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ABSTRACT: The goal of this study was to determine the in vivo functions of the synaptic proteins neurexins and neuroligins in embryonic vascular system development using zebrafish as animal model.
In the present study, we show that the knockdown of the α-form of neurexin 1a induces balance defects and reduced locomotory activity, whereas β-neurexin 1a and neuroligin 1 morphants present defects in sprouting angiogenesis and vascular remodeling, in particular in the caudal plexus and subintestinal vessels. Coinjection of low doses of morpholinos for β-neurexin 1a and neuroligin 1 together or in combination with morpholinos targeting the -heparin--binding isoforms of vascular endothelial growth factor A (encoded by the VEGFAb gene) recapitulates the observed abnormalities, suggesting synergistic activity of these molecules. Similar coinjection experiments with morpholinos, targeting the enzyme heparan sulfate 6-O-sulfotransferase 2, confirm the presence of a functional correlation between extracellular matrix maturation and β-neurexin 1a or neuroligin 1.
Our data represent the first in vivo evidence of the role of neurexin and neuroligin in embryonic blood vessel formation and provide insights into their mechanism of action.
Arteriosclerosis Thrombosis and Vascular Biology 04/2012; 32(7):1563-72. · 6.37 Impact Factor
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ABSTRACT: The dysmorphogenic activity of the triazole fungicide triadimefon (FON) has been evaluated using Xenopus laevis development. Embryos, exposed to 500 μM FON during early neurulation phases (stages 13-17, Nieuwkoop and Faber), were allowed to develop until stage 47. Larvae revealed serious craniofacial defects, bent forebrain, and abnormal hindbrain segmentation. CRABP and CYP26 (markers related to retinoic acid homeostasis) gene and protein expression and protein distribution have been evaluated at stage 17 and at the end of the branchial arch morphogenesis (stage 27) by real-time PCR, western blot and whole-mount immunostaining. A significant increase of CYP26 transcript has been observed at both embryonic stages. A co-localization of the two markers has been observed at the cephalic region. Embryos exposed to FON showed abnormal distribution of positive tissues. Due to the strict similarity of these results with those previously described in rodents, a FON-related alteration of mechanism conserved during vertebrate evolution is suggested.
Reproductive Toxicology 01/2011; 31(4):486-93. · 3.23 Impact Factor
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ABSTRACT: Neuroligins constitute a family of transmembrane proteins localized at the postsynaptic side of both excitatory and inhibitory synapses of the central nervous system. They are involved in synaptic function and maturation and recent studies have linked mutations in specific human Neuroligins to mental retardation and autism. We isolated the human Neuroligin homologs in Danio rerio. Next, we studied their gene structures and we reconstructed the evolution of the Neuroligin genes across vertebrate phyla. Using reverse-transcriptase polymerase chain reaction, we analyzed the expression and alternative splicing pattern of each gene during zebrafish embryonic development and in different adult organs. By in situ hybridization, we analyzed the temporal and spatial expression pattern during embryonic development and larval stages and we found that zebrafish Neuroligins are expressed throughout the nervous system. Globally, our results indicate that, during evolution, specific subfunctionalization events occurred within paralogous members of this gene family in zebrafish.
Developmental Dynamics 02/2010; 239(2):688-702. · 2.54 Impact Factor
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ABSTRACT: Identification and characterization of a bovine cDNA and the corresponding gene coding for a novel protein structurally related to Zar1, therefore called Zar1-like, are here reported for the first time. Structure of Zar1-like is similar to Zar1 gene, nevertheless they are located on distinct chromosomes. We demonstrated that the new gene as well as its genomic context are conserved along the whole vertebrate lineage. Analysis of the deduced protein primary structure showed a high conservation, among vertebrates, of the C-terminal region, where the putative presence of both zinc finger motifs and classical nuclear localization signals is also shared with Zar1. Bovine Zar1-like and the only two other available mRNA leader sequences (human and chicken) exhibit a number of upstream AUGs, suggesting that they are likely to be regulated at translational level. Expression patterns of the cattle transcripts show that Zar1-like is absent in early stages of embryo development, whereas Zar1 is expressed in matured oocytes and in in vitro produced pre-implantation embryos. In adult tissues Zar1-like transcript expression appears to be less restricted than Zar1, nevertheless, at least in bovine, both mRNAs are co-expressed in gonads, raising the question of a possible functional link.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 07/2008; 150(2):233-9. · 1.92 Impact Factor
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ABSTRACT: This study was conducted in order to analyse gene-expression alterations in rat embryos following exposure to triazoles, using an easy-handling approach. Triazole derivatives have been shown to alter the morphology of cranio-facial structures and to induce abnormalities in hindbrain patterning and neural crest cell migration. Specification of hindbrain segments is regulated by retinoic acid and the hox code. Krox20 was chosen as molecular marker for its specific distribution in the anterior neural tube. In fact, this zinc-finger protein is expressed in rhombomere 3 and 5. Mis-regulation of Krox20 levels have shown to induce severe alterations in the correct patterning of the rhomboencephalon and the derived structures. In order to analyse Krox20 mRNA levels in rat embryos exposed in vitro to the triazole derivative triadimefon, a semi-quantitative approach utilising the competitive RT-PCR was chosen. A lambda phage-based plasmid construct that could compete with target and internal standard gene at the same time during enzymatic reaction was generated. Results were confirmed by real-time RT-PCR analysis on the same samples. Our data show a down-regulation of Krox20 transcript levels after exposure to the triazole derivative, implying a key role of this molecule in the pathogenic pathway induced by triazole exposure.
Toxicology Letters 04/2007; 169(3):196-204. · 3.23 Impact Factor
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ABSTRACT: Prion protein (PrP) and PrP-related proteins have been identified in reptiles, amphibians, and fishes by means of cDNA cloning, genome database searching and comparative genomics. However, no studies have been reported so far on the expression of PrP at the protein level in those animals. This report presents a procedure to obtain and purify recombinant PrP from Xenopus laevis expressed in Escherichia coli as a fusion protein in which mature PrP (residues 21-194) is linked to a 35-amino acid N-terminal extension containing a hexahistidine stretch. The protein was used to raise and purify by affinity chromatography anti-Xenopus PrP polyclonal antibodies which were suitable to detect the presence of PrP in Xenopus brain by Western blot. This is the first report of a positive identification of PrP in amphibian at the protein level. Anti-Xenopus PrP antibodies do not cross react with PrP from different sources (human, bovine, sheep, and turtle). Similarly, Xenopus PrP do not react with anti-turtle PrP(143-248) antibodies.
Protein Expression and Purification 05/2006; 46(2):489-94. · 1.59 Impact Factor
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ABSTRACT: We report evidence from cDNA isolation and expression analysis as well as analyses of genome, expressed sequence tag (EST), cDNA and expression databases for a new gene named SPRN (shadow of prion protein). SPRN comprises two exons, with the open reading frame (ORF) contained within exon 2, and codes for a protein of 130-150 amino acids named Shadoo (Japanese shadow), predicted to be extracellular and GPI-anchored. The SPRN gene was found in fish (zebrafish, Fugu) and mammals (mouse, rat, human). Conservation of order and transcription orientation of two proximal genes between fishes and mammals strongly indicates gene orthology. Sequence comparison shows: a highly conserved N-terminal signal sequence; Arg-rich basic region containing up to six tetrarepeats of consensus XXRG (where X is G, A or S); a hydrophobic region of 20 residues with strong homology to PrP; a less conserved C-terminal domain containing a conserved glycosylation motif; and a C-terminal peptide predicted to be a signal sequence for glycophosphotidylinositol (GPI)-anchor attachment. Fish Shadoos (Sho) show well conserved sequences (identity 54%) over 106 amino acids (zebrafish length), and conservation among the mammalian sequences is very high (identity 81-96%). The fish and mammalian sequences are also well conserved, particularly for zebrafish, to beyond the end of the hydrophobic sequence (identity 41-53%, 78 amino acids, zebrafish length). The overall structure appears closely related to prion proteins (PrPs), although the C-terminal domains of Shos are quite different from those of PrPs, for which conformational changes in mammals are implicated in disease. The structural similarity is particularly interesting given recent reports of three new genes with similarities to PrPs found in Fugu (PrP-like, PrP-461/stPrP-1 and stPrP-2) and other fish, but for which direct evolution to higher vertebrate PrPs is unlikely and for which no other mammalian homologues have been found. Database information indicates expression of SPRN in embryo, brain and retina of mouse and rat, hippocampus of human, and in embryo and retina of zebrafish, and we directly confirmed a strikingly specific expression of the mammalian (human, mouse, rat) transcripts in whole brain. This result together with some common structural features led to the suggestive hypothesis of a possible functional link between mammalian PrP and Sho proteins.
Gene 10/2003; 314:89-102. · 2.34 Impact Factor
