探討斑馬魚Nogo-B訊號傳遞對於斑馬魚肝臟發育所扮演的角色

Abstract

Nogo-b也就是大家所熟知的「漿膜蛋白-4b」，在血管受傷後的保護機制中扮演著相當重要的角色。在哺乳類中，Nogo-b會特別表現在肝臟的膽管和非實質細胞裡。肝臟的纖維化和肝內膽管癌(ICC屬於較罕見原發性的肝腫瘤，具有極高度的轉移能力)都會造成Nogo-b表現量的下降。血液中Nogo-b的含量，可以作為肝硬化的有效指標。同時，Nogo-b也能抑制肝臟星狀細胞(HSC)的活性。對於肝細胞，Nogo-b會促進肝細胞的增殖以及肝臟的再生。這些資料讓我們想知道Nogo-b/Nogo受體訊號途徑是否參與斑馬魚肝臟的生長。目前，Nogo-b在肝臟發育中其功能也還未被釐清。 在本篇研究當中，我們收集已培養24、48和72小時的斑馬魚胚胎，透過反義寡核苷酸(MO)抑制和原位雜交技術來分析Nogo-b/Nogo受體對於肝細胞形成和分化的重要性。藉由pHH3免疫染色的結果可以看到抑制Nogo-b的表現能夠阻礙肝細胞增殖。此外，收集注射過Nogo-b-MO的斑馬魚胚胎進行原位雜交分析與對照組比較，結果可以看到Foxa3、Prox1a和Lfabp的表現量有所下降。另一方面，Nogo-b在斑馬魚體內的訊號傳遞，和哺乳類相似，包括三個配位體和四個受體，透過實驗我們確定降低Nogo-b以及其受體的NgR和兩個輔助受體p75和TROY的表現也能夠使斑馬魚的肝臟縮小。因此，我們推測Nogo-B 、NgR、p75和TROY可能形成複合體去影響斑馬魚肝臟的發育。

Nogo-B, also known as Reticulon 4B, plays important roles in vascular injuries. In mammals, Nogo-B is expressed specifically in cholangiocytes and non-parenchymal cells in the liver. Nogo-B expression is down-regulated with the progression of liver fibrosis and in intrahepatic cholangiocarcinoma (ICC), a relatively rare type of primary liver cancer with highly malignant behavior. Circulating Nogo-B may be an effective indicator for liver cirrhosis. Nogo-B inhibition could diminish hepatic stellate cell (HSC) activation. Nogo-B expression in hepatocytes facilitates hepatocyte proliferation and liver regeneration. These data prompt us to consider whether Nogo-B/Nogo receptor signaling participates in the development of the zebrafish liver. So far, its function in the liver development is not understood. In this study, we characterized the roles of Nogo-B/Nogo receptor in hepatic formation and differentiation by morpholino oligonucleotide (MO) knockdown and whole-mount in situ hybridization with several markers in zebrafish embryos from 24 to 72 hpf. Knockdown of Nogo-B inhibited the proliferation of liver by pHH3 immunostaining. In addition, Nogo-b morphants showed lower expression of foxa3, prox1a and lfabp compared to control MO injected embryos by in situ hybridization. On the other hand, Nogo signaling in zebrafish, like that in mammals, involves three ligands and four receptors. We identified that knockdown of Nogo-B and its receptor-NgR and two coreceptors, p75 and TROY, all decreased liver size during early development. Thus, we hypothesized that Nogo-B/NgR, p75, and TROY will form a complex to be involved in zebrafish liver formation.