Mammalian tumor xenografts induce neovascularization in zebrafish embryos.

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein–positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/ tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis

Identification and spatial distribution of the mRNA encoding an egg envelope component of the Cyprinid zebrafish, Danio rerio, homologous to the mammalian ZP3(ZPC)

Using degenerate reverse transcription polymerase chain reaction (RT-PCR) techniques we have isolated a cDNA encoding a putative component of the zebrafish Danio rerio egg chorion, homologous to the mammalian ZP3 (ZPC). The predicted protein (zfZPC) has a calculated molecular mass of 58.4 kDa and contains a signal peptide (located in the N-terminal region) composed of 11 hydrophobic amino acid residues followed by a signal peptide cleavage site. The zfZPC contains the ZP domain, a characteristic amino acid sequence shared by all ZP proteins of the mammalian zona pellucida and of both amphibian and bird egg envelope components. The zfZPC also exhibits certain unique features including five N-terminal Q-rich tandem repeats presumably involved in the hardening of the chorion after the fertilization of the egg and a long C-terminal tail containing two potential sites of N-linked type glycosylation. RT-PCR and in situ hybridization revealed a restricted pattern of tissue distribution: the gene encoding zfZPC is transcribed only in the growing oocyte of sexually mature female fish

Acute effects of whole-body vibrations on the fatigue induced by multiple repeated sprint ability test in soccer players

Background: We tested the hypothesis that whole-body vibration (WBV) positively affects the fatigue process ensuing from repeated bouts of maximal efforts, as induced by repeated sprints' ability (RSA). Eleven male soccer players performed three sets of six repeated shuttle sprints (40 meters). Methods: Eleven male soccer players (age 23.6±4.5 years) were cross-randomized to perform WBW before RSA and during the recovery between sets (WBV-with) or to warm-up and passive recovery between sets (WBV-without). The effects of WBV were quantified by sprint time (ST) and blood lactate concentration (LA), collected up to 15 min after completion of tests. Results: ST during RSA showed a better maintenance of performance in the WBV-with compared to WBV-without condition in all three sets, reaching a statistical significance between-groups during the 2nd and 3rd set (P<0.05). No significant differences in ST over the sets were detected in WBV-with, whereas a significant decrease was observed in the WBV-without condition (P<0.001). LA recovered significantly faster from the 9th to 15th minute of recovery in WBV-with as compared to WBV-without (P<0.05). Conclusions: These findings would indicate that WBV performed during recovery between RSA sets can delay the onset of muscle fatigue resulting in a better maintenance of sprint performance

ADAP2 in heart development: a candidate gene for the occurrence of cardiovascular malformations in NF1 microdeletion syndrome

Background Cardiovascular malformations have a higher incidence in patients with NF1 microdeletion syndrome compared to NF1 patients with intragenic mutation, presumably owing to haploinsufficiency of one or more genes included in the deletion interval and involved in heart development. In order to identify which genes could be responsible for cardiovascular malformations in the deleted patients, we carried out expression studies in mouse embryos and functional studies in zebrafish. Methods and results The expression analysis of three candidate genes included in the NF1 deletion interval, ADAP2, SUZ12 and UTP6, performed by in situ hybridisation, showed the expression of ADAP2 murine ortholog in heart during fundamental phases of cardiac morphogenesis. In order to investigate the role of ADAP2 in cardiac development, we performed loss-of-function experiments of zebrafish ADAP2 ortholog, adap2, by injecting two different morpholino oligos (adap2-MO and UTR-adap2-MO). adap2-MOs-injected embryos (morphants) displayed in vivo circulatory and heart shape defects. The molecular characterisation of morphants with cardiac specific markers showed that the injection of adap2-MOs causes defects in heart jogging and looping. Additionally, morphological and molecular analysis of adap2 morphants demonstrated that the loss of adap2 function leads to defective valvulogenesis, suggesting a correlation between ADAP2 haploinsufficiency and the occurrence of valve defects in NF1-microdeleted patients. Conclusions Overall, our findings indicate that ADAP2 has a role in heart development, and might be a reliable candidate gene for the occurrence of cardiovascular malformations in patients with NF1 microdeletion and, more generally, for the occurrence of a subset of congenital heart defects

P316Expression and functional role of Ccdc80 in developing heart and in cardiomyopathies.

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Characterization of the AP-1 μ1A and μ1B adaptins in zebrafish (Danio rerio)

Protein transport between the trans-Golgi network and endosomes is mediated by transport vesicles formed by the adaptor-protein complex AP-1, consisting of the adaptins γ1, β1, μ1, σ1. Mammalia express μ1A ubiquitously and isoform μ1B in polarized epithelia. Mouse γ1 or μ1A 'knock out's revealed that AP-1 is indispensable for embryonic development. We isolated μ1A and μ1B from Danio rerio. Analysis of μ1A and μ1B expression revealed tissue-specific expression for either one during embryogenesis and in adult tissues in contrast to their expression in mammalia. μ1B transcript was detected in organs of endodermal derivation and "knock-down" experiments gave rise to embryos defective in formation of intestine, liver, and pronephric ducts. Development ceased at 7-8 dpf. μ1B is not expressed in murine liver, indicating loss of μ1B expression and establishment of alternative sorting mechanisms during mammalian development

The zebrafish prospero homolog prox1 is required for mechanosensory hair cell differentiation and functionality in the lateral line

Background: The lateral line system in zebrafish is composed of a series of organs called neuromasts, which are distributed over the body surface. Neuromasts contain clusters of hair cells, surrounded by accessory cells. Results: In this report we describe zebrafish prox1 mRNA expression in the migrating primordium and in the neuromasts of the posterior lateral line. Furthermore, using an antibody against Prox1 we characterize expression of the protein in different cell types within neuromasts, and we show distribution among the supporting cells and hair cells. Conclusion: Functional analysis using antisense morpholinos indicates that prox1 activity is crucial for the hair cells to differentiate properly and acquire functionality, while having no role in development of other cell types in neuromasts

Zebrafish as a Model to Investigate Dynamin 2-Related Diseases

Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down the zebrafish dnm2a, producing defects in muscle morphology. Finally, we expressed two mutated DNM2 mRNA by injecting zebrafish embryos with human mRNAs carrying the R522H mutation, causing CNM, or the G537C mutation, causing CMT. Defects arose especially in secondary motor neuron formation, with incorrect branching in embryos injected with CNM-mutated mRNA, and total absence of branching in those injected with CMT-mutated mRNA. Muscle morphology in embryos injected with CMT-mutated mRNA appeared less regularly organized than in those injected with CNM-mutated mRNA. Our results showing, a continuum between CNM and CMTDIB phenotypes in zebrafish, similarly to the human conditions, confirm this animal model to be a powerful tool to investigate mutations of DNM2 in vivo

Characterization of the Neuroligin gene family expression and evolution in zebrafish

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

FAS/FASL are dysregulated in chordoma and their loss-of-function impairs zebrafish notochord formation

Chordoma is a rare malignant tumor that recapitulates the notochord phenotype and is thought to derive from notochord remnants not correctly regressed during development. Apoptosis is necessary for the proper notochord development in vertebrates, and the apoptotic pathway mediated by Fas and Fasl has been demonstrated to be involved in notochord cells regression. This study was conducted to investigate the expression of FAS/FASL pathway in a cohort of skull base chordomas and to analyze the role of fas/fasl homologs in zebrafish notochord formation. FAS/FASL expression was found to be dysregulated in chordoma leading to inactivation of the downstream Caspases in the samples analyzed. Both fas and fasl were specifically expressed in zebrafish notochord sorted cells. fas and fasl loss-of-function mainly resulted in larvae with notochord multi-cell-layer jumps organization, larger vacuolated notochord cells, defects in the peri-notochordal sheath structure and in vertebral mineralization. Interestingly, we observed the persistent expression of ntla and col2a1a, the zebrafish homologs of the human T gene and COL2A1 respectively, which are specifically up-regulated in chordoma. These results demonstrate for the first time the dysregulation of FAS/FASL in chordoma and their role in notochord formation in the zebrafish model, suggesting their possible implication in chordoma onset