Studies On HMGB, Thymosin B-4 And Rack1 Gene In Early Development And Phylogenetic Evolution Of Amphioxus, Branchiostoma Belcheri Tsingtaunese

Amphioxus, a cephalochordate, widely considered as the closest living invertebrate relatives 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 vertebrate. Here three genes (HMGB, Thymosin-β4, RACK1) were cloned from amphioxus, and the expression patterns and the phylogenetic analysis were performed. The amphioxus systematic evolution analysis on mitochondrion genes was also studied. These data give insight into the development mechanism and evolution of the amphioxus and vertebrates.We isolated a cDNA clone encoding HMGB in amphioxus, by random screening of amphioxus neurula cDNA library. The cDNA clone is 833 bp long and contains a 666 bp open reading frame, which encoded a putative protein of 222 aa (GenBank accession number: AY578709). The further analysis shows that a full-length AmphiHMGB cDNA has been identified here. NCBI database BLAST search indicated that the nucleotide sequence shared high sequence similarity with HMGB homologues from other species. At the protein level, amphioxus HMGB shows identities of 41, 49. 50, 50, 50, 50 and 50% with the HMG protein of sea urchin, lamprey, rainbow trout, frog, chicken, mouse and human respectively. AmphiHMGB shared higher sequence identities with the homologue in vertebrates. The putative protein sequence contains four domains: HMG-box A, HMG-box B, basic region, acidic carboxyl terminal tail and a linker, all of which are conserved in HMGsuperfamily members. Thus, our data from AmphiHMGB further confirm that HMG proteins are highly conserved in evolution.Phylogenetic analysis was performed on 16 homologues genes from vertebrate and invertebrate species by the neighbor-joining method. Results of phylogenetic analysis indicated that amphioxus HMGB falls outside the vertebrate clade. Our data further suggest that HMG gene duplication occurs near the base of the vertebrate gene family clade. Southern blotting results also showed that there might be only one copy of AmphiHMGB in Qingdao amphioxus.AmphiHMGB expression was detectable from fertilization through the 72-hr larva stages. The mRNA transcripts were conspicuous in the cytoplasm of zygote and in blastomeres at the cleavage stage. At the blastula AmphiHMGB was expressed at a low lever. Its expression was detected throughout the endomesoderm at the gastrula stage. At the 9.5-hr early neurula stage, AmphiHMGB was mainly expressed in the neural plate and presumptive notochord. As the embryo develops, transcripts of AmphiHMGB remain in the neural plate, notochord and mesoderm, differentiating paraxial mesoderm. At the 16-hr neurula stage, AmphiHMGB transcripts were detected in the cerebral vesicle, neural tube, notochord, developing somites, and endoderm. Expression continues in the cerebral vesicle, neural tube, epithelium of the gut and pharynx until at least the 72-hr stage. No signal was detectable in the ectoderm and the resulting epidermis. In consistent with our theory that only a single AmphiHMGB appears in amphioxus, Northern hybridization only detected one band in all embryonic stages. In summary, our data are similar to those of other HMGB vertebrate homologues. The developmental expression pattern of AmphiHMGB indicates that it might be involved in differentiation of neural ectoderm, mesoderm and endoderm in this animal and have important roles in embryonic development.Of more than 5000 clones sequenced from an amphioxus neurula cDNA library, one clone encoded a peptide of 45-amino-acid, and its putative protein sequence shared high identities with the known thymosin-p4 proteins both in vertebrates and invertebrates, ranging between 62% and 82% in amino acid sequence. By comparing its deduced amino acid sequence with known thymosin-p4 proteins, itcontained the conserved helix-actin binding-regions. The LKKTETQEK, a signature sequence, showed a high degree of conservation in all these organisms. We then named this cDNA clone as amphioxus thymosin-fi4 (GenBank accession number: AY 03788).Phylogenetic analysis was performed on the orthologues from vertebrate and invertebrate species by the neighbor-joining analysis. Amphioxus thymosin-p4 failed outside the vertebrate clade and was at the base of the vertebrate gene family clade, indicating that it may represent an independent branch, which is accordant with the traditional view of animal taxology.The expression of amphioxus thymosin-/34 was consistently detected during development from fertilized egg through larval stage. Thymosin-/i4 gene transcript was detected in fertilized eggs and the cleavage stage embryos. By late-gastrula stage, intense thymosin-fi4 expression appeared in the differentiating mesentoderm and then in the neural plate, paraxial mesoderm and the wall of archenterons. The transcript of thymosin-f54 was continually detected in the neural tube, developing myotomes of somites and alimentary canal throughout the later embryonic development. During 24-h to 36-h larval stages, the thymosin-/34 was markedly expressed in myotomes as two dorsal irregular but continuous stripes along the anteroposterior axis, as well as in neural tube, the alimentary canal and differentiating pharynx. By late larval stage, amphioxus thymosin-[H strongly expressed in nascent branchias whereas decreased in the dorsal muscle. In situ study also showed that thymosin-P4 was expressed in neural tube, pharynx, midgut diverticulus, blood vessel and the wall of body spaces including subdermal lymph space and the spaces between myotomes in adult animal. There was no transcript signal of amphioxus thymosin-ji4 in the originally ectoderm and later epidermis.To study the temporal expression pattern of amphioxus thymosin-f$4. Northern blot analysis was carried out. The transcript of thymoun-^4 was detected in most embryonic developmental stages. The mRNA level was increased slightly from the gastrula stage. In summary, our study demonstrated that amphioxus thymosin-fl4 might be related to multiple developmental events and support the notion ofthymosin-p4 as an important regulator gene for patterning the chordate embryo.A clone containing partial sequence of AmphiRACKl was found during a large scale sequencing of randomly picked clones from an amphioxus neurula cDNA library. Its complete open reading frame was obtained using 3'-race reaction (GenBank accession number AY954690). The cDNA insert is 1023 bp long and contains a 951 bp open reading frame, which encoded a putative protein of 317 aa. NCBI database BLAST search indicated that the nucleotide sequence shared high sequence similarity with RACK1 homologues from other species. The predicted protein shows identities of 70%, 83% with theRACKl protein of drosophila, lamprey and 81% with zebrafish, frog, chicken, mouse and human respectively. AmphiRACKl also has seven WD40 repeats, which are conserved in RACK members. Thus, our data from AmphiRACKl further confirmed that RACK1 proteins are highly conserved in evolution.Phylogenetic analysis was performed using 21 RACK1 homologues from vertebrate and invertebrate species by the neighbor-joining method. Results of phylogenetic analysis indicated that amphioxus RACK1 falls outside the vertebrate clade.The spatial and temporal expression pattern of AmphiRACKl was investigated by whole mount in situ hybridization followed by histological sectioning. Maternal AmphiRACKl transcripts were detected in the cytoplasm of the zygote and blastomeres at the early cleavage stage, but not at blastula and gastrula stage. By early neurula stage, strong AmphiRACKl expression was detected at the neuroectoderm, dorsal mesoderm and the wall of archenteron. From the 12-hr neurula to knife-shaped larva, transcripts of AmphiRACKl appeared in cerebral vesicle, neural tube, somites, splanchnic mesoderm and the epithelium of gut. The gene expression was marked at the borderline area of somites. Expression of AmphiRACKl maintained in the cerebral vesicle, neural tube, the epithelium of the gut and pharynx until at least the 72-hr stage. AmphiRACKl expression was also detected in the epithelium of midgut diverticulus and gut, wheel organ and gill blood vessels in the transverse sections of adult animal. Weak expression of AmphiRACKl was also detectable intestis and the epithelium of gills.A comparative study of expression patterns of AmphiRACKl in the malformed embryos and larvae caused by lithium treatment was performed. In such animals, a diverse expression pattern from observed in normal early development was revealed. AmphiRACKl transcripts were looked stronger and with increased dorsal area at 12-hr neurula stage. Later, the segmented expression of AmphiRACKl in somites became blurry and letdown at the borderline of somites. There was no visible borderline of somites in knife-shaped larva. Meantime, the expression in cerebral vesicle and neural tube was also decreased and disappeared. The data of in site indicated that lithium chloride treatment affected AmphiRACKl expression in the development of somites and central nervous system in amphioxus. There was also visible exterior morphological change between the normal and lithium treated embryos under microscope. At 11 to 12-hr neurula stages, the dorsal of lithium treated embryos looked thicker. At 16hr and 2Ohr neurula stage, 6-9 somites were distinctly observed but the somites of lithium treated embryos showed mistiness. Our study revealed that AmphiRACKl might be related to multiple developmental events similar to its vertebrate homologs. Lithium chloride treatment embryo affects AmphiRACKl expression pattern, the diverse expression appears in somitogenesis and neurogenesis of amphioxus.Comparisons of mitochondrial genes are useful for modeling genome evolution and for phylogenetic inference. Here we clone four genes in amphioxus: cytochrome b, cytochrome c oxidase subunit I, cytochrome c oxidase subunit II and cytochrome c oxidase subunit III. All sequences were processed and analyzed with ClustalxJ.8, DNA Star Megalign, Mega and DAMBE software. Through sequences analysis, the result shows the four genes sequences shows strong A/T bias, the average content of A+T is higher than G+C. Transition is higher than transversion among the nucleotide substitution of the three amphioxus sequences while the transversion is higher than transition among the different group species sequences. And the nucleotide substitution occurs mainly at the third location of the codon. Through Mega software, some useful statistical data set could be obtained and the phylogenetic trees wereconstructed by three methods (NJ, ME, MP). The species Branchiostoma lanceolatum and Branchiostoma floridae formed a branch, they are the similar evolutionary groups. Branchiostoma belcheri tsingtaunese formed a branch by oneself, and is much more original than the former. And the divergence time was estimated.In total, the present study provided new information to the phylogeny of amphioxus on molecular level.