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PubMed2006

The microtubule-severing protein Spastin is essential for axon outgrowth in the zebrafish embryo.

Hereditary spastic paraplegia (HSP) is a collection of neurological disorders characterized by developmental failure or degeneration of motor axons in the corticospinal tract and progressive lower limb spasticity. SPG4 mutations are the most common cause of autosomal dominant HSP and Spastin (the SPG4 gene product) is a microtubule severing protein that shares homology with katanin, the microtubule severing activity of which promotes axon growth in cultured neurons. Given the sequence and functional similarity between spastin and katanin, we hypothesized that spastin promotes the dynamic disassembly and remodelling of microtubules required for robust, properly directed motor axon outgrowth. To investigate this hypothesis, we cloned the zebrafish spg4 orthologue and used morpholino antisense oligonucleotides directed against the translation start site and the intron 7-8 splice donor site to knock down spastin function in the developing zebrafish embryo. Reduced spg4 function caused dramatic defects in motor axon outgrowth without affecting the events driving the initial specification of motor neurones. Other neuronal subtypes also exhibited a requirement for spg4 function, since spg4 knock down caused both widespread defects in neuronal connectivity and extensive CNS-specific apoptosis. Our results reveal a critical requirement for spastin to promote axonal outgrowth during embryonic development, and they validate the zebrafish embryo as a novel model system to dissect the pathogenetic mechanisms underlying HSP. Taken together with other recent studies, our findings suggest that axon outgrowth defects may be a common feature of childhood SPG3A and SPG4 cases.

Adenosine Triphosphatases↗

PubMed2000

Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo.

Definition of cell fates along the dorso-ventral axis depends on an antagonistic relationship between ventralizing transforming growth factor-beta superfamily members, the bone morphogenetic proteins and factors secreted from the dorsal organizer, such as Noggin and Chordin. The extracellular binding of the last group to the bone morphogenetic proteins prevents them from activating their receptors, and the relative ventralizer:antagonist ratio is thought to specify different dorso-ventral cell fates. Here, by taking advantage of a non-genetic interference method using a specific competitive inhibitor, the Lefty-related gene product Antivin, we provide evidence that cell fate along the antero-posterior axis of the zebrafish embryo is controlled by the morphogenetic activity of another transforming growth factor-beta superfamily subgroup--the Activin and Nodal-related factors. Increasing antivin doses progressively deleted posterior fates within the ectoderm, eventually resulting in the removal of all fates except forebrain and eyes. In contrast, overexpression of activin or nodal-related factors converted ectoderm that was fated to be forebrain into more posterior ectodermal or mesendodermal fates. We propose that modulation of intercellular signalling by Antivin/Activin and Nodal-related factors provides a mechanism for the graded establishment of cell fates along the antero-posterior axis of the zebrafish embryo.

Activins↗

PubMed1996

Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo.

As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations that affect the formation and function of the cardiovascular system. The cardiovascular system is particularly amenable for screening in the transparent zebrafish embryo because the heart and blood vessels are prominent and their function easily examined. We have classified the mutations affecting the heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci, miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia. Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor coordination between the chambers with regard to the timing of contraction. The mutations identified in this screen point to discrete and critical steps in the formation and function of the heart and vasculature.

Animals↗

PubMed2001

Brain-derived neurotrophic factor gene organization and transcription in the zebrafish embryo.

The gene encoding zebrafish brain-derived neurotrophic factor (BDNF) was cloned from a PAC genomic DNA library. The entire transcription unit was contained in two independently isolated clones that together encompass 120 kb of genomic DNA. The intron/exon organization of the zebrafish gene was found to be identical to that of the mammalian gene but only one promoter has so far been identified. The associated 5' exon is 67% identical to exon 1c of the rat BDNF gene. A search of the 5' flank of the cloned promoter for sequence similarities with known transcription factor binding sites revealed potential AP-1, CREB, and SP1 binding sites. Fusion constructs containing the cloned promoter and 1.7 kb of 5' flank and an enhanced green fluorescent protein reporter that becomes membrane-anchored were injected into 1-8 cell stage embryos. Expression was seen in notochord, muscle, epithelial and endothelial cells of the 1-day-old embryo in consonance with the endogenous gene. These results demonstrate that the cloned promoter mediates cell-specific expression.

Animals↗

PubMed1990

Development of spinal neurons and tracts in the zebrafish embryo.

We have analyzed pathfinding by growth cones in the spinal cord of the early zebrafish embryo, because it is an extremely simple system. At 18-20 hours of development the spinal cord contains approximately 18 lateral and presumably post-mitotic cell bodies per hemisegment. Of these 8-11 have projected growth cones by 18 hr of development and fall into five classes of neurons (Bernhardt et al., J. Comp. Neurol, preceding paper), including a set of mechanosensory (RB) neurons, three classes of interneurons (DoLA, ascending commissural, and VeLD), and previously characterized primary motor neurons (Eisen et al., '86: Nature 320:269-271). Of these five classes we analyzed pathfinding by the RB, DoLA, early ascending commissural, and VeLD neurons. These neurons are distinguishable at the earliest stages of axonogenesis based on the location of their somata and the number and initial directionality of their growth cones. In each case they follow stereotyped, cell-specific pathways to reach their termination sites. Up through larval stages exuberant axons have not been observed. The longitudinal axons of each neuronal class form bundles in the early cord. This apparently occurs because growth cones extend in close association with the longitudinal axons of the same neuronal class. At later stages spatially discrete commissural tracts are found in the cord suggesting that commissural growth cones may follow earlier commissural axons as well.

Animals↗

PubMed1999

Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signalling.

The specification of different muscle cell types in the zebrafish embryo requires signals that emanate from the axial mesoderm. In previous studies we and others have shown that overexpression of different members of the Hedgehog protein family can induce the differentiation of two types of slow-twitch muscles, the superficially located slow-twitch fibres and the medially located muscle pioneer cells. Here we have investigated the requirement for Hedgehog signalling in the specification of these distinct muscle cell types in two ways: first, by characterising the effects on target gene expression and muscle cell differentiation of the u-type mutants, members of a phenotypic group previously implicated in Hedgehog signalling, and second, by analysing the effects of overexpression of the Patched1 protein, a negative regulator of Hedgehog signalling. Our results support the idea that most u-type genes are required for Hedgehog signalling and indicate that while such signalling is essential for slow myocyte differentiation, the loss of activity of one signal, Sonic hedgehog, can be partially compensated for by other Hedgehog family proteins.

Animals↗

PubMed2004

Melatonin stimulates cell proliferation in zebrafish embryo and accelerates its development.

All vertebrates show a dramatic circadian rhythm in circulating melatonin with high levels at night and very low levels during daytime. In adults, melatonin is thought to synchronize other circadian rhythms and regulate seasonal rhythms in photoperiodic animals by acting on specific G-protein coupled receptors. The role of melatonin in development is unknown, even though melatonin receptors appear to be more highly expressed in developing embryos and neonates than in adults. In this study on zebrafish embryos, we describe a role for melatonin in increasing cell proliferation and accelerating development. We propose that melatonin has a role in extending the safe limit of proliferation rate at night to allow more rapid development when potentially damaging ultraviolet light is absent.

Animals↗

PubMed1994

The fates of the blastomeres of the 16-cell zebrafish embryo.

We present a fate map for the 16-cell-stage blastomeres of the zebrafish embryo Brachydanio rerio. We injected high molecular weight fluorescent dextran into cleavage-stage cells to observe the contributions of the descendants of the first 16 cells to the adult. The patterns derived from these early cells are similar, but not identical among different embryos. Furthermore, two-color injections showed that sister blastomeres at the 16-cell stage regularly contribute to different sets of adult structures. A few of the scored tissues could not be mapped in a manner consistent with the predicted axes. Other tissues were mapped to several of the 16 blastomeres. Many of the tissues map with high probability to a few 16-cell-stage blastomeres. Thus, based on 112 injections in 56 different embryos, we have constructed a fate map by assigning probabilities for the contribution of each blastomere to each of 31 tissues in the 26-hour embryo.

Animals↗

PubMed2001

Oxygen deprivation causes suspended animation in the zebrafish embryo.

Continuous exposure to oxygen is essential for nearly all vertebrates. We found that embryos of the zebrafish Danio rerio can survive for 24 h in the absence of oxygen (anoxia, 0% O2). In anoxia, zebrafish entered a state of suspended animation where all microscopically observable movement ceased, including cell division, developmental progression, and motility. Animals that had developed a heartbeat before anoxic exposure showed no evidence of a heartbeat until return to terrestrial atmosphere (normoxia, 20.8% O2). In analyzing cell-cycle changes of rapidly dividing blastomeres exposed to anoxia, we found that no cells arrested in mitosis. This is in sharp contrast to similarly staged normoxic embryos that consistently contain more than 15% of cells in mitosis. Flow cytometry analysis revealed that blastomeres arrested during the S and G2 phases of the cell cycle. This work indicates that survival of oxygen deprivation in vertebrates involves the reduction of diverse processes, such as cardiac function and cell-cycle progression, thus allowing energy supply to be matched by energy demands.

Anaerobiosis↗

PubMed1998

Real-time measurements of calcium dynamics in neurons developing in situ within zebrafish embryos.

We have developed a non-invasive technique to measure intracellular calcium ([Ca2+]i) in neurons growing within intact embryos of the zebrafish (Danio rerio). A single blastomere was injected with a calcium-sensitive fluorescent dye (Calcium Green dextran) between the 32- and 128-cell stage and the embryo imaged between 16 h and 20 h postfertilisation using laser scanning confocal microscopy. Labelled nerve cells from embryos preinjected with dye and dissociated at 16 h showed a fluorescence increase (66+/-22%; n=11) in response to depolarisation with KCl confirming that the dye remained intracellular and was sensitive to calcium. In addition, fluorescence changes in activated muscle cells of intact embryos showed that the dye was capable of responding to [Ca2+]i changes in vivo. Imaging of dye loaded cells over 30-min periods in embryos between 16 and 20 h revealed that the majority of neurons within the brain and spinal cord did not show spontaneous fluorescence changes distinguishable from noise. However, a subset of neurons within the ventral spinal cord exhibited spontaneous, repetitive [Ca2+]i oscillations which may have a functional significance during neuronal development.

Animals↗

PubMed2004

Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO.

One of the most frequently detected pharmaceuticals in environmental water samples is the anti-rheumatic drug, diclofenac. Despite its increasing environmental significance, investigations concerning the effects of this drug on the early developmental stages of aquatic species are lacking up to now. To determine the developmental toxicity and proteotoxicity of this drug on the growing fish embryos, eggs of zebrafish were exposed to six concentrations of diclofenac (0, 1, 20, 100, 500, 1000, and 2000 microg l(-1)) using DMSO as solvent. Early life stage parameters such as egg and embryo mortality, gastrulation, somite formation, movement and tail detachment, pigmentation, heart beat, and hatching success were noted and described within 48- and 96-h of exposure. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in both the diclofenac-treated and respective DMSO controls. Results showed no significant inhibition in the normal development until the end of 96 h for all exposure groups. However, there was a delay in the hatching time among embryos exposed to 1000 and 2000 microg l(-1). Late-hatched embryos (108 h) did not differ morphologically from normally hatched embryos. The mortality and average heart rate data did not show significant differences for all embryos in both diclofenac-treated and DMSO control groups. No significant malformations were likewise noted among all developing embryos throughout the exposure period. The levels of heat shock proteins in diclofenac-treated and control embryos did not differ significantly. DMSO control embryos, on the other hand, showed a concentration-dependent increase in hsp 70 levels. We suggest possible modulating effect of diclofenac in DMSO-triggered expression of stress proteins and this might have a possible repercussion on the use of DMSO as solvent in any toxicity assay. Since the present data indicate no significant embryotoxicity and proteotoxicity induced by diclofenac and due to the fact that the concentrations of diclofenac used in the present study is up to 2000-fold higher than the concentrations detected in the environment, it is unlikely that this drug would pose a hazard to early-life stages of zebrafish.

Analysis of Variance↗

PubMed1998

Characterization of two frizzled8 homologues expressed in the embryonic shield and prechordal plate of zebrafish embryos.

We have isolated and characterized two complete cDNA clones, Zfz8a and Zfz8b, which encode zebrafish Frizzled (Fz) homologues. The predicted protein sequences, spanning 579 and 576 amino acid residues for ZFz8a and ZFz8b, respectively, were highly homologous (78%) to each other and contained an extracellular cysteine-rich domain and seven transmembrane domains that are well conserved in Fz receptor protein members. In comparison with other Fz family members, ZFz8a and ZFz8b showed the highest homology with mouse Fz8 (MFz8), sharing 84 and 76% amino acid identity, respectively. The presence of Zfz8a and Zfz8b transcripts was detected by in situ hybridization in zebrafish embryos from the 512 cell stage, and their appearance in the future dorsal region could be observed before embryos reached the 30% epiboly stage. At shield stage, Zfz8a transcripts were expressed in both epiblast and shield whereas expression of Zfz8b was only detected in the embryonic shield. During gastrula stages, both Zfz8a and Zfz8b transcripts were found in anterior dorsal regions of the involuting mesendoderm (future prechordal plate). By the 2- to 3-somite stage, expression of both Zfz8a and Zfz8b was restricted to the prechordal plate and prospective anterior neurectoderm, although expression of the Zfz8a gene was no longer present in the most anterior portion of the prechordal plate, the polster. In one-eyed pinhead mutant embryos, which lack prechordal plate, both Zfz8a and Zfz8b transcripts were reduced, confirming the prechordal plate specificity of Zfz8a and Zfz8b gene expression. These results provide an additional evidence supporting the role of Wnt signaling in organizer-mediated axial patterning.

Amino Acid Sequence↗

PubMed2005

The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.

Certain aggressive melanoma cell lines exhibit a dedifferentiated phenotype, expressing genes that are characteristic of various cell types including endothelial, neural, and stem cells. Moreover, we have shown that aggressive melanoma cells can participate in neovascularization in vivo and vasculogenic mimicry in vitro, demonstrating that these cells respond to microenvironmental cues and manifest developmental plasticity. To explore this plasticity further, we transplanted human metastatic melanoma cells into zebrafish blastula-stage embryos and monitored their behavior post-transplantation. The data show that human metastatic melanoma cells placed in the zebrafish embryo survive, exhibit motility, and divide. The melanoma cells do not form tumors nor integrate into host organs, but instead become scattered throughout the embryo in interstitial spaces, reflecting the dedifferentiated state of the cancer cells. In contrast to the fate of melanoma cells, human melanocytes transplanted into zebrafish embryos most frequently become distributed to their normal microenvironment of the skin, revealing that the zebrafish embryo contains possible homing cues that can be interpreted by normal human cells. Finally, we show that within the zebrafish embryo, metastatic melanoma cells retain their dedifferentiated phenotype. These results demonstrate the utility of the zebrafish embryonic model for the study of tumor cell plasticity and suggest that this experimental paradigm can be a powerful one in which to investigate tumor-microenvironment interactions.

Animals↗

PubMed2001

Apoptosis in the developing zebrafish embryo.

Apoptosis is a major part of the normal development of many organ systems and tissues. The zebrafish (Danio rerio) has become a useful model for studying early development, and recent advances in techniques used to label apoptotic cells have made it possible to visualize apoptotic cells in this model system. We have used the in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to describe the temporal and spatial distribution of apoptotic cells during normal development of the zebrafish embryo from 12 to 96 h postfertilization. By counting labeled apoptotic cells, we have demonstrated transient high rates of cell death in various structures during development, and we have correlated these peaks with previously described developmental changes in these structures. Our analysis has focused on the nervous system and associated sensory organs including the olfactory organ, retina, lens, cornea, otic vesicle, lateral line organs, and Rohon-Beard neurons. Apoptosis is also described in other non-neural structures such as the notochord, somites, muscle, tailbud, and fins.

Animals↗

PubMed1998

beta-lactamase as a marker for gene expression in live zebrafish embryos.

In this report we describe the development of a sensitive assay for gene expression in zebrafish embryos using beta-lactamase as a reporter gene. We show that injection of a green fluorescent substrate for beta-lactamase allows the detection of reporter gene expression in live embryos. The beta-lactamase enzyme catalyzes the hydrolysis of the substrate, thereby disrupting fluorescence resonance energy transfer from the donor to the acceptor dye in the molecule. As a result, a blue fluorescent product is produced and retained specifically in cells within which the enzyme is expressed. beta-Lactamase is therefore suitable for monitoring spatially restricted patterns of gene expression in the early embryo. We suggest that this new reporter system provides a major advancement in sensitivity over the existing methods for monitoring gene expression in vivo during early embryogenesis.

Animals↗

PubMed2001

Tyrosinase gene expression in zebrafish embryos.

The enzyme tyrosinase is required for the conversion of tyrosine into the pigment melanin. Thus, tyrosinase gene expression is a useful marker for studying the differentiation of melanin-expressing cells during embryogenesis. We describe the spatiotemporal pattern of transcription of the tyrosinase gene and the presence of active enzyme in whole embryos of the zebrafish, Danio rerio. At 16.5 h post-fertilisation the tyrosinase gene is transcribed in the dorsal extremity of the developing retinal pigment epithelium, approximately 7 h before visible pigmentation. Shortly thereafter, transcription in neural crest-derived melanocytes is first observed dorsolateral to the mesencephalon and diencephalon and the posterior hindbrain/anterior spinal cord. A wave of gene activation and cell migration is then observed moving towards the posterior of the animal. DOPA staining for tyrosinase activity shows the presence of active enzyme in embryos at least 3 h before visible pigmentation.

Animals↗

PubMed2002

Expression of three spalt (sal) gene homologues in zebrafish embryos.

Three homologues of the Drosophilaregion-specific homeotic gene spalt (sal) have been isolated in zebrafish, sall1a, sall1b and sall3. Phylogenetic analysis of these genes against known salDNA sequences showed zebrafish sall1aand sall1b to be orthologous to other vertebrate sal-1 genes and zebrafish sall3to be orthologous to other vertebrate sal-3 genes, except Xenopus sall3. Phylogenetic reconstruction suggests that zebrafish sall1a and sall1bresulted from a gene duplication event occurring prior to the divergence of the ray-finned and lobe-finned fish lineages. Analysis of the expression pattern of the zebrafish sal genes shows that sall1a and sall3 share expression domains with both orthologous and non-orthologous vertebrate sal genes. Both are expressed in various regions of the CNS, including in primary motor neurons. Outside of the CNS, sall1a expression is observed in the otic vesicle (ear), heart and in a discrete region of the pronephric ducts. These analyses indicate that orthologies between zebrafish sal genes and other vertebrate sal genes do not imply equivalence of expression pattern and, therefore, that biological functions are not entirely conserved. However we suggest that, like other vertebrate sal genes, zebrafish sal genes have a role in neural development. Also, expression of zebrafish sall1a in the otic vesicle, heart sac and the pronephric ducts of zebrafish embryos is possibly consistent with some of the abnormalities seen in Sall1-deficient mice and in Townes-Brocks Syndrome, a human disorder which is caused by mutations in the human spalt gene SALL1.

Animals↗

PubMed2000

Development of spontaneous glycinergic currents in the Mauthner neuron of the zebrafish embryo.

We used whole cell and outside-out patch-clamp techniques with reticulospinal Mauthner neurons of zebrafish embryos to investigate the developmental changes in the properties of glycinergic synaptic currents in vivo from the onset of synaptogenesis. Miniature inhibitory postsynaptic currents (mIPSCs) were isolated and recorded in the presence of TTX (1 microM), kynurenic acid (1 mM), and bicuculline (10 microM) and were found to be sensitive to strychnine (1 microM). The mIPSCs were first observed in 26-29 h postfertilization (hpf) embryos at a very low frequency of approximately 0.04 Hz, which increased to approximately 0.5 Hz by 30-40 hpf, and was approximately 10 Hz in newly hatched (>50 hpf) larvae, indicating an accelerated increase in synaptic activity. At all embryonic stages, the amplitudes of the mIPSCs were variable but their means were similar ( approximately 100 pA), suggesting rapid formation of the postsynaptic matrix. The 20-80% rise times of mIPSCs in embryos were longer (0.6-1.2 ms) than in larvae (approximately 0.3 ms), likely due to slower diffusion of glycine at the younger, immature synapses. The mIPSCs decayed with biexponential (tau(off1) and tau(off2)) time courses with a half-width in 26-29 hpf embryos that was longer and more variable than in older embryos and larvae. In 26- to 29-hpf embryos, tau(off1) was approximately 15 ms and tau(off2) was approximately 60 ms, representing events of intermediate duration; but occasionally long mIPSCs were observed in some cells where tau(off1) was approximately 40 ms and tau(off2) was approximately 160 ms. In 30-40 hpf embryos, the events were faster, with tau(off1) approximately 9 ms and tau(off2) approximately 40 ms, and in larvae, events declined somewhat further to tau(off1) approximately 4 ms and tau(off2) approximately 30 ms. Point-per-point amplitude histograms of the decay of synaptic events at all stages resulted in the detection of similar single channel conductances estimated as approximately 45 pS, indicating the presence of heteromeric glycine receptors (GlyRs) from the onset of synaptogenesis. Fast-flow (1 ms) application of a saturating concentration of glycine (3-10 mM) to outside-out patches obtained at 26-29 hpf revealed GlyR currents that decayed biexponentially with time constants resembling the values found for intermediate and long mIPSCs; by 30-40 hpf, the GlyR currents resembled fast mIPSCs. These observations indicate that channel kinetics limited the mIPSC duration. Our data suggest that glycinergic mIPSCs result from the activation of a mixture of fast and slow GlyR subtypes, the properties and proportion of which determine the decay of the synaptic events in the embryos.

Animals↗