斑馬魚 ADP-ribosylation-like factor-6 interacting protein 在 neural crest 發育之角色

Abstract

ADP-ribosylation-like factor-6 interacting protein (Arl6ip) 是Arl6 的interacting protein。Arl6屬於small ADP ribosylation fator GTP-binding proteins，在intracellular traffic 中為主要的 regulators。Fan et al. (2004) 證實當 Arl6 缺失時會產生人類遺傳疾病 Bardet-Biedl syndrome type 3 (BBS3) 。BBS3是一種 multisystemic disorder會同時發生 obesity, blindness, polydactyly, renal abnormalities 和 cognitive impairment 的症狀。此外當 neural crest migration被抑制時，也會造成 BBS 中的craniofacial dysmorphology 和腸道神經缺失的 Hirschsprung’s disease (Tobin et al., 2008)，代表與BBS相關的基因必定和 neural crest 的發育有著密切的關係。雖然 已知 Arl6ip 在 in vitro 的功能可能和造血細胞maturation中蛋白質運送、membrane trafficking、細胞訊息傳遞有關，但是在 in vivo 胚胎發育的功能目前並沒有相關研究指出，因此我們想知道當 Arl6ip 缺失時是否同樣會造成 BBS，並且 Arl6ip 是否參與了 neural crest 的發育。本篇研究中我們在斑馬魚胚胎利用專一性的antisense morpholino oligonucleotides (MO) 抑制 Arl6ip轉譯，neural crest衍生物如軟骨, cranial ganglia, peripheral neurons 和心臟的發育出現嚴重缺失，和 BBS 症狀非常相似。Whole mount in situ hybridization 結果中可以看到早期 neural plate border specifiers 如 msxb, dlx3b 和 pax3 表現並沒有產生異常；但 neural crest specification genes 如 foxd3, snail1b和 sox10 則嚴重減少，顯示出 Arl6ip 為 neural crest 特化所必要的因子，但並不影響 neural crest induction。Arl6ip 缺失胚胎中表現在遷移中 cranial 和 trunk neural crest cells 的crestin 和 sox10 缺失，代表 Arl6ip 影響了 neural crest 的遷移。最後利用 TUNEL assay 實驗，pre-migratory的 neural crest 發生了不正常的 apoptosis 現象，表示Arl6ip 影響了pre-migratory neural crest 的 survival。此外在 Arl6ip 缺失胚胎中，可以看到心臟不正常的 looping 和心跳緩慢的表型，而心臟發育異常的情形可能是由 cardiac pre-migratory neural crest cells 缺失所造成。綜合以上結果， Al6ip影響了 neural crest 的特化, migration及survival，同時也為 neural crest derivatives 分化所必須。本篇也是首次來討論arl6ip 在neural crest 發育時所扮演的功能。

ADP-ribosylation-like factor-6 interacting protein (Arl6ip) is an interacting protein of Arl6 which is one of the small ADP ribosylation factor GTP-binding proteins that are major regulators in intracellular traffic. Fan et al. (2004) identified Arl6 as the gene underlying Bardet-Biedl syndrome type 3 (BBS3), a multisystemic disorder characterized by obesity, blindness, polydactyly, renal abnormalities and cognitive impairment. Inhibition of neural crest migration underlies craniofacial dysmorphology and Hirschsprung’s disease in BBS (Tobin et al., 2008). It means that BBS associated genes must involve in neural crest development. Although the in vitro function of Arl6ip gene is suggested as protein transport, membrane trafficking, or cell signaling during hematopoietic maturation, the in vivo roles that alr6ip plays in BBS and neural crest development are totally unknown. Here, we demonstrated that when Arl6ip function was lost by injecting the zebrafish embryos an antisense morpholino oligonucleotides (MO) which inhibited Arl6ip mRNA translation specifically, the neural crest derivatives, such as cartilage, cranial ganglia, peripheral neurons, and heart, were defective. These defects are similar to Bardet-Biedl syndrome. The expressions of neural plate border specifiers, msxb, dlx3b, and pax3 were normal, but the expression of neural crest specification genes, foxd3, snail1b, and sox10 were reduced, implicating Arl6ip is essential for neural crest specification, but not for neural crest induction. Furthermore, crestin and sox10, which were expressed in the cranial and trunk migrating neural crest cells, were also decreased, suggesting that Arl6ip is required for neural crest migration. In addition, apoptosis was apparent occurrence in the pre-migratory neural crest cells, indicating a critical role for arl6ip in the survival of neural crest cells. Interestingly, we noticed that the hearts of the Arl6ip-MO-injected embryos were failure to undergo normal looping and the function of heart was depressed. This defective heart may result from the loss of cardiac pre-migratory neural crest cells. Taken together, we conclude that Arl6ip is not only required for neural crest specification, survival, and migration, but also for neural crest derivatives differentiation. This is the first report that demonstrates the in vivo function of Arl6ip during neural crest development.