Amphioxus expresses both vertebrate-type and invertebrate-type dopamine D1 receptors

The cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates, which makes it an ideal organism for studying the molecular basis of the evolutionary transition from invertebrates to vertebrates. The biogenic amine, dopamine regulates many aspects of motor control in both vertebrates and invertebrates, and in both cases, its receptors can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. We have recently characterized one of these receptors, AmphiD1/β, which was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors, but functionally appeared to be a vertebrate-type dopamine D₁ receptor. Here, we report on the cloning of two further dopamine D₁ receptors (AmphiAmR1 and AmphiAmR2) from adult amphioxus cDNA libraries and their pharmacological characterisation subsequent to their expression in cell lines. AmphiAmR1 shows closer structural similarities to vertebrate D₁-like receptors but shows some pharmacological similarities to invertebrate “DOP1” dopamine D₁-like receptors. In contrast, AmphiAmR2 shows closer structural and pharmacological similarities to invertebrate “INDR”-like dopamine D₁-like receptors.     

Identification and tissue-specific expression of amphioxus GM2 activator protein gene from amphioxus Branchiostoma belcheri.

An amphioxus cDNA, AmphiGM2AP, encoding GM2 activator protein was isolated from the gut cDNA library of Branchiostoma belcheri. It is 907 bp long, and its longest open reading frame codes for a precursor protein consisting of 242 amino acid residues with a signal peptide of 14 amino acids. The deduced amino acid sequence includes a conserved domain typical of GM2APs between residues 53 and 224, a single N-linked glycosylation site at position 65 and 8 conserved cysteines. Phylogenetic analysis showed that amphiGM2AP forms a club together with invertebrate GM2APs, indicating that AmphiGM2AP is evolutionarily closely related to invertebrate GM2APs rather than vertebrate ones. Both Northern blotting and in situ hybridization histochemistry analyses revealed a tissue-specific expression pattern of AmphiGM2AP in adult amphioxus with the strongest expression in the digestive system, which is in contrast to the widespread expression pattern of human, mouse and sheep GM2AP genes. It is suggested that AmphiGM2AP is possibly involved in the take-in of digested food components like lipid molecules.     

AmphiD1/β, a dopamine D1/β-adrenergic receptor from the amphioxus <Emphasis Type="Italic">Branchiostoma floridae</Emphasis>: evolutionary aspects of the catecholaminergic system during development

Catecholamine receptors mediate wide-ranging functions in vertebrates and invertebrates but are largely unknown in invertebrate chordates such as amphioxus. Catecholaminergic cells have been described in amphioxus adults, but few data are known about the transmembrane signal transduction pathways and the expression pattern of related receptors during development. In Branchiostoma floridae, we cloned a full-length cDNA (AmphiD1/β) that corresponds to the dopamine D1/β receptor previously cloned from a related species of amphioxus, Branchiostoma lanceolatum, but no expression studies have been performed for such receptor in amphioxus. In B. floridae, AmphiD1/β encodes a polypeptide with typical G-protein-coupled receptor features, characterized by highest sequence similarity with D1 dopamine and β-adrenergic receptors. The expression of AmphiD1/β mRNA in different regions of the cerebral vesicle corresponds to that of D1-like receptors in vertebrate homologous structures. Furthermore, in situ experiments show that during development, the expression in the nervous system is restricted to cells located anteriorly. A further expression was found in larvae at the level of the endostyle, but it has no counterpart in the predominant expression domains of vertebrate dopamine and/or adrenergic receptor genes. At the same time, we compared the dopaminergic system, consisting of AmphiTH-expressing cells, with the AmphiD1/β expression. In conclusion, the identification of the AmphiD1/β receptor provides further basis for understanding the evolutionary history of the dopaminergic system at the transition from invertebrates and vertebrates.     

Eleven new putative aminergic G-protein coupled receptors from Amphioxus (<Emphasis Type="Italic">Branchiostoma floridae</Emphasis>): identification,sequence analysis and phylogenetic relationship

We have identified eleven novel aminergic-like G-protein coupled receptor (GPCRs) sequences (named AmphiAmR1-11) by searching the genomic trace sequence database for the amphioxus species, Branchiostoma floridae. They share many of the structural motifs that have been used to characterize vertebrate and invertebrate aminergic GPCRs. A preliminary classification of these receptors has been carried out using both BLAST and Hidden Markov Model analyses. The amphioxus genome appears to express a number of D1-like dopamine receptor sequences, including one related to insect dopamine receptors. It also expresses a number of receptors that resemble invertebrate octopamine/tyramine receptors and others that resemble vertebrate alpha-adrenergic receptors. Amphioxus also expresses receptors that resemble vertebrate histamine receptors. Several of the novel receptor sequences have been identified in amphioxus cDNA libraries from a number of tissues.     

Origin and evolution of vertebrate ABCA genes: a story from amphioxus

Previous studies showed that the vertebrate ABCA subfamily, one subgroup of the ATP-binding-cassette superfamily, has evolved rapidly in terms of gene duplication and loss. To further uncover the evolutionary history of the ABCA subfamily, we characterized ABCA members of two amphioxus species (Branchiostoma floridae and B. belcheri), the closest living invertebrate relative to vertebrates. Phylogenetic analysis indicated that these two species have the same set of ABCA genes (both containing six members). Five of these genes have clear orthologs in vertebrate, including one cephalochordate-specific duplication and one vertebrate-specific duplication. In addition, it is found that human orthologs of amphioxus ABCA1/4/7 and its neighboring genes mainly localize on chromosome 1, 9, 19 and 5. Considering that most of analyzed amphioxus genes have clear orthologs in zebrafish, we conclude these four human paralogous regions might derive from a common ancestral region by genome duplication occurred prior to teleost/tetrapod split. Therefore, the present results provide new evidence for 2R hypothesis.     

Genes "waiting" for recruitment by the adaptive immune system: the insights from amphioxus

In seeking evidence of the existence of adaptive immune system (AIS) in ancient chordate, cDNA clones of six libraries from a protochordate, the Chinese amphioxus, were sequenced. Although the key molecules such as TCR, MHC, Ig, and RAG in AIS have not been identified from our database, we demonstrated in this study the extensive molecular evidence for the presence of genes homologous to many genes that are involved in AIS directly or indirectly, including some of which may represent the putative precursors of vertebrate AIS-related genes. The comparative analyses of these genes in different model organisms revealed the different fates of these genes during evolution. Their gene expression pattern suggested that the primitive digestive system is the pivotal place of the origin and evolution of the AIS. Our studies support the general statement that AIS appears after the jawless/jawed vertebrate split. However our study further reveals the fact that AIS is in its twilight in amphioxus and the evolution of the molecules in amphioxus are waiting for recruitment by the emergence of AIS.     

文昌鱼sfy1基因的克隆及其在早期发育中的表达

文昌鱼是公认现存最接近于脊椎动物的一种头索动物,具有与脊椎动物相似但简单得多的身体图式[1],因而是研究脊椎动物发育机制起源和进化的宝贵材料,也是发育生物学的经典实验模型之一.近年来,人们在对果蝇和脊椎动物发育分子机制的研究取得了一系列重大突破之后,利用发育调控基     

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.     

文昌鱼AmphiRab23b基因的克隆、进化分析及表达模式

Hedgehog信号通路在胚胎发育过程中发挥着重要作用, 同时与多种肿瘤的发生密切相关。Rab23蛋白在Hedgehog信号通路中扮演着十分重要的角色。目前关于文昌鱼Rab23同源基因的研究仅局限于佛罗里达文昌鱼(Branchiostoma floridae)基因组中的注释。文章首次克隆了中国文昌鱼(Branchiostoma belcheri) Rab23b基因 (AmphiRab23b)cDNA全序列 , 对其演译的蛋白序列进行了序列比对、进化树分析以及基因时空表达分析。研究结果显示, 文昌鱼AmphiRab23b基因的 cDNA总长为2 062 bp (包括UTR区), 其中开放阅读框 (Open reading frame, ORF) 714 bp, 编码237个氨基酸; 虽然在进化树中不属于脊椎动物Rab23进化支, 但是AmphiRab23具有保守的Rab23_lke结构域, 暗示该基因在进化过程中可能在功能上是保守的。时空表达的研究结果进一步显示, AmphiRab23b基因在胚胎发育中的神经板和消化道中表达, 与其脊椎动物同源基因的表达模式相似, 这说明该基因可能对文昌鱼神经系统和消化道的发育有重要作用。     

The single amphioxus Mox gene: insights into the functional evolution of Mox genes,somites, and the asymmetry of amphioxus somitogenesis

Mox genes are members of the "extended" Hox-cluster group of Antennapedia-like homeobox genes. Homologues have been cloned from both invertebrate and vertebrate species, and are expressed in mesodermal tissues. In vertebrates, Mox1 and Mox2 are distinctly expressed during the formation of somites and differentiation of their derivatives. Somites are a distinguishing feature uniquely shared by cephalochordates and vertebrates. Here, we report the cloning and expression of the single amphioxus Mox gene. AmphiMox is expressed in the presomitic mesoderm (PSM) during early amphioxus somitogenesis and in nascent somites from the tail bud during the late phase. Once a somite is completely formed, AmphiMox is rapidly downregulated. We discuss the presence and extent of the PSM in both phases of amphioxus somitogenesis. We also propose a scenario for the functional evolution of Mox genes within chordates, in which Mox was co-opted for somite formation before the cephalochordate-vertebrate split. Novel expression sites found in vertebrates after somite formation postdated Mox duplication in the vertebrate stem lineage, and may be linked to the increase in complexity of vertebrate somites and their derivatives, e.g., the vertebrae. Furthermore, AmphiMox expression adds new data into a long-standing debate on the extent of the asymmetry of amphioxus somitogenesis.     

文昌鱼tropomyosin基因的克隆、进化分析及其胚胎发育与成体中的表达模式

Tropomyosin是一种分布广泛而且在进化上十分保守的蛋白,是肌肉形成和收缩过程中重要的调节蛋白质。通过RT-PCR和RACE技术得到文昌鱼tropomyosin基因全长,编码一个含284个氨基酸残基的蛋白质,将文昌鱼Tropomyosin和在其他物种中的同源物进行比对建树,发现其在功能域上高度保守并且只有一个拷贝,符合动物分类学中各物种的进化地位。胚胎整体原位杂交实验得知,tropomyosin在文昌鱼早期发育的表达,最早从原肠胚末期神经胚早期开始,定位于分化中的中内胚层。到神经胚期,tropomyosin的表达出现在发育中的体节和脊索中。随着发育的进行,tropomyosin的表达稳定地集中在体节、脊索处。到72h幼虫阶段,tropomyosin的表达仍然在肌节内。成体的切片原位杂交结果显示,tropomyosin在肌节中的表达大幅度下调,而在神经管细胞、脊索和腮区腮瓣处仍然可以检测到明显的表达,在外胚层和表皮内没有发现杂交信号。研究结果表明,tropomyosin的表达与文昌鱼肌节、肌肉以及神经索的发生相关,参与文昌鱼胚胎躯体模式的构建,而且在成体的生命活动中发挥重要作用。     

Identification and tissue-specific expression of a promethin-like homolog in amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

Promethins have been shown to be present in the vertebrates examined so far, yet little is known to date about them in invertebrates. Here we isolated a cDNA encoding a promethin-like homolog from the gut cDNA library of the amphioxus Branchiostoma belcheri, a cephalochordate occupying a nodal position transient from invertebrates to vertebrates. It contained a 504 bp open reading frame corresponding to a protein of 167 amino acids. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic analysis showed that B. belcheri promethin-like homolog was located at the base of the vertebrate counterparts, suggesting that it represents the archetype of vertebrate promethins. Both Northern blotting and in situ hybridization histochemistry revealed a tissue-specific expression pattern of promethin-like gene, like that of mammalian promethins. This is the first report on invertebrate promethin-like homolog, paving the way for further insights into the evolution and function of promethins.     

The amphioxus Hairy family: differential fate after duplication

Vertebrate Hairy genes are highly pleiotropic and have been implicated in numerous functions, such as somitogenesis, neurogenesis and endocrine tissue development. In order to gain insight into the timing of acquisition of these roles by the Hairy subfamily, we have cloned and studied the expression pattern of the Hairy gene(s) in amphioxus. The cephalochordate amphioxus is widely believed to be the living invertebrate more closely related to vertebrates, the genome of which has not undergone the massive gene duplications that took place early during vertebrate evolution. Surprisingly, we have isolated eight Hairy genes from the 'pre-duplicative' amphioxus genome. In situ hybridisation on amphioxus embryos showed that Hairy genes had experienced a process of subfunctionalisation that is predicted in the DDC model (for duplication-degeneration-complementation). Only the summation of four out of the eight Amphi-Hairy genes expression resembles the expression pattern of vertebrate Hairy genes, i.e. in the central nervous system, presomitic mesoderm, somites, notochord and gut. In addition, Amphi-Hairy genes expression suggest that amphioxus early somites are molecularly prefigured in an anteroposterior sequence in the dorsolateral wall of the archenteron, and the presence of a midbrain/hindbrain boundary. The expansion of the amphioxus Hairy subfamily request for caution when deducing the evolutionary history of a gene family in chordates based in the singularity of the amphioxus genome. Amphioxus may resemble the ancestor of the vertebrates, but it is not the ancestor, only its closest living relative, a privileged position that should not assume the freezing of its genome.     

Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region

On the basis of developmental gene expression, the vertebrate central nervous system comprises: a forebrain plus anterior midbrain, a midbrain-hindbrain boundary region (MHB) having organizer properties, and a rhombospinal domain. The vertebrate MHB is characterized by position, by organizer properties and by being the early site of action of Wnt1 and engrailed genes, and of genes of the Pax2/5/8 subfamily. Wada and others (Wada, H., Saiga, H., Satoh, N. and Holland, P. W. H. (1998) Development 125, 1113-1122) suggested that ascidian tunicates have a vertebrate-like MHB on the basis of ascidian Pax258 expression there. In another invertebrate chordate, amphioxus, comparable gene expression evidence for a vertebrate-like MHB is lacking. We, therefore, isolated and characterized AmphiPax2/5/8, the sole member of this subfamily in amphioxus. AmphiPax2/5/8 is initially expressed well back in the rhombospinal domain and not where a MHB would be expected. In contrast, most of the other expression domains of AmphiPax2/5/8 correspond to expression domains of vertebrate Pax2, Pax5 and Pax8 in structures that are probably homologous - support cells of the eye, nephridium, thyroid-like structures and pharyngeal gill slits; although AmphiPax2/5/8 is not transcribed in any structures that could be interpreted as homologues of vertebrate otic placodes or otic vesicles. In sum, the developmental expression of AmphiPax2/5/8 indicates that the amphioxus central nervous system lacks a MHB resembling the vertebrate isthmic region. Additional gene expression data for the developing ascidian and amphioxus nervous systems would help determine whether a MHB is a basal chordate character secondarily lost in amphioxus. The alternative is that the MHB is a vertebrate innovation.