斑馬魚胚Citron Kinase選殖、基因表現及功能分析

Abstract

Citron kinase (CRIK) 屬於絲胺酸/酥胺酸磷酸激酶，並為已活化Rho的下游作用蛋白之一。若於體外培養之哺乳細胞中過度表現喪失功能之CRIK突變種會造成細胞分裂的缺陷，此結果顯示CRIK會影響細胞分裂，其於細胞分裂溝及神經組織之聚集更增加其調控細胞分裂及神經發育之可能性。在果蠅發育過程CRIK雖為各類組織細胞質分裂所必需，而CRIK基因剔除老鼠胚胎早期可發育並出生，但於出生後伴隨明顯神經細胞分裂缺陷和凋亡而死亡。CRIK基因可能在不同物種間有差異或有功能相近之同源基因存在，而導致其在不同物種之功能差異，為探討CRIK在發育過程的基因表現及其功能，我們在模式動物斑馬魚中以in silica cloning和RACE方式選殖出斑馬魚CRIK基因(zcrik)，比較分析其與其他物種CRIK蛋白質序列，利用RT-PCR偵測到成魚腦、眼睛、腎臟、胰臟、卵巢及睪丸組織中皆有zcrik的表現，在原位雜合試驗(in situ hybridization)中可見在發育24小時胚中zcrik表現多在細胞具有高度增殖的區域，如神經先驅細胞等，此現象與先前發表在老鼠表現模式相類似。為了進一步探討zcrik在斑馬魚發育的功能，我們顯微注射morpholino oligonucleotides (MO)至一胞期胚來抑制zcrik mRNA之轉譯，結果顯示zcrik-MO處理過之胚在發育六小時內死亡率較對照組高，並於早期細胞分裂時期有分裂不完整與不正常分裂情形發生，此分裂嚴重受干擾的胚亦在之後細胞遷移過程產生障礙，使得胚無法繼續正常發育而死亡，而存活之胚在受精後24小時觀察其外觀上有明顯無頭和神經管消失等缺陷發生，此結果和先前研究中指出CRIK對神經細胞分裂發育有影響相呼應，再則此等胚之缺陷與過度腹部化(ventralized)的胚型態相類似，由此推測z CRIK在胚發育時期不僅具有調節細胞分裂與神經發育的功能，對於斑馬魚的原腸胚發育過程及背腹部化亦有一定程度的影響。

Citron kinase (CRIK) belongs to an evolutionarily conserved family of serine-threonine kinases and is a target molecule for activated Rho. CRIK has been suggested to be involved in regulating cytokinesis in mammalian cells due to the inhibition of cytokinesis by overexpressing dominant negative CRIK. It accumulates at the cleavage furrow and neuronal tissues that implies its roles in early development and neurogenesis. In Drosophila melanogaster, CRIK is essential for normal cell division in all tissue of Drosophila, while the knock out citron kinase allows early embryonic development, but shows defective neurogenesis in mice. To further study the role of CRIK in development, we utilized the recently developed vertebrate model, zebrafish. In this study, we first isolated the zebrafish citron kinase (zcrik) by in silica cloning and RACE experiments. Secondly, the zcrik expression patterns were examined using in situ hybridization and RT-PCR. We found that zcrik mRNA was detected by RT-PCR in adult zebrafish tissues including brain, eye, ovary, testis, kidney, spleen and gill. Whole-mount in situ hybridization of different developmental stages of zebrafish embryos revealed that zcrik expressed in the entire embryo until 18 hour post fertilization and the expression of zcrik mRNA was limit to the proliferate neuroblasts and tissues that is consistent with the expression pattern in mice. Lastly, to evaluate the functions of CRIK in zebrafish development, we applied etopic expression analysis and morpholino oligonucleotides (MO) knockdown to the developing zebrafish embryos. Functional analysis results demonstrated that gain or loss of function of zCRIK resulted defects in cytokinesis, epiboly formation and embryonic death. In addition, the MO-knockdown resulted in severe defects in CNS with little or no head structure formation, which are the characteristics of ventralized embryos at 24 hpf. These results suggest that, in addition to cytokinesis and neuronal development, citron kinase is also essential for gastrulation progressing and may be involved in dorsal-ventral patterning during development.