Characterization of kinesin superfamily4 (kif4) and kinesin superfamily7 (kif7) in teleost development
Student thesis: Master's Thesis
Kinesins are molecular motor proteins that transport organelles, protein complexes, mRNAs and chromosomes along microtubules through ATP hydrolysis in a highly regulated manner. Here we report the characterization of two members of kinesin-4, kinesin superfamily7 (kif7) and kinesin superfamily4 (kif4), in teleost development. Estuarine medaka (Oryzias melastigma) and zebrafish (Danio rerio) were selected as the teleost model organisms for this study. To investigate kif7 in teleost development, we cloned the estuarine medaka kif7 and characterized it in comparison with the zebrafish kif7. The cloned kif7 has an open reading frame of 3783bp and is deduced to encode a polypeptide of 1261 amino acids that shares 49–77% identity to other homologues. Both medaka and zebrafish kif7s were shown to be expressed in all embryonic stages and adult tissues examined with higher expression level in the testis and ovary. Whole-mount in situ hybridization revealed that the expression of kif7 was ubiquitous during early stage of embryonic development, but became more restrictive and localized to the brain, fin bud and eye at later development in both teleosts. However, kif7 was expressed in the atrium of the heart of zebrafish but not medaka. Thus, it was suggested that kif7 might have similar functions in the development of brain, fin and eye, but have divergent functions in the heart development in these two teleosts. As medaka and zebrafish are divergent teleosts, genetic information from these distantly related teleosts would be a useful tool for comparative studies of development and evolution in animals. In zebrafish, kif7 was reported to be the homologue of the Drosophila costal2 (cos2). Recent studies in Drosophila showed that Cos2 was involved in Hedgehog (Hh) protein signaling by controlling the activity of Cubitus interruptus (Ci) directly, and part of this regulation was through the formation of a microtubule-binding Hedgehog signaling complex (HSC) with Ci, Fused (Fu) and Suppressor of Fused (Su(Fu)). As kif7 was shown to be expressed in the brain of zebrafish and Hedgehog signaling is well-known to be important in central nervous system (CNS) development, knockdown of kif7 in zebrafish embryo through morpholino injection was performed and the brain morphology of the kif7 morphant was examined. Slight disruption of the brain structure with changes in the brain molecular markers of dl2x, ngn1 and zp-50 was observed in the zebrafish kif7 morphant. Hedgehog signaling has also been shown to be important in the testis development of mice, but no information about hh signaling in teleost testis development is available. Thus, in this study, we also examine the gene expression profile of the hh signaling members including sonic hedgehog (shh), tiggy winkle (twhh) desert hedgehog (dhh), gli1, gli2, gli2b, gli3 (the vertebrate homologues of Drosophila Ci), fused (fu), suppressor of fused (su(fu)), kif7, patched1 (ptc1) and patched2 (ptc2) in the testis of adult zebrafish. The result revealed that high levels of gli2, gli3, fused, su(fu), kif7, ptc1 and ptc2, and moderate levels of shh, dhh, gli1 and gli2b transcripts were observed in zebrafish testis. Testis of zebrafish treated with cyclopamine showed slight decrease in the expression levels of the hh target genes, gli1 and ptc1. The existence of all these hh signaling members in the zebrafish testis and the reduction in hh target genes after cyclopamine treatment suggested that the hh signaling system might be conserved in both embryo and adult testis of zebrafish. Kif4s have been shown to be a mitotic kinesin and localize predominantly to the nucleus and perform important functions in multiple steps cell division. Although many experiments have been done to examine the role of Kif4s, all of the studies in vertebrates were limited to in vitro data and no in vivo information concerning the roles of Kif4 in vertebrate is available. In this research, I characterized the zebrafish kif4 during embryonic development. I showed that zebrafish kif4 was expressed in proliferating cells and its protein products had a cell-cycle regulated dynamic localization in nucleus during mitosis. The localization of zebrafish kif4 in prophase and metaphase was similar to that of as that of HKIF4; while their cellular localization became different in telophase. This indicated that zebrafish kif4 and HKIF4 might have similar function in early stages of mitosis, but have some divergent functions in later stages. I also demonstrated that loss of kif4 in vivo by morpholino injection caused epiboly arrest and early embryonic lethality of zebrafish. Our in vivo data of zebrafish kif4 indicated that kif4 might have a function in driving epibolic movement and embryo survival. Divergent results were obtained from the two kinesins studied especially in the morpholino knockdown. Loss of kif7 only led to mild disruption of the brain structure while blocking of kif4 caused server phenotype and even embryonic lethality in zebrafish. Our study clearly demonstrated the divergent functions of kinesins in teleost development.
- Osteichthyes, Kinesin