Amphioxus Early Development The Gene Amphimdp And Brp-like

Amphioxus, a cephalochordate, shares a common ancestry with vertebrates. Although amphioxus and vertebrates split about 55()million years ago and the amphioxus body structure is simpler than the vertebrates, it resembles the common ancestor of amphioxus and vertebrate. However, the amphioxus genome has not escaped evolution. In the 550 million years evolution, the genome of amphioxus is not duplicated but some independent genes are duplicated. After duplication, the duplicated genes may survive and acquire novel function or they may lose in the genome, which is similar to the event that occurs in the vertebrates. So amphioxus is an important animal model for phylogenetic analysis including chordate developmental mechanisms and gene evolution.Here, we report two genes related to the developmental mesoderm of amphioxus. One is AmphiMdp and the other is brp-like. The transcripts of AmphiMdp and its orthologous genes in vertebrates are in the similar regions. However, the amphioxus brp-like gene and its orthologous gene in zebrafish, a model of vertebrate, are expressed in the different regions. So the function of the AmphiMdp and its orthologous genes are kept during the evolution but the function of brp-like and its orthologous genes are not.In a large scale sequencing of an amphioxus neurula cDNA library, more than 5000 clones are analysed. Among them a partial sequence of AmphiMdp is found. This sequence is 1136bp Jong, and there is an open reading frame in it. However, with the further analysis with blastX in NCBI, the predicted protein sequence is shorter than the orthologousand there is no in-frame stop codon upstream the ATG. So the sequence of 5' end is absent. With the 5'RACE, about 600bp long sequence is obtained. Compared it with the original sequence, there is 132bp long overlap between the new obtained sequence and the original sequence. Analysis of the extended sequence with blastX in NCBI and the soft Clone, we obtain the whole open reading frame and there is an in-frame stop codon in the upstream. The MDP77 in chick, 0 -taxilin in human and predicted protein of AmphiMdp, have the similar special structure, the coiled-coil domain. So AmphiMdp may have the same function with the orthologous in the vertebrate.Southern Blotting indicates that amphioxus might have only one copy of AmphiMdp gene in its genome.The spatial and temporal expression pattern of AmphiMdp is analysed by whole mount in situ hybridisation followed by sectioning. The gene doesn't express itself before 6 hours after fertilized. And faint AmphiMdp expression is first detected in mid-gastrula stage. By the time embryogenesis has proceeded to the early neurula stage, intense AmphiMdp expression is detected in the forming somites. As the somite formation extends to the posterior, AmphiMdp expression extends in the same way. The expression of AmphiMdp in somites is maintained during somite formation. In addition expression is down-regulated from anterior lo posterior and eventually disappeares in the formed somites. The transcripts are only detected in the most posterior region in 48h larva. In 72h larva no AmphiMdp transcripts are detected in the amphioxus larvae.Early studies report that the AmphiMRF2 is a member of the MyoD or myogenic regulatory factor (MRF) gene family. MyoD genes are expressed in developing paraxial mesoderm. somites, derivatives of somites, and skeletal muscles in vertebrates. It is considered that theMyoD gene plays a major role in controlling the events leading to skeletal muscle development. The result of the in situ hybridization of the AmphiMdp shows that the location of the gene transcripts mimics that of the AmphiMRF2 gene.In situ hybridizations are carried with two probes: AmphiMRF2 and AmphiMdp in the LiCl treated amphioxus embryos and larvae. LiCl in 300 mM, 10 min or 100 mM, 30 min is supplied at l?8-cells stage. The expression patterns of the two genes are similar in the normal amphioxus embryos. The expressional leveJ and of the two genes is increased in the treated embryos at the stage ofneurula. And at the same time, the spatial expression of both genes, which is limited to the mesoderm region in the normal embryos, is expanded and expressed in the mesoderm and endoderm in LiCl treated embryos. The expression pattern of the AmphiMRF2 and AmphiMdp are similar, both in the normal embryos and in the treated embryos. So the AmphiMdp is a muscle development related gene, and it may function in the muscle development.We also reported another mesoderm related gene brp — like. Phylogenetic analysis of nineteen genes with high identities to this putative protein from Genbank indicates that amphioxus gene falls outside the cluster of the vertebrate genes, neither belonging to the branch of human brain protein239AB or human brain protein239FB. So we named it brp-like gene. The putative brp-like protein in Branchiostoma belcheri is 60% and 56% identical to human brain protein239AB and human brain pro-tein239FB in Homo sapiens, 51% identical to the orthologous protein in D. rerio, and it is 45%, 44% identical to similar proteins in Drosophila melanogaster and Anopheles fiambiae. The putative brp-like protein in amphioxus has a higher similarity to the orthologous proteins in vertebrates than those ininvertebrates. It may be the ancestor of the human brain 239AB and FB.Embryonic developmental expression of amphioxus brp-like gene is analyzed by whole-mount in situ hybridization and a series of histological sections. Transcripts of amphioxus brp-like gene appear from the early neurula to the 26-h larval stages, but are not detectable before the early neurula stage. At about 10 hours-post-fertilized neurula stage, weak brp-like expression is first detected in the wall of the archenteron. Intense expression is revealed in the differentiating presumptive notochord, the presumptive pharynx and gut at the following neurular stages. Soon thereafter, transcripts of brp-like seem to be down-regulated and disappear in the formed notochord. However, gene expression continues in the dorsal epithelium of the pharynx and gut and the developing gills throughout the neurular stages. It remains up to 26-h early larval stages and later disappears. Expression of brp-like is never detected in the cerebral vesicle and/or the anterior of the neural plate during early neurulation. The expression pattern of brp-like is quite different to its orthologous-human brain 239AB and FB.To find out what is the brain protein genes function in vertebrates, we have done the in situ hybridization with the orthologous of the brp-like gene in zebrafish, which is a model organism in vertebrate developmental biology. The transcripts are detectable in blastomeres of two-cell stage and in the following stages, but are absent in the yolk cell. At 90% epiboly. the transcripts are mostly collected at the brain anlagen. They are also observed in optic primordium, retina, differentiating spinal cord, tail bud, somites, and differentiating brain in the following developmental stages of zebrafish. And at the 26-somite stage, a high level expression of the gene is detected in the eyes, telencephalon. diencephalon, midhrain and hindbrain. andmoderate level in the otic placode. So the orthologous gene of the amphioxus brp-like gene in zebrafish may have the function in the differentiating central nervous system.Compared with the results of the in situ hybridization of the amphioxus brp-like gene and its orthologous in the zebrafish, we find the expression patterns are different. Variant expression patterns may imply the different functions of the two genes. From the phylogenetic analysis, the amphioxus brp-like has the closer relationship to vertebrate orthologous. However, it is not expressed in the central nervous system (CNS) and its anlage, neural plate, which suggests there is no relation between the gene and the development of CNS in amphioxus. So the function of brain protein genes may have changed during the organism evolution.The Sec61 complex, which locates at endoplasmic reticulum(ER), is known to play a crucial role in promoting translocation at the ER membrane. Sec61 complex consists of Sec61 a , Sec61 3 and Sec61 Y in mammalian. By sequencing the Qingdao amphioxus neurulae cDNA library. We obtains the open reading frame sequence of sec61 Y . The deduced amino acid sequence is analyzed and compared with other vertebrate and invertebrate Sec61 Y proteins. The results shows that the Sec61 Y protein is conserved in evolution and will offer more evidence for study of the evolution of vertebrate.In the dissertation we first report the clones, phylogenetic analysis, and gene expression partterns of the two amphioxus genes, AmphiMdp and brp —like, which are bolh related to the development of the mesoderm. The AmphiMdp, whose expression pattern is similar to its orthologous in the vertebrates, is expressed in the paraxial mesoderm. However, the othologous gene of brp —like, which is expressed in the axial mesoderm. is expressed in the different region in the zebrafish. Sowe predicted that the function of the brp-like is changed in the evolution. And what we have done here will reveal the mechanic of the molecule development and provide more evidences for the diversity and conservation of the gene function in the evolution.