探討Wnt5b和Wntless對於調控斑馬魚下顎軟骨發育之分子機制

Abstract

Wnt蛋白由其所製造的細胞中，分泌並結合至目標細胞後的訊息傳導，在體軸生成、神經發育與器官生成中扮演著非常重要的角色。而Wntless (Wls)則是在少數被發現的膜蛋白中，被研究出具有調控Wnt分泌進而活化Wnt訊號傳遞的膜蛋白。儘管抑制Wnt訊號傳遞會造成各種斑馬魚早期胚胎發育的缺陷，但即便是抑制了來自母體中Wls的表現，也無法引起失去各種Wnt訊號所產生的集合性的缺陷。因此，我認為Wls在斑馬魚早期胚胎的發育中，對於Wnt的調控是具有選擇性的。在已知的研究中，Wnts和Fgfs被證實具有調控早期發育的功能。但對於Wnt或Fgf在局部的組織中的作用、或是其兩者之間是否有相互的調控關係，並沒有太多相關且詳細的研究。因此，在我一開始的研究中，首先發現在Wls的突變種 (mutants)、morphants和Wnt5b的突變種的斑馬魚中，Fgf3的表現量是有明顯的下降的，但在wnt9a和wnt11的morphants中則是沒有改變的。除此之外，利用顯微注射的方式將全長的fgf3 mRNA打入斑馬魚胚胎中，則可以解救Wls和Wnt5b突變所引起的下顎軟骨缺陷，但fgf8則否。另外在免疫組織螢光染色的結果中，我也發現Wnt5b的分泌在受精後的48 小時是受到Wls所調控的，但Wnt11則否。這實驗結果指出在斑馬魚早期胚胎發育中，Wls並非參與了所有Wnt的分泌。最後，在我的實驗中證實Wls是透過調控細胞增生的方式來影響下顎軟骨的發育，而不是透過調控細胞凋亡的途徑。因此，我得到的結論是:斑馬魚下顎軟骨的發育，是Wnt5b經過Wls的調控而分泌出其製造細胞，進而增加Fgf3的表現來調控細胞增生，以達到下顎軟骨正常發育之目的。

Secreted Wnt molecules play a pivotal role in cell-cell communications during the development of multicellular organisms. The transmembrane protein Wntless (Wls) is one of the few proteins that are identified to control the secretion of active Wnt signaling. Although loss of Wnt signaling resulted in diverse developmental defects during zebrafish embryonic development, loss of maternal or zygotic Wls activity in embryos caused no obvious collective loss-of-Wnt phenotypes, suggesting that Wls exists selectivity on different Wnt molecules during zebrafish embryonic development. To date, Wnts and Fgfs have been reported to regulate various tissues development, but relatively little is known about how regional Wnt or Fgf activities are established and how they interact in any given developmental event. Here, I have investigated the Wnt-mediated craniofacial cartilage development in zebrafish and found that fgf3 expression is down-regulated in wls mutants and wls morpholino (MO)-injected embryos (morphants) and in wnt5b mutants, but no fgf3 expression alternation is observed in wnt9a and wnt11 morphants. In addition, introducing full-length fgf3 mRNAs but not fgf8 can rescue the Wls or Wnt5b deficiency-induced jaw cartilage defects. Immuno-histochemical staining also revealed that endogenous Wnt5b, but not Wnt11, required Wls for its secretion during the jaw development at 48 hpf in zebrafish embryos, indicating that Wls is not involved in every Wnt secretion events in zebrafish cells. Moreover, cell proliferation was affected while apoptosis was normal in the developing jaw of wls morphants. Together, Wnt5b requires Wls for secretion and regulates the proliferation of chondrogenic cells through fine-tuning the expression of fgf3 during embryonic jaw cartilage development.