵴細胞會沿著神經管（neural tube）與骨節（sclerotome）之間空隙的腹側路徑（ventral pathway）遷徙到達腹側體節，並分化為神經元與神經膠原細胞（neurons and glial cells）；於第21-22期時，神經嵴細胞先分化成黑色素母細胞（melanoblasts）後，再沿著皮肌節（dermamyotome）與外胚層（ectoderm）之間的背側路徑（dorsolateral pathway）遷徙，隨後侵入表皮再分化成黑色素細胞。而烏骨雞胚胎於發育過程中，黑色素母細胞的遷移會同時進入背側與腹側路徑，故導致其色素過度沈澱。由於研究指出黑色素母細胞的遷徙與增殖，可受到Endothelin 3（EDN3）調控，因此，本論文之研究目的為探討於神經嵴細胞遷徙時期，EDN3與Endothelin receptors B2（EDNRB2）於烏骨雞以及L2品系土雞之表現與分佈狀況，並瞭解EDN3於色素過度沉澱現象所扮演之角色。結果顯示，烏骨雞胚胎EDN3之表現，於神經嵴細胞遷徙時期均顯著高於同一時期L2品系土雞胚胎之表現量，且烏骨雞胚胎之EDNRB2於開始進行背側遷徙之第22期與第28期亦均高於L2胚胎之表現。EDN3於L2胚胎表現位置幾乎都集中於背側路徑；反之，於烏骨雞胚胎中，EDN3表現位置遍佈於背側路徑與腹側路徑周邊之組織。將小鼠EDN3-RCAS反轉錄病毒注射入L2品系土雞胚胎體節內，可讓小鼠EDN3於雞胚中表現，且黑色素母細胞可侵入受感染之雞胚體節內部，但孵化至第14天時，並未出現類似烏骨雞色素過度沉澱之現象。利用蛋白質體學之分析方法，比對絲羽烏骨雞與L2品系胚胎發育到第22期以及第28期胚胎所表現之蛋白質。結果顯示在兩品種間表現量具顯著差異之蛋白質於第22期胚胎有30個，而第28期有59個蛋白質，其中於第22期可鑑定出身分之蛋白質有12個，而第28期則有24個。於烏骨雞胚胎內表現量高的蛋白質功能包括了氧化還原反應、細胞增殖、細胞老化、粒線體內蛋白質輸送以及大分子胜肽聚合等。本研究確認EDN3為造成絲羽烏骨雞之黑色素母細胞出現腹側遷移之關鍵因子，但後續分化成黑色素細胞以及維持黑色素細胞生長，可能還需要其他因素輔助以形成色素過度沉澱，而在神經嵴細胞遷徙時期，於絲羽烏骨雞與L2胚胎內表現具顯著差異之蛋白質則可提供進一步研究之方向。

Hyperpigmentation is one of the obvious characteristics in Silky fowl. It is caused by large numbers of melanocytes existing in connective tissues, such as the mesenteries and the peritoneumin adult Silky. Melanocytes are derived from a group of pluripotent cells called the neural crest cells (NCCs). In chicken embryos at the stages 15-16, the NCCs migrate along the ventral pathway between the neural tube and sclerotome and enter the ventral somties, and then differentiate into neurons and glial cells. At the stages 21-22, the NCCs, commencing to differentiate into the melanoblasts, migrate along the dorsolateral pathway between dermamyotome and ectoderm, and then invade into epidermis and differentiate into the melanocytes. Whereas in Silky, the melanoblasts migrate not only along the dorsolateral pathway but also theventral pathway at the stages 21-22, which could result in hyperpigmentation.It has been reported that Endothelin 3 (EDN3) could regulate the migration and survival of melanoblasts expressingEDN3receptor,Endothelin receptors type B2 (EDNRB2). Therefore, the objectives of this research were to investigate the expression profiles of EDN3 and EDNRB2 at the NCC-migration waves in Silky and L2 chicken embryos, and to examine the role of EDN3 in the phenomenon of hyperpigmentation. The RT-PCR resultsdemonstrated that the expression of EDN3 was significantly higher in the Silky embryos at the three stages examined. In addition, the Silky embryos at the stages 22 and 28 expressed significantly higher EDNRB2 than L2 embryos did. EDN3 was expressed at the dorsolateral pathway in L2 embryos at the stage 28, while in Silky,EDN3 was expressed around the somites located in both dorsolateral and ventral pathways. The mouse EDN3-RCAS retroviruswas constructed and injected into the somites of L2 embryo.Expression of mouse EDN3 was detected in the injected L2 embryos and it was found that the melanoblasts had migrated into the somites of infected L2 embryos. However, no hyperpigmentation was observed after incubation of the infected embryos to day 14. The differentially expressed proteins between Silky and L2 embryos at the stages 22 and 28 were analyzed by proteomic approach. The results showed that 30 and 59 protein spots were significantly different between Silky and L2 embryos at the stages 22 and 28, respectively, and from which, 12 and 24 proteins wereidentified individually.Functions of the identified proteins in Silky include redox regulations, cell proliferation, cellular ageing, mitochondrial protein import and macromolecular assembly. In conclusions, EDN3 plays a crucial role in directing the ventral pathway of the melanoblasts in Silky embryo. However, except for EDN3, other factors might also be required to support the differentiation of melanoblasts and maintenance of survival and proliferation of melanocytes. Additionally, the identified proteins significantly differentially expressed between Silky and L2 embryos at NCCs migration stages might participate the phenomena of hyperpigmentation, which is worth further studying.