Lancelet Hedgehog Signaling Pathway Important Member Of The Rbx1 Gene And Expression Of Tlr2 Protein

Amphioxus, a cephelachodate, widely considered as the closest living invertebrate relative of the vertebrates, is a key organism for understanding evolution of developmental mechanism. Its body plan is similar to but simpler than vertebrates. The study of amphioxus will be certainly helpful to elucidate the mechanism of development and evolution of vertebrates. Here we cloned three important genes in hedgehog pathway: Amphi-ptch, Amphi-smo, Amphi-gli, which is an important network in development and pathology and an AmphiRbx1 gene related cell differentiation from amphioxus(Branchiostoma belcheri tsingtauense).The expression patterns and the phylogenetic analysis were performed. In addition, the expression of Toll-like receptor (TLR) protein were performed in amphioxus. These data provide insight into the development mechanisms and evolution of the amphioxus and vertebrates.We got the Amphi-ptch, Amphi-smo, Amphi-gli fragments through RT-PCR with degenerate primers designed in conserved areas of nucleic acid according to the analysis from database of Branchiostoma floridae genome (http://www.genome.jp)and GenBank(www.ncbi.nlm.nih.gov). They encoded putative proteins of 374aa, 151aa and 134aa respectively. Through NCBI database BLAST search, we found that these putative proteins share high sequence identities and similarities with respective homologues from other species and the identities are high to 44%-57%, 47%-67% and 90%-94% repectively. This shows that the components of hedgehog pathway are conserved evolutionally at least in sequence. Phylogenetic analysis was performed on homologues proteins from vertebrate and invertebrate species by the neighbor-joining method. Results of phylogenetic analysis indicated that three putative proteins was an independent clade, branched away at the base of the vertebrate group, which is accordance with the traditional view of animal taxology.In situ hybridization results showed Amphi-ptch was expressed initially in presumptive notochord and differentiating endomesoderm at 12h neurula stage. At 14h neurula stage, the transcription of Amphi-ptch was detected in notochord, forming somites and alimentary tract endoderm. This expression pattern maintained during the neurula stages and the expression of Amphi-ptch disappeared in notochord and was just detected in forming somites and differentiating endoderm epidermis in alimentary tract at 20h neurula. This expression pattern continued until 48h larva. Amphi-smo was first detected in differentiation endomesoderm at 10h early neurula stage. Then Amphi-smo expressed in forming somites and alimentary tract endoderm at 14h neurula. At 20h neurula stage, the expression of Amphi-smo decreased in almost formed somites and still existed in differentiating endoderm epidermis in alimentary tract. This expression pattern maintained to 36h and the expression of Amphi-smo was detected jut on the right wall of alimentary epidermis at 48h. The expression pattern of Amphi-gli was very similar to that of Amphi-ptch. Amphi-gli was expressed initially in presumptive notochord and differentiating endomesoderm at 12h neurula stage. At 14h neurula stage, the transcription of Amphi-gli was detected in notochord, forming somites and alimentary tract endoderm. This expression pattern maintained during the neurula stages and the expression of Amphi-gli disappeared in notochord and was just detected in forming somites and differentiating endoderm epidermis in alimentary tract at 20h neurula. This expression pattern continued until 48h larva. Our data suggested that the hedgehog signial pathway played an important role in the development and differentiation of endomesoderm in amphioxus.A clone containing complete open reading frame(ORF) of Rbx1 was found during a large scale sequencing of randomly picked clones from an amphioxus neurula cDNA library. The cDNA insert was 726bp long, encoding a putative protein of 111aa (GenBank accession number: DQ866806). The NCBI database BLAST search indicated that the putative protein shared high sequence identity and sililarity with Rbx1 homologues from other species. Sequence analysis showed that the putative protein contained a conserved RING box domain. So we called the putative protein AmphiRbx1. Results of phylogenetic analysis indicated that AmphiRbx1 fell in the lineage of the Rbx1 and in this lineage, AmphiRbx1 was an independent clade, branched away at the base of the vertebrate group, which is accordance with the traditional view of animal taxology. In situ hybridization and RT-PCR analysis showed that AmphiRbx1 was distinctly expressed in all developmental stages. AmphiRbx1 transcripts were predominant in the cytoplasm at the zygote and cleavage stages. But the expression decreased at blastula stage. In gastrula, the expression of AmphiRbx1 appeared in the differentiating endomesoderm. By about 9.5 h neurula stage, AmphiRbx1 mRNA was detected in neural plate, the presomitic mesoderm, presumptive notochord mesoderm and differentiating archenteron. As development proceeds, the expression was detected in the forming somites, gut, gills and tail bud. However, the expression of AmphiRbx1 was down-regulated after notochord and somites were formed. At the early larval stages, it was maintained in the differentiating gills and tail bud. In adult amphioxus, the transcripts were found in the epithelial cells of gut, peri-branchial epithelial cells, lateral and internal ciliary band, notochord, muscle, ovary and testis. In summary, our data suggested that the expression of AmphiRbx1 in the cells of forming organ and in actively proliferating tissues, such as the developing neural tube, notochord, somites, gut, brancialarches and pharynx indicated that AmphiRbx1 may be related to cell division and differentiation during embryogenesis in amphioxus, but the precise role of AmphiRbx1 in the development of amphioxus needed to be further elucidated.We first studied the expression of Toll-like receptor2 (TLR2) in adult amphioxus by immunohistochemistry and the results in mice were positive control. Our data showed that in amphioxus, the expression of TLR2 protein was detected in hepatic cecum, gut, gill, dorsal neural cord, coelomic dells and epidermis. Weak expressional signal in ovaries could be observed but on in testes. The expression of TLR2 was the strongest in the epithelium of gut and hepatic cecum. TLR2 protein located in the microvilli of epithelial cells, especially in the endocytosis cesicles locating near the inner side plasma membrane of the epithelial cells. In dorsal neural cord, TLR2 expressed in neurons near the central neural tube. In each branchial bar, the positive signal of expression was obvious in the gill blood vessels, the coelomic cells, the pigmented epithelial band and skeletal axis of the septum of bar. The TLR2 protein was also detected in the endostyle, especially in the secretory vesicles located at the top of the gland cells. The coelomic wall and macrophage in coelom as well as the endostyle paquette were also with positive staining. In addition, we could find the distinct expression in the epidermis and macrophage in the subdermal lymph spaces. In summary,our data suggested that there was TLRs proteins in amphioxus and this protein had a high identity with vertebrate TLR2 at least. Moreover, TLR2 protein in amphioxus was not only related with development but also with the innate immunity in terms of the expression in skins, the coelomic wall and macrophage in coelom, epithelium of gut and gill. These indicated that TLR2 played an important role in innate immunity of amphioxus and applied a new clue to the related study.