Study on the mitotic machinery using zebrafish embryogenesis

Abstract

Cell division culminates with mitosis in which duplicated chromosomes are equally segregated to two daughter cells to maintain genomic integrity. To finely regulate the mitotic procedures, many mitotic kinases, phosphatases, and motor proteins are involved. In this study, I aim to discover the conserved function of mitotic machineries such as Aurora-A kinase and Kif18a motor protein using zebrafish embryogenesis as a model system. Aurora-A is a serine/threonine kinase, which regulates many intricate processes during mitosis in a phosphorylation-dependent manner. Despite the emerging interest for clinical applications, our understanding on Aurora-A function at the organism level is still limited. Here, I have discovered the function of Aurora-A in zebrafish embryo. Using morpholino (MO) technology, I showed that Aurora-A morphant zebrafishes were unhealthy displaying cell death and growth retardation with short and bended trunks. The developmental defects might be attributable to the abnormal mitotic progression, manifested by monopolar and/or disorganized spindle formation and mitotic arrest, accompanying spindle assembly checkpoint activation. Cell death in the absence of Aurora-A was partially rescued by p53 MO co-injection. Taken together, I elucidate that zebrafish Aurora-A is essential for mitotic progression and its depletion induces p53-dependent cell death, which can provide insight into the molecular mechanism of anti-cancer drug targeted for Aurora-A. Chromosome movements in mitosis are directed by microtubules which are regulated by motor proteins. Kif18a is a kinesin-8 family motor protein, which has been identified as a suppressor of chromosome movements. It is known to regulate microtubule dynamics such as pre-anaphase chromosome oscillations and chromosome congression through two regulation modules: N-terminal catalytic motor domain and C-terminal tail domain. Tail domain in kinesin-8 is known to have controversies whether it has microtubule depolymerase activity or not. Despite of the important role of Kif18a in in vitro and cell-line data, there are still limited knowledge on the physiological role of Kif18a. Therefore, I attempted to dissect the role of two different Kif18a knockout zebrafishes: the C-terminus disrupted one using ZFN (zinc finger nuclease) technique and the motor targeted one using CRISPR/Cas9 technique. As Kif18a C-terminal domain is not required for the viability, fertility, and mitosis, I am now generating motor domain targeted Kif18a mutant zebrafish using a CRISPR/Cas9 knockout method. I suggest that this study, using zebrafish, provides the knowledge on the evolutionary conserved role and regulation mechanism of mitotic machineries.