The Function Of Bockl In The Development Of The Zebrafish Hindbrain Vasculature

Bockl is a novel501aa type1transmembrane aspartic protease related to the pepsin and retroviral aspartic protease families. Based upon previous studies postulate that Bacel can modulates myelination in the central and peripheral nervous system,it’s also paly a key role in the development of nervous system.as yet,there are no many studies on bockl in zebrafish, the section of bockl expression in zebrafish has different on mouse, which led to temporarily unable to predict the possible existence of substrates and metabolic pathways involved.Formation of the vertebrate vasculature is a complex process, which is on the basis of the preexisting vascular sprouting, migration to formate the new blood vessels. Therefore angiogenesis closely related to this process with many vascular-related disease, it’s also plays a key role in the embryos of development. The cranial vasculature is essential for the survival and development of the central nervous system and is important in stroke and other brain pathologies.Cranial vessels form in a reproducible and evolutionarily conserved manner. The cardiovascular system of zebrafish bears85%homology to that of human on the level of genes and the early development of that is very similar with human. So the researches can provide theoretic bases for clinical therapeutics, which are to study the development of vascular and the function of important factors in the process using zebrafish.In recent years, angiogenesis of the molecular mechanisms make many great progress. Blood vessels and nerve fibres course throughout the body in an orderly pattern, often alongside one another. Although superficially distinct, the mechanisms involved in wiring neural and vascular networks seem to share some deep similarities. Recent genetic insights show that They use similar signals and principles to differentiate, grow and navigate towards their targets. Moreover, the vascular and nervous systems cross-talk. in the research of chicken and zebrafish, more and more similar signals involved in the development of vessels and nerves system.Here, we firstly cloned bockl gene of zebrafish, showed bockl expression patterns during the different embryogenesis stages by whole mount in situ hybridization.the expression of bockl was detected in the head.we further investigated it in transgenic line:Tg(flk1:GFP×gatal:DsRed), in which flkl:GFP marks endothelial cells and gatal:DsRed marks blood cells. Then we knocked down the translation of endogenous bockl by injecting antisense morpholino oligonucleotide in the1-2cell stage. Then morphology analysis of vessel by confocal projection reveals that down-regulation of bockl indeed has defects in development of the zebrafish hindbrain vasculature at36hpf, especially in the formation of BA and CtAs.but bockl mRNA rescued the phenotypes of bockl morphants. and overexpression of bockl resulted in display excessive branch points leading to unconnected endothelial tips compared with those in control embryos. The development of orderly pattern was disorganized.Blood vessels and nerves use similar signals and principles to differentiate, grow and navigate towards their targets. Moreover, the vascular and nervous systems cross-talk.bockl geng regulate myelination in the central and peripheral nervous system. And vascular endothelial growth factor (VEGF) is a nerve-derived arteriotropic guidance signal for small arteries; its expression in, and secretion from, Schwann cells attracts and induces arteries,but not veins, to track alongside the nerve fibres.VEGF also play a key role in the endothelial cell migration and vascular sprouting. We observed that the expression of VEGF markers(vegfa,vegfc,flt4,dII4) and Schwann cells markers(gfap) were in low level after knocking down the endogenous bockl by in situ hybridization.but in the embryos was co-inject bockl MO and vegfa mRNA, We observed that vegfa mRNA partly rescued the phenotypes of bockl morphants. It showed that bockl has effect on the development of the zebrafish hindbrain vasculature, especially in the formation of BA and CtAs. Bockl has interaction with VEGF signaling. maybe has indirect effect on blood vessel through Schwann cells. Subsequently, In order to study the mechanism about interaction of bockl and vegfa each other. we further identify what is the tissue of bockl expression. So we use transgenic line:Tg(flkl:GFP x gatal:DsRed), in which flkl:GFP marks endothelial cells,gata1:DsRed marks blood corpuscle. To Collect the head of zebrafish embryos at30hpf, Sorting cells with green and red fluorescence by FACS, identify whether bockl expression in the blood vessel or blood corpuscle by RT-PCR.but it has no target band..This result identify there is no bacel expression in the blood vessel and blood corpuscle. Further identify what is the tissue of bockl expression, we need do more wok to get the answer. Our study will provide a new approaches for monitoring, prevention and therapy of stroke, cerebral hemorrhage and other brain pathologies.