Expression of AmphiNaC, a new member of the amiloride-sensitive sodium channel related to degenerins and epithelial sodium channels in amphioxus

Degenerins and amiloride-sensitive Na+ channels form a new family of cationic ion channels (DEG/NaC). DEG/NaC family emerged as common denominator within a metazoan mechanosensory apparatus. In this study, we characterized a new member of such family in amphioxus, Branchiostoma floridae. The AmphiNaC cDNA sequence encodes a protein showing amino acid residues characteristic of DEG/NaC family, such as two hydrophobic domains surrounding a large extracellular loop that includes cystein-rich domains; nevertheless its predicted sequence is quite divergent from other family members. AmphiNaC is expressed at early larval stage in some putative sensory epidermal cells in the middle of the body and in neurons of the posterior cerebral vesicle, as well as in some ventrolateral and mediolateral neurons of the neural tube. In late larvae, AmphiNaC expression is maintained in some neurons of the neural tube, and it is expressed in putative sensory epidermal cells of rostrum and mouth. The analysis of AmphiNaC gene expression pattern suggests that it might be involved in neurotransmission and sensory modulation

Characterization, developmental expression and evolutionary features of the huntingtin gene in the amphioxus Branchiostoma floridae

<p>Abstract</p> <p>Background</p> <p>Huntington's disease is an inherited neurodegenerative disorder that is caused by the expansion of an N-terminal polyQ stretch in the huntingtin protein. In order to investigate the hypothesis that huntingtin was already involved in development of the nervous system in the last common ancestor of chordates, we isolated and characterised the huntingtin homologue from the amphioxus <it>Branchiostoma floridae</it>. In the present paper the amphioxus general term must be referred to <it>Branchiostoma floridae</it>.</p> <p>Results</p> <p>In this report, we show that the exon-intron organization of the amphioxus huntingtin gene is highly conserved with that of other vertebrates species. The AmphiHtt protein has two glutamine residues in the position of the typical vertebrate polyQ tract. Sequence conservation is greater along the entire length of the protein than in a previously identified <it>Ciona </it>huntingtin. The first three N-terminal HEAT repeats are highly conserved in vertebrates and amphioxus, although exon rearrangement has occurred in this region. <it>AmphiHtt </it>expression is detectable by in situ hybridization starting from the early neurula stage, where it is found in cells of the neural plate. At later stages, it is retained in the neural compartment but also it appears in limited and well-defined groups of non-neural cells. At subsequent larval stages, <it>AmphiHtt </it>expression is detected in the neural tube, with the strongest signal being present in the most anterior part.</p> <p>Conclusion</p> <p>The cloning of amphioxus huntingtin allows to infer that the polyQ in huntingtin was already present 540 million years ago and provides a further element for the study of huntingtin function and its evolution along the deuterostome branch.</p

A study of neural-related microRNAs in the developing amphioxus

<p>Abstract</p> <p>Background</p> <p>MicroRNAs are small noncoding RNAs regulating expression of protein coding genes at post-transcriptional level and controlling several biological processes. At present microRNAs have been identified in various metazoans and seem also to be involved in brain development, neuronal differentiation and subtypes specification. An approach to better understand the role of microRNAs in animal gene expression is to determine temporal and tissue-specific expression patterns of microRNAs in different model organisms. Therefore, we have investigated the expression of six neural related microRNAs in amphioxus, an organism having an important phylogenetic position in terms of understanding the origin and evolution of chordates.</p> <p>Results</p> <p>In amphioxus, all the microRNAs we examined are expressed in specific regions of the CNS, and some of them are correlated with specific cell types. In addition, miR-7, miR-137 and miR-184 are also expressed in endodermal and mesodermal tissues. Several potential targets expressed in the nervous system of amphioxus have been identified by computational prediction and some of them are coexpressed with one or more miRNAs.</p> <p>Conclusion</p> <p>We identified six miRNAs that are expressed in the nervous system of amphioxus in a variety of patterns. miR-124 is found in both differentiating and mature neurons, miR-9 in differentiated neurons, miR-7, miR-137 and miR-184 in restricted CNS regions, and miR-183 in cells of sensory organs. Therefore, such amphioxus miRNAs may play important roles in regional patterning and/or specification of neuronal cell types.</p

The synapsin gene family in basal chordates: evolutionary perspectives in metazoans

<p>Abstract</p> <p>Background</p> <p>Synapsins are neuronal phosphoproteins involved in several functions correlated with both neurotransmitter release and synaptogenesis. The comprehension of the basal role of the synapsin family is hampered in vertebrates by the existence of multiple synapsin genes. Therefore, studying homologous genes in basal chordates, devoid of genome duplication, could help to achieve a better understanding of the complex functions of these proteins.</p> <p>Results</p> <p>In this study we report the cloning and characterization of the <it>Ciona intestinalis </it>and amphioxus <it>Branchiostoma floridae </it>synapsin transcripts and the definition of their gene structure using available <it>C. intestinalis </it>and <it>B. floridae </it>genomic sequences. We demonstrate the occurrence, in both model organisms, of a single member of the synapsin gene family. Full-length synapsin genes were identified in the recently sequenced genomes of phylogenetically diverse metazoans. Comparative genome analysis reveals extensive conservation of the SYN locus in several metazoans. Moreover, developmental expression studies underline that synapsin is a neuronal-specific marker in basal chordates and is expressed in several cell types of PNS and in many, if not all, CNS neurons.</p> <p>Conclusion</p> <p>Our study demonstrates that synapsin genes are metazoan genes present in a single copy per genome, except for vertebrates. Moreover, we hypothesize that, during the evolution of synapsin proteins, new domains are added at different stages probably to cope up with the increased complexity in the nervous system organization. Finally, we demonstrate that protochordate synapsin is restricted to the post-mitotic phase of CNS development and thereby is a good marker of postmitotic neurons.</p

Expression of the tissue specific transcription factor Pit-1 in the lancelet, Branchiostoma lanceolatum

Lancelets, known also as amphioxus, are protochordates that share common archetypal features with vertebrates. Recently, several developmental and molecular biology studies have pointed out homologies between anatomical structures of lancelets and vertebrates. We have studied the head region of the lancelet, Branchiostoma lanceolatum, by means of scanning electron microscopy, immunocytochemistry, and Western blotting techniques, to localize the pituitary-specific transcription factor, Pit-1. Immunoreactive Pit-1 protein has been found in cells of two typical structures of the lancelets, the Ko\ua8lliker\u2019s and Hatschek\u2019s pits. Moreover, the frontal eye complex, neurons, and the rostral nerves show Pit-1 immunoreactivity. A band of 33 kilodaltons has been resolved in lancelet extracts by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis and after Western blotting, the bands have been probed by a monoclonal antibody to rat Pit-1. Our results demonstrate that Pit-1 is expressed in both neurones and receptosecretory epithelial cells of adult lancelets, and that the cells lining the two pits display ultrastructural and immunocytochemical features typical of chemoreceptosecretory/olfactory- and adenohypophyseal-like structures

The tissue-specific transcription factor Pit-1 is expressed in the spinal cord of the lancelet, Branchiostoma lanceolatum

The spinal cord of the lancelet Branchiostoma lanceolatum was studied by using a monoclonal antibody to the rat tissue-specific transcription factor, Pit-1. Our previous studies have demonstrated Pit-1 immunoreactivity in different nervous and endocrine structures of the head region of adults and in the rostral central nervous system (CNS) of larval lancelet. Our present results show the presence of Pit-1-like protein in dorso-lateral nerve cells and ependymocytes of the adult spinal cord. Using double immunofluorescence techniques, we have revealed the coexistence of the glial fibrillary acidic protein (GFAP) with Pit-1 in groups of laterally located ependymocytes. The occurrence of GFAP, a specific marker of mammalian astrocytes and radial glia, in some lancelet ependymocytes confirms that glial elements are also present in protochordates. Furthermore, other ependymocytes, located in the roof of the central canal and containing Pit-1-like protein exclusively, could be considered as ependymal tanycytes