The amphioxus rab GDP-dissociation inhibitor (GDI) gene is neural-specific: implications for the evolution of chordate rab GDI genes.

The rab GDP-dissociation inhibitor (rab GDI) proteins are involved in the regulation of vesicle-mediated cellular transport. We isolated the amphioxus rab GDI gene, analyzed its expression during amphioxus development, and performed a phylogenetic analysis of the rab GDI family. In contrast to the two major rab GDI forms in mammals, the alpha and beta forms, there is only one rab GDI isoform in amphioxus. Our analysis indicates that the occurrence of the alpha and beta forms of rab GDI preceded the divergence of lineages leading to birds and mammals, and that the amphioxus rab GDI may have evolved directly from the common ancestor of both forms. While the mammalian rab GDI beta-genes are ubiquitously expressed, the rab GDI alpha genes are predominantly expressed in neural tissues. The expression analysis of the amphioxus rab GDI gene shows predominantly neural expression similar to that of the mammalian rab GDI alpha form, suggesting that the ancestral expression pattern of chordate rab GDI was neural. In addition, the chicken rab GDI beta-like gene also shows neural-specific expression, which indicates that the neural expression was retained in both early postduplication alpha and beta isoforms and that a novel function associated with ubiquitous expression may have evolved uniquely in mammals. These results reveal a likely scenario of functional divergence of the rab GDI genes after duplication of the ancestral gene. A similar pattern of evolution, in which one of the duplicated genes retained a role similar to that of the ancestral one while other genes were recruited into novel roles, was also observed in the analysis of chordate Otx and hedgehog genes. In the rab GDI, hedgehog, and Otx gene families, the gene retaining the ancestral role shows a lower rate of sequence evolution than its counterpart, which was recruited for a novel function.     

Identification and expression of amphioxus beta-microseminoprotein (MSP)-like gene encoding an ancient and rapidly evolving protein in chordates

The cDNA encoding beta-microseminoprotein-like (beta-MSPL) was identified from the gut cDNA library of amphioxus. It contains a 336 bp open reading frame corresponding to a deduced protein of 111 amino acids and has eight cysteines conserved and located at the same positions as those in the vertebrate beta-MSPs. At amino acid level, it shares 12-20% similarity to the vertebrate beta-MSPs, and seems lacking the signal peptide at the N-terminus. This not only confirms that beta-MSP is a rapidly evolving protein during phylogeny, but also provides further data on the degree of diversity between species of this protein. RT-PCR and Northern blotting show that amphioxus beta-MSPL is expressed in all tissues examined, suggesting that beta-MSPL plays a fundamental role. However, in situ hybridization reveals that positive hybridization signals were present in all blastomeres of the embryos from 4-cell to gastrula stages, while its expression is restricted exclusively to notochord, somites and primitive gut in neurulae and larvae, and disappears in the ectoderm including the neural tube differentiated from the ectoderm. This suggests that beta-MSPL is possibly involved in the differentiation of ectoderm during embryonic development of cephalochordate amphioxus though it is ubiquitously expressed in embryos prior to gastrula stage and in the adult animal.     

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.     

Identification of up-regulated genes in amphioxus neurula and the expression of AmphiFABP

Amphioxus is a good model organism for understanding the origin and developmental mechanism of vertebrates owing to its important evolutionary position. During the developmental process of amphioxus embryo, the neurula is a crucial stage because of neural tube and notochord formation as well as somite emergence at this stage. In order to isolate genes up-regulated at the neurula stage, we constructed an 11-hour neurula subtracted cDNA library of amphioxus Branchiostoma belcheri and sequenced 204 ESTs representing 82 contigs. Comparative analysis revealed that 55% of those contigs were homologous to various known genes while 45% of them had no significant similarity to any known genes. Those observations imply that the un-identified ESTs might contain some new genes which are involved in the development of amphioxus neurula. Real-time quantitative PCR (RTqPCR) indicated that the expression levels of 14 genes are up-regulated after gastrulation among 20 assayed genes. Of those up-regulated genes, we further cloned and sequenced the full-length of fatty acid binding protein gene (AmphiFABP). The deduced protein sequence was similar to that of vertebrate brain FABP and heart FABP, and in situ hybridization displayed that AmphiFABP, similar to their vertebrate cognates, was expressed not only in nervous system but also in embryonic somite and gut, hinting a multifunctional property of AmphiFABP in amphioxus.     

Genes expressed in the amphioxus notochord revealed by EST analysis

The notochord cell of the cephalochordate amphioxus adult is unique due to the occurrence of myofilaments in the cytoplasm. The present EST (expressed sequence tag) analysis targeted mRNAs of the amphioxus notochord to determine genes that are expressed there. Notochord cells were isolated from Branchiostoma belcheri adults, from which a cDNA library was constructed. Analysis of a set of 257 ESTs (both 5' and 3' ends) showed that about 11% of the cDNAs are related to muscle genes, while 9% of them are genes for extracellular matrix proteins associated with formation of the notochordal sheath. The muscle-related genes included actin, tropomyosin, troponin I, myosin regulatory light chain, myosin light chain kinase, myosin heavy chain, calmodulin, calponin, calcium vector protein, creatine kinase, muscle LIM protein, and SH3-binding glutamate-rich protein, suggesting that vertebrate skeletal and smooth muscle-type genes are simultaneously expressed in the amphioxus notochord. Nucleotide sequences of cDNAs for actin, tropomyosin, troponin I, and a few others were completely determined to substantiate the conclusions. The chordate muscle-type actin is distinguishable from the cytoplasmic-type actin by the usage of amino acid residues at 20 diagnostic positions. Interestingly, analysis of the usage of amino acid residues at these positions showed that the "amphioxus notochord actin" is a unique intermediate between muscle-type and cytoplasmic-type actins. These results strongly suggest that the notochord of adult amphioxus is a mechanical swimming organ and its role is quite different from the role of the vertebrate embryonic notochord, which functions as a source of signals required for body plan formation.     

Identification,Evolution and Expression of an Insulin-Like Peptide in the Cephalochordate Branchiostoma lanceolatum

Insulin is one of the most studied proteins since it is central to the regulation of carbohydrate and fat metabolism in vertebrates and its expression and release are disturbed in diabetes, the most frequent human metabolic disease worldwide. However, the evolution of the function of the insulin protein family is still unclear. In this study, we present a phylogenetic and developmental analysis of the Insulin Like Peptide (ILP) in the cephalochordate amphioxus. We identified an ILP in the European amphioxus Branchiostoma lanceolatum that displays structural characteristics of both vertebrate insulin and Insulin-like Growth Factors (IGFs). Our phylogenetic analysis revealed that amphioxus ILP represents the sister group of both vertebrate insulin and IGF proteins. We also characterized both temporal and spatial expression of ILP in amphioxus. We show that ilp is highly expressed in endoderm and paraxial mesoderm during development, and mainly expressed in the gut of both the developing embryo and adult. We hypothesize that ILP has critical implications in both developmental processes and metabolism and could display IGF- and insulin-like functions in amphioxus supporting the idea of a common ancestral protein.     

Amphioxus allantoicase: molecular cloning, expression and enzymatic activity

Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.     

Verification and tissue-specific expression of nifU-like gene from the amphioxus Branchiostoma belcheri.

A nifU-like gene exhibiting similarity to nifU of nitrogen fixation gene cluster was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri. Both RT-PCR and Northern blotting as well as in situ hybridization histochemistry verified that the cDNA represents an amphioxus nifU-like gene rather than a microbial contaminant. The nifU-like gene encodes a protein of 164 amino acid residues including a highly-conserved U-type motif (C-X(26)-C-X(43)-C), and shares 66-86% identity to NifU-like proteins from a variety of species including vertebrates, invertebrates and microbes. It is expressed in a tissue-specific manner in the digestive system including epipharyngeal groove, endostyle, hepatic caecum and hindgut and in the gill, ovary and testis. Taken together, it is highly likely that NifU-like protein plays some tissue-dependent and critical role in amphioxus.