157 research outputs found
Onecut is a direct neural-specific transcriptional activator of Rx in Ciona intestinalis
AbstractRetinal homeobox (Rx) genes play a crucial and conserved role in the development of the anterior neural plate of metazoans. During chordate evolution, they have also acquired a novel function in the control of eye formation and neurogenesis. To characterize the Rx genetic cascade and shed light on the mechanisms that led to the acquisition of this new role in eye development, we studied Rx transcriptional regulation using the ascidian, Ciona intestinalis. Through deletion analysis of the Ci-Rx promoter, we have identified two distinct enhancer elements able to induce Ci-Rx specific expression in the anterior part of the CNS and in the photosensory organ at tailbud and larva stages. Bioinformatic analysis highlighted the presence of two Onecut binding sites contained in these enhancers, so we explored the role of this transcription factor in the regulation of Ci-Rx. By in situ hybridization, we first confirmed that these genes are co-expressed in the same cells. Through a series of in vivo and in vitro experiments, we then demonstrated that the two Onecut sites are responsible for enhancer activation in Ci-Rx endogenous territories. We also demonstrated in vivo that Onecut misexpression is able to induce ectopic activation of the Rx promoter. Finally, we demonstrated that Ci-Onecut is able to promote Ci-Rx expression in the sensory vesicle. Together, these results support the conclusion that in Ciona embryogenesis, Ci-Rx expression is under the control of the Onecut transcription factor and that this factor is necessary and sufficient to specifically activate Ci-Rx through two enhancer elements
Characterization of a new variant DNA (cytosine-5)-methyltransferase unable to methylate double stranded DNA isolated from the marine annelid worm Chaetopterus variopedatus
AbstractThe enzyme S-adenosylmethionine-DNA (cytosine-5)-methyltransferase has been identified, first time for invertebrates, in embryos of the marine polychaete annelid worm Chaetopterus variopedatus. The molecule has been isolated from embryos at 15 h of development. It is a single peptide of about 200 kDa molecular weight, cross-reacting with antibodies against sea urchin DNA methyltransferase. The enzymatic properties of the molecule are similar to those of Dnmt1 methyltransferases isolated from other organisms, but with the peculiarity to be unable to make ‘de novo’ methylation on double stranded DNA
Evolution of anterior Hox regulatory elements among chordates
<p>Abstract</p> <p>Background</p> <p>The <it>Hox </it>family of transcription factors has a fundamental role in segmentation pathways and axial patterning of embryonic development and their clustered organization is linked with the regulatory mechanisms governing their coordinated expression along embryonic axes. Among chordates, of particular interest are the <it>Hox </it>paralogous genes in groups 1-4 since their expression is coupled to the control of regional identity in the anterior nervous system, where the highest structural diversity is observed.</p> <p>Results</p> <p>To investigate the degree of conservation in <it>cis</it>-regulatory components that form the basis of <it>Hox </it>expression in the anterior nervous system, we have used assays for transcriptional activity in ascidians and vertebrates to compare and contrast regulatory potential. We identified four regulatory sequences located near the <it>CiHox1, CiHox2 </it>and <it>CiHox4 </it>genes of the ascidian <it>Ciona intestinalis </it>which direct neural specific domains of expression. Using functional assays in <it>Ciona </it>and vertebrate embryos in combination with sequence analyses of enhancer fragments located in similar positions adjacent to <it>Hox </it>paralogy group genes, we compared the activity of these four <it>Ciona cis</it>-elements with a series of neural specific enhancers from the amphioxus <it>Hox1-3 </it>genes and from mouse <it>Hox </it>paralogous groups 1-4.</p> <p>Conclusions</p> <p>This analysis revealed that Kreisler and Krox20 dependent enhancers critical in segmental regulation of the hindbrain appear to be specific for the vertebrate lineage. In contrast, neural enhancers that function as <it>Hox </it>response elements through the action of Hox/Pbx binding motifs have been conserved during chordate evolution. The functional assays reveal that these <it>Hox </it>response <it>cis</it>-elements are recognized by the regulatory components of different and extant species. Together, our results indicate that during chordate evolution, <it>cis</it>-elements dependent upon Hox/Pbx regulatory complexes, are responsible for key aspects of segmental <it>Hox </it>expression in neural tissue and appeared with urochordates after cephalochordate divergence.</p
MARINE-EXPRESS: taking advantage of high throughput cloning and expression strategies for the post-genomic analysis of marine organisms
Background: The production of stable and soluble proteins is one of the most important steps prior to structural and functional studies of biological importance. We investigated the parallel production in a medium throughput strategy of genes coding for proteins from various marine organisms, using protocols that involved recombinatorial cloning, protein expression screening and batch purification. This strategy was applied in order to respond to the need for post-genomic validation of the recent success of a large number of marine genomic projects. Indeed, the upcoming challenge is to go beyond the bioinformatic data, since the bias introduced through the genomes of the so called model organisms leads to numerous proteins of unknown function in the still unexplored world of the oceanic organisms. Results: We present here the results of expression tests for 192 targets using a 96-well plate format. Genes were PCR amplified and cloned in parallel into expression vectors pFO4 and pGEX-4T-1, in order to express proteins N-terminally fused to a six-histidine-tag and to a GST-tag, respectively. Small-scale expression and purification permitted isolation of 84 soluble proteins and 34 insoluble proteins, which could also be used in refolding assays. Selected examples of proteins expressed and purified to a larger scale are presented. Conclusions: The objective of this program was to get around the bottlenecks of soluble, active protein expression and crystallization for post-genomic validation of a number of proteins that come from various marine organisms. Multiplying the constructions, vectors and targets treated in parallel is important for the success of a medium throughput strategy and considerably increases the chances to get rapid access to pure and soluble protein samples, needed for the subsequent biochemical characterizations. Our set up of a medium throughput strategy applied to genes from marine organisms had a mean success rate of 44% soluble protein expression from marine bacteria, archaea as well as eukaryotic organisms. This success rate compares favorably with other protein screening projects, particularly for eukaryotic proteins. Several purified targets have already formed the base for experiments aimed at post-genomic validation
Occurrence and neuroendocrine role of D-aspartic acid and N-methyl-D-aspartic acid in Ciona intestinalis
AbstractProbes for the occurrence of endogenous D-aspartic acid (D-Asp) and N-methyl-D-aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D-Asp is synthesized from L-Asp by an aspartate racemase. Then, D-Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin-releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction
Natural Variation of Model Mutant Phenotypes in Ciona intestinalis
BACKGROUND: The study of ascidians (Chordata, Tunicata) has made a considerable contribution to our understanding of the origin and evolution of basal chordates. To provide further information to support forward genetics in Ciona intestinalis, we used a combination of natural variation and neutral population genetics as an approach for the systematic identification of new mutations. In addition to the significance of developmental variation for phenotype-driven studies, this approach can encompass important implications in evolutionary and population biology. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report a preliminary survey for naturally occurring mutations in three geographically interconnected populations of C. intestinalis. The influence of historical, geographical and environmental factors on the distribution of abnormal phenotypes was assessed by means of 12 microsatellites. We identified 37 possible mutant loci with stereotyped defects in embryonic development that segregate in a way typical of recessive alleles. Local populations were found to differ in genetic organization and frequency distribution of phenotypic classes. CONCLUSIONS/SIGNIFICANCE: Natural genetic polymorphism of C. intestinalis constitutes a valuable source of phenotypes for studying embryonic development in ascidians. Correlating genetic structure and the occurrence of abnormal phenotypes is a crucial focus for understanding the selective forces that shape natural finite populations, and may provide insights of great importance into the evolutionary mechanisms that generate animal diversity
The genome of the sea urchin Strongylocentrotus purpuratus
We report the sequence and analysis of the 814-megabase genome of the sea urchin Strongylocentrotus
purpuratus, a model for developmental and systems biology. The sequencing strategy combined
whole-genome shotgun and bacterial artificial chromosome (BAC) sequences. This use of BAC clones,
aided by a pooling strategy, overcame difficulties associated with high heterozygosity of the genome.
The genome encodes about 23,300 genes, including many previously thought to be vertebrate
innovations or known only outside the deuterostomes. This echinoderm genome provides an
evolutionary outgroup for the chordates and yields insights into the evolution of deuterostomes
Patterning the ascidian nervous system: structure, expression and transgenic analysis of the CiHox3 gene
12 pages, 8 figures.Hox genes play a fundamental role in the establishment of chordate body plan, especially in the anteroposterior patterning of the nervous system. Particularly interesting are the anterior groups of Hox genes (Hox1-Hox4) since their expression is coupled to the control of regional identity in the anterior regions of the nervous system, where the highest structural diversity is observed. Ascidians, among chordates, are considered a good model to investigate evolution of Hox gene, organisation, regulation and function. We report here the cloning and the expression pattern of CiHox3, a Ciona intestinalis anterior Hox gene homologous to the paralogy group 3 genes. In situ hybridization at the larva stage revealed that CiHox3 expression was restricted to the visceral ganglion of the central nervous system. The presence of a sharp posterior boundary and the absence of transcript in mesodermal tissues are distinctive features of CiHox3 expression when compared to the paralogy group 3 in other chordates. We have investigated the regulatory elements underlying CiHox3 neural-specific expression and, using transgenic analysis, we were able to isolate an 80 bp enhancer responsible of CiHox3 activation in the central nervous system (CNS). A comparative study between mouse and Ciona Hox3 promoters demonstrated that divergent mechanisms are involved in the regulation of these genes in vertebrates and ascidians.M. M. was supported by HFSP and EEC Marie Curie
postdoctoral fellowships and by an EEC Biotechnology Network
grant (#BIO4 CT-960378). This work was also funded in part by Core
MRC Programme support and an EEC Biotechnology Network grant
(#BIO4 CT-960378) to R. K.Peer reviewe
Control of the expression of DNA methyltransferase during sea urchin embryo development.
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