Defects In Zebrafish Embryos Research Articles

Benzo[a]pyrene exposure influences the cardiac development and the expression of cardiovascular relative genes in zebrafish (Daniorerio) embryos

It is reported that the most abundant polycyclic aromatic hydrocarbons (PAHs) in weathered crude oils are cardiotoxic. However, the action mechanism of PAHs on vertebrate cardiovascular development and disease is unclear. In the present study, the cardiac morphology and functioning of zebrafish embryos exposed to benzo[a]pyrene [B(a)P], as a high-ring PAHs, for 72h were observed and determined. The results showed that B(a)P exposure resulted in cardiac developmental defects in zebrafish embryos. Significant changes in expression level of multiple genes potentially critical for regulating the B(a)P-induced cardiovascular developmental defects were also found. A gene network regulating cardiac development perturbed by B(a)P exposure was identified and established by computational analysis and employment of some databases. The information from the network could provide a clue for further mechanistic studies explaining molecular events regulating B(a)P-mediated cardiovascular defects and consequences.

Noninvasive Imaging of Ethanol‐Induced Developmental Defects in Zebrafish Embryos Using Optical Coherence Tomography

In this article, we report the use of optical coherence tomography for noninvasive cross-sectional real-time imaging of ethanol-induced developmental defects in zebrafish embryos larvae. For ethanol concentration of over 300 mM, developmental defects of eye (shrinkage and retinal abnormalities), malformation of the notochord and ataxia arising due to the toxic effects of ethanol were observed in OCT images from 3 days post fertilization onwards. The results suggest that OCT could be a valuable tool for noninvasive assessment of birth defects in small animal systems.

Ptenb Mediates Gastrulation Cell Movements via Cdc42/AKT1 in Zebrafish

Phosphatidylinositol 3-kinase (PI3 kinase) mediates gastrulation cell migration in zebrafish via its regulation of PIP2/PIP3 balance. Although PI3 kinase counter enzyme PTEN has also been reported to be essential for gastrulation, its role in zebrafish gastrulation has been controversial due to the lack of gastrulation defects in pten-null mutants. To clarify this issue, we knocked down a pten isoform, ptenb by using anti-sense morpholino oligos (MOs) in zebrafish embryos and found that ptenb MOs inhibit convergent extension by affecting cell motility and protrusion during gastrulation. The ptenb MO-induced convergence defect could be rescued by a PI3-kinase inhibitor, LY294002 and by overexpressing dominant negative Cdc42. Overexpression of human constitutively active akt1 showed similar convergent extension defects in zebrafish embryos. We also observed a clear enhancement of actin polymerization in ptenb morphants under cofocal microscopy and in actin polymerization assay. These results suggest that Ptenb by antagonizing PI3 kinase and its downstream Akt1 and Cdc42 to regulate actin polymerization that is critical for proper cell motility and migration control during gastrulation in zebrafish.

Noonan syndrome gain-of-function mutations inNRAScause zebrafish gastrulation defects

SUMMARYNoonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome.

Elevated glucose induces congenital heart defects by altering the expression of tbx5, tbx20, and has2 in developing zebrafish embryos

Maternal diabetes increases the risk of congenital heart defects in infants, and hyperglycemia acts as a major teratogen. Multiple steps of cardiac development, including endocardial cushion morphogenesis and development of neural crest cells, are challenged under elevated glucose conditions. However, the direct effect of hyperglycemia on embryo heart organogenesis remains to be investigated. Zebrafish embryos in different stages were exposed to D-glucose for 12 or 24 hr to determine the sensitive window during early heart development. In the subsequent study, 6 hr post-fertilization embryos were treated with either 25 mmol/liter D-glucose or L-glucose for 24 hr. The expression of genes was analyzed by whole-mount in situ hybridization. The highest incidence of cardiac malformations was found during 6-30 hpf exposure periods. After 24 hr exposure, D-glucose-treated embryos exhibited significant developmental delay and diverse cardiac malformations, but embryos exposed to L-glucose showed no apparent phenotype. Further investigation of the origin of heart defects showed that cardiac looping was affected earliest, while the specification of cardiac progenitors and heart tube assembly were complete. Moreover, the expression patterns of tbx5, tbx20, and has2 were altered in the defective hearts. Our data demonstrate that elevated glucose alone induces cardiac defects in zebrafish embryos by altering the expression pattern of tbx5, tbx20, and has2 in the heart. We also show the first evidence that cardiac looping is affected earliest during heart organogenesis. These research results are important for devising preventive and therapeutic strategies aimed at reducing the occurrence of congenital heart defects in diabetic pregnancy.

Dok-7 promotes slow muscle integrity as well as neuromuscular junction formation in a zebrafish model of congenital myasthenic syndromes

The small signalling adaptor protein Dok-7 has recently been reported as an essential protein of the neuromuscular junction (NMJ). Mutations resulting in partial loss of Dok-7 activity cause a distinct limb-girdle subtype of the inherited NMJ disorder congenital myasthenic syndromes (CMSs), whereas complete loss of Dok-7 results in a lethal phenotype in both mice and humans. Here we describe the zebrafish orthologue of Dok-7 and study its in vivo function. Dok-7 deficiency leads to motility defects in zebrafish embryos and larvae. The relative importance of Dok-7 at different stages of NMJ development varies; it is crucial for the earliest step, the formation of acetylcholine receptor (AChR) clusters in the middle of the muscle fibre prior to motor neuron contact. At later stages, presence of Dok-7 is not absolutely essential, as focal and non-focal synapses do form when Dok-7 expression is downregulated. These contacts however are smaller than in the wild-type zebrafish, reminiscent of the neuromuscular endplate pathology seen in patients with DOK7 mutations. Intriguingly, we also observed changes in slow muscle fibre arrangement; previously, Dok-7 has not been linked to functions other than postsynaptic AChR clustering. Our results suggest an additional role of Dok-7 in muscle. This role seems to be independent of the muscle-specific tyrosine kinase MuSK, the known binding partner of Dok-7 at the NMJ. Our findings in the zebrafish model contribute to a better understanding of the signalling pathways at the NMJ and the pathomechanisms of DOK7 CMSs.

Interaction with LC8 Is Required for Pak1 Nuclear Import and Is Indispensable for Zebrafish Development

Pak1 (p21 activated kinase 1) is a serine/threonine kinase implicated in regulation of cell motility and survival and in malignant transformation of mammary epithelial cells. In addition, the dynein light chain, LC8, has been described to cooperate with Pak1 in malignant transformation of breast cancer cells. Pak1 itself may aid breast cancer development by phosphorylating nuclear proteins, including estrogen receptor alpha. Recently, we showed that the LC8 binding site on Pak1 is adjacent to the nuclear localization sequence (NLS) required for Pak1 nuclear import. Here, we demonstrate that the LC8-Pak1 interaction is necessary for epidermal growth factor (EGF)-induced nuclear import of Pak1 in MCF-7 cells, and that this event is contingent upon LC8-mediated Pak1 dimerization. In contrast, Pak2, which lacks an LC8 binding site but contains a nuclear localization sequence identical to that in Pak1, remains cytoplasmic upon EGF stimulation of MCF-7 cells. Furthermore, we show that severe developmental defects in zebrafish embryos caused by morpholino injections targeting Pak are partially rescued by co-injection of wild-type human Pak1, but not by co-injection of mutant Pak1 mRNA disrupting either the LC8 binding or the NLS site. Collectively, these results suggest that LC8 facilitates nuclear import of Pak1 and that this function is indispensable during vertebrate development.

Ewing Sarcoma Fusion Protein EWSR1/FLI1 Interacts with EWSR1 Leading to Mitotic Defects in Zebrafish Embryos and Human Cell Lines

The mechanism whereby the fusion of EWSR1 with the ETS transcription factor FLI1 contributes to malignant transformation in Ewing sarcoma remains unclear. We show that injection of human or zebrafish EWSR1/FLI1 mRNA into developing zebrafish embryos leads to mitotic defects with multipolar and disorganized mitotic spindles. Expression of human EWSR1/FLI1 in HeLa cells also results in mitotic defects, along with mislocalization of Aurora kinase B, a key regulator of mitotic progression. Because these mitotic abnormalities mimic those observed with the knockdown of EWSR1 in zebrafish embryos and HeLa cells, we investigated whether EWSR1/FLI1 interacts with EWSR1 and interferes with its function. EWSR1 coimmunoprecipitates with EWSR1/FLI1, and overexpression of EWSR1 rescues the mitotic defects in EWSR1/FLI1-transfected HeLa cells. This interaction between EWSR1/FLI1 and EWSR1 in Ewing sarcoma may induce mitotic defects leading to genomic instability and subsequent malignant transformation.

Treatment with small interfering RNA affects the microRNA pathway and causes unspecific defects in zebrafish embryos

MicroRNAs (miRNAs) are generated from primary transcripts through sequential processing by two RNase III enzymes, Drosha and Dicer, in association with other proteins. This maturation is essential for their function as post-transcriptional regulators. Notably, Dicer is also a component of RNA-induced silencing complexes, which incorporate either miRNA or small interfering RNA (siRNA) as guides to target specific mRNAs. In zebrafish, processed miRNAs belonging to the miR-430 family have previously been shown to promote deadenylation and degradation of maternal mRNAs during early embryogenesis. We show that injection of one-cell-stage zebrafish embryos with siRNA causes a significant reduction in the endogenous levels of processed miR-430 and other miRNAs, leading to unspecific developmental defects. Coinjection of siRNA with preprocessed miR-430 efficiently rescued development. This indicates that the abnormalities generally observed in siRNA-treated zebrafish embryos could be due to inhibition of miR-430 processing and/or activity. Our results also suggest that the miRNA pathway in mammals, under some experimental or therapeutic conditions, may be affected by siRNA.

Tip‐1 induces filopodia growth and is important for gastrulation movements during zebrafish development

Wnt signaling is essential during animal development and also plays important roles in pathological conditions. Two mayor pathways have been described: the beta-catenin-dependent canonical (or classical) pathway and the beta-catenin-independent non-canonical Wnt pathway. Recent binding studies suggest links between the small PDZ protein TIP-1 (Tax-1 interacting protein) to components of both Wnt pathways. We have cloned and characterized the zebrafish tip-1 gene. Whole mount in situ hybridization and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) indicated that zebrafish tip-1 is present as a maternal RNA and is ubiquitously expressed during early development. After 24 h of development, tip-1 expression was high in the central nervous system (CNS) whereas only weak expression was detected in the caudal regions of the zebrafish embryo. Tip-1 knockdown using antisense morpholino oligonucleotides, as well as ectopic tip-1 expression, led to elongation defects in zebrafish embryos and larvae. Both knockdown and overexpression of tip-1 resulted in a widened goosecoid (gsc) expression domain in shield stage embryos, led to an abbreviated prechordal plate, and to reduced convergent extension movements during gastrulation. We constructed a green fluorescence protein (GFP)/TIP-1 fusion protein which, when expressed in cultured fibroblasts (ZF4-cells), induced filopodia growth. Our observations indicate a role for TIP-1 in gastrulation movements and in filopodia growth induction.

Mycophenolic Acid Is a Potent Inhibitor of Angiogenesis

Angiogenesis is necessary for the vascularization of a tumor, providing essential nourishment for tumor growth, and the progression and metastasis of cancer cells.1 Although tremendous efforts and resources have been dedicated to discovering angiogenesis inhibitors, very few candidates have been approved by FDA for therapeutic applications. Currently, Avastin (bevacizumab), a humanized monoclonal antibody, and Macugen (Pegaptanib), a pegylated oligonucleotide aptamer, both directed against vascular endothelial growth factor (VEGF) pathway, are two of the approved drugs for clinical use. There is still a great need to develop small molecule drugs for antiangiogenesis purpose. One approach to broaden the discovery platform is to identify antiangiogenic activities from existing drugs that have well defined toxicity and pharmacokinetics. Once a known drug is demonstrated to inhibit angiogenesis, it would move into the clinic trails more rapidly. Because most known drugs have well defined structures and defined biological targets, it is also possible to further improve the compound structure or select the target as a new entry point for angiogenesis-based studies and therapies. Currently there are more than 2000 known drugs that can be used for re-purposing applications. It would be desirable to have a single in vivo assay allowing rapid screening of a large number of compounds for new activities such as angiogenesis inhibition. Recently, the zebrafish has been shown to be a useful model organism for this type of approach.2,3 Zebrafish blood vessels form by angiogenic sprouting and appear to use the same pathways necessary for blood vessel growth in mammals.4 Using transgenic embryos expressing GFP in vessels, the embryonic blood vessels in live zebrafish can be directly …

The Developmental Neurotoxicity of Fipronil: Notochord Degeneration and Locomotor Defects in Zebrafish Embryos and Larvae

Fipronil is a phenylpyrazole insecticide designed to selectively inhibit insect gamma-aminobutyric acid (GABA) receptors. Although fipronil is often used in or near aquatic environments, few studies have assessed the effects of this neurotoxicant on aquatic vertebrates at sensitive life stages. We explored the toxicological effects of fipronil on embryos and larvae using the zebrafish (Danio rerio) experimental model system. Embryos exposed to fipronil at nominal concentrations at or above 0.7 microM (333 mug/l) displayed notochord degeneration, shortening along the rostral-caudal body axis, and ineffective tail flips and uncoordinated muscle contractions along the body axis in response to touch. This phenotype closely resembles zebrafish locomotor mutants of the accordion class and is consistent with loss of reciprocal inhibitory neurotransmission by glycinergic commissural interneurons in the spinal cord. Consistent with the hypothesis that notochord degeneration may be due to abnormal mechanical stress from muscle tetany, the expression patterns of gene and protein markers specific to notochord development were unaffected by fipronil. Moreover, the degenerative effects of fipronil (1.1 microM) were reversed by coexposure to the sodium channel blocker MS-222 (0.6mM). The notochord effects of fipronil were phenocopied by exposure to 70 microM strychnine, a glycinergic receptor antagonist. In contrast, exposure to gabazine, a potent vertebrate GABA(A) antagonist, resulted in a hyperactive touch response but did not cause notochord degeneration. Although specifically developed to target insect GABA receptors with low vertebrate toxicity, our results suggest that fipronil impairs the development of spinal locomotor pathways in fish by inhibiting a structurally related glycine receptor subtype. This represents an unanticipated and potentially novel mechanism for fipronil toxicity in vertebrates.

Ciliary Signaling

Most vertebrate cells have a primary cilium. Recent evidence indicates that, unlike the cilia of unicellular organisms that confer motility, those on vertebrate cells may be waving about in the extracellular environment picking up signals. In mice, mutations that prevent cilia formation produce developmental defects similar to those in animals with disrupted Hedgehog signaling. Corbit et al. now report that Smoothened proteins (Smo), which are receptors for the morphogen Hedgehog, could be detected on cells of the ventral node of three-somite-stage mouse embryos, especially on the primary cilium. Such localization of Smo was enhanced in various cultured cells treated with an activating ligand for Smo and was lost in cells treated with a Smo antagonist. The authors used mutants lacking key residues in a motif required for ciliary localization to show that activation of a reporter gene in cells transfected with the receptor required localization of Smo to the cilium. Similarly, such mutant Smo molecules were unable to rescue morphological defects in Zebrafish embryos lacking endogenous Smo. Precisely why Smo needs to be at the cilium to function remains to be explored. The authors suggest that it may need to be there to interact with an activator or that signaling complexes formed with other components of the Hedgehog signaling pathway may also accumulate at the cilium. K. C. Corbit, P. Aanstad, V. Singla, A. R. Norman, D. Y. R. Stainier, J. F. Reiter, Vertebrate Smoothened functions at the primary cilium. Nature 437 , 1018-1021 (2005). [PubMed]

A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline

genesisVolume 30, Issue 3 p. 183-185 ArticleFree Access A zebrafish vasa morphant abolishes Vasa protein but does not affect the establishment of the germline Arie K. Braat, Arie K. Braat Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsSearch for more papers by this authorSandra van de Water, Sandra van de Water Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsSearch for more papers by this authorJeroen Korving, Jeroen Korving Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsSearch for more papers by this authorDanica Zivkovic, Corresponding Author Danica Zivkovic [email protected] Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsHubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The NetherlandsSearch for more papers by this author Arie K. Braat, Arie K. Braat Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsSearch for more papers by this authorSandra van de Water, Sandra van de Water Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsSearch for more papers by this authorJeroen Korving, Jeroen Korving Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsSearch for more papers by this authorDanica Zivkovic, Corresponding Author Danica Zivkovic [email protected] Hubrecht Laboratory, The Netherlands Institute for Developmental Biology, Utrecht, The NetherlandsHubrecht Laboratory, The Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The NetherlandsSearch for more papers by this author First published: 23 July 2001 https://doi.org/10.1002/gene.1060Citations: 40AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. LITERATURE CITED Braat AK, Water van de S, Goos H, Bogerd J, Zivkovic D. 2000. Vasa protein expression and localization in the zebrafish. Mech Dev 95: 271– 274. Braat AK, Zandbergen T, Water van de S, Goos HJTh, Zivkovic D. 1999. Characterization of zebrafish primordial germ cells: Morphology and early distribution of vasa RNA. Dev Dyn 216: 153– 167. Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. 2000. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 408: 325– 330. Gruidl ME, Smith PA, Kuznicki KA, McCrone JS, Kirchner J, Roussell DL, Bennett K. 1996. Multiple potential germ-line helicases are components of the germ-line specific P granules of Caenorhabditis elegans. Proc Natl Acad Sci USA 93: 13837– 13842. Hay B, Ackerman L, Barbel S, Jan LY, Jan YN. 1988a. Identification of a component of Drosophila polar granules. Development 103: 625– 640. Hay B, Jan LY, Jan YN. 1988b. A protein component of Drosophila polar granules is encoded by vasa and has extensive sequence similarity to ATP-dependent helicases. Cell 55: 577– 587. Ikenishi K, Tanaka TS. 1997. Involvement of the protein of Xenopus vasa homolog (Xenopus vasa-like gene 1, XVLG1) in the differentiation of primordial germ cells. Dev Growth Differ 39: 625– 633. Knaut H, Pelegri F, Bohmann K, Schwarz H, Nusslein-Volhard C. 2000. Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification. J Cell Biol 149: 875– 888. Kuznicki KA, Smith PA, Leung-Chiu WMA, Estevez AO, Scott HC, Bennett KL. 2000. Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans. Development 127: 2907– 2916. Lasko PF, Ashburner M. 1988. 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Citing Literature Volume30, Issue3July 2001Pages 183-185 ReferencesRelatedInformation

Double–Stranded RNA Induces Specific Developmental Defects in Zebrafish Embryos

Treatment with double-stranded RNA (dsRNA) has been shown to interfere with the function of specific genes in various invertebrate species. However, it has not yet been reported that this technique can be applied to vertebrates as well. We have investigated whether dsRNA treatment will inhibit gene function in zebrafish embryos. By microinjecting dsRNA corresponding to three genetically characterised genes we produced embryonic defects that were similar to the known mutant phenotypes of these loci. The efficiency of inducing specific defects (20–30%) was about 10-fold higher than in experiments with antisense RNA. We also observed that the level of the endogenous mRNA in zebrafish embryos was substantially reduced throughout the embryo following dsRNA injection. However, the interference of gene function showed a strong dependence on the amount of dsRNA. These findings suggest that dsRNA-mediated interference will become an important tool for analysing the functional roles of genes in zebrafish and other vertebrates.

Heat shock produces periodic somitic disturbances in the zebrafish embryo

Environmental influences are known to produce segmental defects in a variety of organisms. In this paper we report upon segmental aberrations produced by brief heat shocks delivered to developing zebrafish embryos. The initial defects in the segmental pattern of somitic boundaries and motoneuron axon outgrowth were usually observed five somites caudal to the somite which was forming at the time of heat shock application. Segmental defects in zebrafish embryos exposed to a single heat shock treatment can occur in a periodic pattern similar to the multiple disturbances observed to occur in chick embryos. These data are discussed with regard to models involving cell cycle synchrony or ‘clock and wavefront’ schemes in the process of somitogenesis.