Wntless蛋白之羧基端功能在斑馬魚胚胎發育扮演角色之探討

Abstract

Wnt ligand是一個在多細胞生物中被廣泛使用之蛋白質訊息分子。Wnt所誘發的訊息傳遞路徑被廣泛使用在眾多生物現象之中，包含胚胎早期發育、幹細胞多型性維持和細胞癌化。對於Wnt訊息所引發之反應已有廣泛的研究，但關於Wnt如何被製造和如何自細胞釋放等的詳細資訊仍有所不足。Wntless蛋白被報導負責將Wnt由高基氏體運送至細胞膜的攜帶蛋白質，Wntless存在與否對於多數Wnt之分泌皆有重大影響。透過研究Wntless，我們即可從側面探索Wnt如何在細胞中被輸送和釋放。儘管數個Wntless的功能性區段已被報導，但Wntless羧基端區段在斑馬魚中所扮演之功能仍然尚未釐清。在本實驗室先前研究中推測Wntless的羧基端150個胺基酸殘基對於其功能扮演重要角色。而在本研究中，我進一步針對Wntless最末端49個胺基酸區塊設計切除版本，並且將受部分切除的mRNAs以顯微注射的方式使其表現在Wntless突變的斑馬魚胚胎中。實驗組中，表現末端缺乏的Wntless和單獨只有最末端兩種版本皆無法拯救wls突變之斑馬魚胚胎，據此我推測Wntless羧基端為其生物功能必須之區段。此外，我使用特定辨識羧基端之抗體來進行免疫螢光染色，發現單獨只剩羧基端的切除版本和切除中間第221至420胺基酸區段的Wntless之細胞內分布與全長的Wntless不同。據此我推測這兩個切除版本的切除區段皆為Wntless在細胞之中被正確運送所必須。

Wnt is a secreted ligand and involved in numerous cell-cell signaling processes, especially in embryo development and tissue homeostasis. Comparing to the understanding of the responding signaling in Wnt-receiving cells, the sorting and secretion of Wnt in Wnt-producing cell is less clear. Wntless (Wls, also known as Gpr177 in mammal) plays a crucial role in carrying Wnts from Golgi to plasma membrane. Although several important functional motifs of Wls have been identified, whether the carboxyl-terminal domain of Wls is functional in zebrafish embryonic development is unknown. I have evaluated the abilities of different truncated Wls proteins in rescuing the inner ear and jaw cartilage defects of wls mutants. I found that neither the carboxyl-terminal 49 amino acids deleted form nor the form containing only the carboxyl-terminal 49 a.a rescue the defects of wls mutants. These data indicate that the carboxyl terminal 49 amino acids are important for Wls function in modulating Wls-mediated development in zebrafish embryo. In addition, utilizing antibody against Wls carboxyl-terminal 49 amino acids, I found the subcellular location of the truncated Wls containing only the carboxyl-terminal 49 a.a or carrying a.a. 221-420 deletion is distinct from endogenous Wls. I suggest these deleted region are both required for Wls subcellular sorting.