選殖以及定性分析FAK在斑馬魚胚發育過程中之角色

Abstract

Focal adhesion kinase (FAK) 為Tyrosine kinase family 之一員，調控細胞的生長和移動。為了了解FAK在胚胎發育中所扮演的角色，我們使用斑馬魚系統作為基因功能研究的動物模式生物。我們先將斑馬魚的FAK基因zfak1a (3159 bp)及zfak1b (3138 bp) 選殖出來，並送入哺乳類細胞（NIH3T3、FAK-/-）當中表現，觀察是否在cell biology、biochemistry、以及function上面也有相似性。我們藉由Morpholino (MO) knock down 內生的 zfak1a 或zfak1b 並探討FAK在斑馬魚發育中的角色。藉由氨基酸序列比對得知斑馬魚的FAK和雞、老鼠、及人類的序列相似度高達85%以上，顯示其是相當保守的一個蛋白。Fak1a/1b 表現在細胞的focal contacts上，並且Fak1a的表現會促進細胞生長、移動、以及能動性的上升。使用MO降低斑馬魚內生Fak1a的表現造成魚體原腸形成過程中的缺陷，藉由間歇性拍攝觀察得知這樣的缺陷是由於細胞移動的能力和方向性失調所造成的。除此之外，降低Fak1a表現的斑馬魚胚體尾部的eve1基因的表現量下降，顯示Fak1a可能參與在決定前後體軸發育的角色當中。斑馬魚 faks參與在原腸形成過程中調控細胞移動，正確的細胞移動被認為是中胚層正常形成的動力。

Focal adhesion kinase (FAK) is a protein tyrosine kinase involved in cell migration, proliferation and cell adhesion. To investigate the role of FAK in embryonic development, we employed a well-established vertebrate model, zebrafish, to proceed genetic study of FAK in vivo. Here we isolated two zebrafish FAK genes, fak1a (3159 bp) and fak1b (3138) bp and expressed in FAK-/- and NIH3T3 mammalian cells to study the outcomes of cell biology, biochemistry and cellular functional level in comparison to those of mouse FAK. Zebrafish fak1a and fak1b showed more than 85% similarity in amino acid sequences to their homologs in chicken, mice and humans. The expression of fak1a could promote cell proliferation, migration, and motility. In addition, we used antisense morpholino oligos (MO) knocking down intrinsic fak1a and fak1b in zebrafish embryos to investigate the role of Fak1a/1b during development. Upon knocking down intrinsic fak1a and fak1b in zebrafish embryos, epiboly and convergence extension defects were prominent. By a means of time-lapse recording assay, we demonstrated that zebrafish fak governed convergent extension movements via regulating cell migration ability and polarization. Also, the expression of eve1 is down regulated upon knocking down fak1a, suggesting that Fak1a may play a role in anterior-posterior (A-P) axial patterning. Together, zebrafish Faks involved in regulating cell migration both in vitro and in vivo, it is been regarded as the driving force during gastrulation cell movement which contributes to normal mesoderm formation.