Fak1a與Wnt5b經由Rac1與Cdc42聯合調控斑馬魚胚早期腔腸化時之細胞遷徙過程

Abstract

黏著斑激酶(focal adhesion kinase，FAK)在許多重要的細胞生理過程上扮演著重要的角色，其中包括了胚胎發育及器官發育等過程。儘管我們已知FAK在胚胎發育過程中的重要性，然究竟FAK如何調控早期胚胎發育，又如何在此一時期與其他訊息分子溝通的研究還是很有限。在本論文中，藉由比較fak1a與1b與人類FAK的氨基酸序列, 我發現兩者有高度的相似性。利用mopholino oligonucleotides (MO)抑制蛋白質生成，我觀察到魚胚(morphants)的外包作用(epiboly), 胚體趨中與延展作用(convergent extension)以及下胚層(hypoblast)細胞的移動都受到明顯的影響。藉由細胞的移植實驗, 我發現fak1a所調控的細胞移動受外界環境所影響，肌凝蛋白(filamentous actin，F-actin)在細胞外包作用時所產生的環狀構造(actin ring)在fak1a缺乏的胚胎是不完整的,進而推測其可能為導致胚外包作用缺失的原因。我同時也利用了CRISPR/Cas9的基因修改技術製作出了fak1a的突變魚(mutant)，但僅有少部分胚有腔腸化缺陷的表型。然而在mutants與mophants我發現相同的基因有上升之趨勢，此顯示相似的細胞傳導路徑受到了影響，更重要的是我發現Wnt5b可能位於fak1a的上游調控下游路徑以調節細胞移動的過程。藉由mRNA拯救的實驗我發現其下游因子是small GTPase，Rac1與CDC42。總結,我首次在斑馬魚胚腔腸化過程中發現了FAK與WNT5b共同藉由調控Rac1與CDC42影響了肌凝蛋白之動態平衡以控制細胞移動現象。

Focal adhesion kinase is known to mediate multiple vital cellular processes and be involved in embryogenesis and organ development. Despite its necessity, how FAK regulates and integrates with other cellular signals during early embryogenesis still remains poorly understood. Here, I first demonstrated the high sequence similarity of zebrafish fak1a and fak1b to human FAK. Using antisense morpholino (MO), I observed that the loss of Fak1a impaired epiboly, convergence and extension and hypoblast cell migration in a non cell-autonomous manner. Furthermore, I showed clear disturbance of the filamentous actin (F-actin) linkage bundles between actin-ring and yolk syncytial nuclei that appeared to affect epiboly in fak1a morphants. Genetic deletion of fak1a using CRISPR/Cas9 mediated gene editing reveals minor gastrulation defects than that of morphants, but some genes were induced both in morphants and mutants. It suggests that similar molecular pathways were affected. More importantly, I found that overexpression of fak1a or wnt5b mRNA could cross rescue convergence defects induced by wnt5b or fak1a MO, respectively. Both Wnt5b and Fak1a appeared to mediate gastrulation via Rac1 and Cdc42, since both small GTPases could synergistically rescue wnt5b and fak1a morphant phenotypes. Taken together, I demonstrate for the first time the missing functional interaction between Wnt and FAK signaling to mediate gastrulation cell movements via precise regulation of Rac1 and Cdc42 activities and subsequent actin dynamics.