Expression Pattern Of Complement-related Genes In Normal And LPS-challenged Zebrafish Embryo/Larva

Complement is a central component of innate immunity. Eggs of most fish are released and fertilized externally, and the resulting embryos/larvae are therefore exposed to a hostile aquatic environment full of potential pathogens. Compared with mammals, the innate immune system, including complement system, is of primary importance in combating infections in fish. The reason is basically the intrinsic inefficiency of the acquired immune response of fish due to its evolutionary status and poikilothermic nature. In addition to that, the acquired immune system of fish embryo/larva is immature compared to the instant innate immune response.Recent years, the studies of zebrafish (Danio rerio) complement system have drawn worldwide attention. Genes of many components involved in the activation and regulation of complement pathways have been successfully cloned. However, many of these complement genes have not yet been studied at the expression level, especially in the embryo/larva stages. Using WISH and qRT-PCR, this study were aimed to examine the expression patterns of several complement components(C3, C4, C9, Bfl, Bf2, BO, RCA2.1 and RCA2.2) and their responses to LPS-induced challenges, unwinding their roles in early development and maturation of complement system in zebrafish.This is the first report showing the expression pattern of complement related genes in zebrafish embryo/larva, especially the expression pattern of two C2/Bf isoform(Bf2 and Bf3) and two complement regulatory genes(RCA2.1 and RCA2.2). The result of WISH demonstrated that both complement and complement regulatory genes displayed maternally localized ubiquitously during early developmental stages(cleavage stage to segmentation stage), and then they exhibited tissue-specific expression patterns in later developmental stages.These genes are mainly expressed in the liver and digestive tract, which are important immune organs in fish. Adult zebrafish also show the same expression patterns. Therefore, we can draw the conclusion that these expression patterns have been built since larva stage (72-96hours post fertilization).C3、C9、Bf1、Bf2. Bf3 genes are all up-regulated in embryos/larvae after LPS injection.while RCA2.1 gene is down-regulated according to qRT-RCR. These data suggest that zebrafish embryos are capable of activating and regulating complement system to protect themselves by pathogenic challenge. The Bf/C2 genes of zebrafish are unique in that their primary structure shows the features intermediate of mammalian Bf and C2, making their identification as Bf or C2 difficult. However, the phylogenetic analysis reveals that zebrafish Bf2 and Bf3 form a clade together with mammalian Bf, whereas zebrafish Bfl clusters together with mammalian C2. When the embryos/larvaes are challenged with LPS, it was found that the contents of Bf2 and Bf3 transcript representing the AP and C3 transcript important in all the activation pathways are both markedly increased soon after segmentation stage(15hpf), while the expression levels of Bfl similer to mammalian C2 is up-regulated by LPS challenge after 48hpf; C4 transcripts representing the CP and LP are not elevated, or even decreased in some period. Thus it is likely that the AP may be more significant than the CP and LP in complement activation during embryonic development of zebrafish.Three Bf/C2 isoforms and two RCA group 2 genes are believed to be generated by gene duplication. However, these genes displayed different expression patterns to other isoforms after LPS injection, suggesting that functional divergence has already occurred.In conclusion, this is the first report showing the expression patterns of complement related genes in zebrafish embryo/larva and its responses to LPS-induced challenge. Zebrafish embryos are capable of activating and regulating complement system to protect themselves by pathogenic challenge and CP is the main pathway of complement activation in the early development of zebrafish embryo/larva, which provides new information for the research of complement in fish.