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PubMed2006

SMC3 knockdown triggers genomic instability and p53-dependent apoptosis in human and zebrafish cells.

BACKGROUND: The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated. RESULTS: Mammalian cells that are SMC3 deficient fail to expand in a clonal population. In order to shed light on the underlying mechanism, experiments were conducted in zebrafish embryos in which cell competence to undergo apoptosis is acquired at specific stages of development and affects tissue morphogenesis. Zebrafish Smc3 is 95% identical to the human protein, is maternally contributed, and is expressed ubiquitously at all developmental stages. Antisense-mediated loss of Smc3 function leads to increased apoptosis in Smc3 expressing cells of the developing tail and notocord causing morphological malformations. The apoptosis and the ensuing phenotype can be suppressed by injection of a p53-specific MO that blocks the generation of endogenous p53 protein. Results in human cells constitutively lacking p53 or BAX, confirmed that a p53-dependent pathway mediates apoptosis in SMC3-deficient cells. A population of aneuploid cells accumulated in zebrafish embryos following Smc3-knockdown whereas in human cells the transient downregulation of SMC3 level lead to the generation of cells with amplified centrosome number. CONCLUSION: Smc3 is required for normal embryonic development. Its deficiency affects the morphogenesis of tissues with high mitotic index by triggering an apoptotic cascade involving p53 and the downstream p53 target gene bax. Cells with low SMC3 level display centrosome abnormalities that can lead to or are the consequence of dysfunctional mitosis and/or aneuploidy. Collectively the data support the view that SMC3 deficiency affects chromosomal stability leading to the activation of p53-dependent mitotic checkpoint.

Amino Acid Sequence↗

PubMed2001

The homeobox protein Six3 interacts with the Groucho corepressor and acts as a transcriptional repressor in eye and forebrain formation.

Six3 is a vertebrate homeobox gene that is expressed in the anterior neural plate and eye anlage. We overexpressed a dominant transcriptional activator or repressor form of Six3 in zebrafish embryos to analyze their effect on eye and forebrain formation. RNA injection of the activator form of Six3 into zebrafish embryos caused reduction of the expression domains for rx2, pax2, and emx1 in the anterior neural plate, resulting in eye and forebrain hypoplasia. On the other hand, overexpression of the repressor form of Six3 or wild-type Six3 showed phenotypes opposite to those of the activator form. We found that Six3 has eh1-related motifs, motifs crucial for transcriptional repression function of Drosophila engrailed which plays a role in tethering the Groucho corepressor to the promoters. We isolated one of the zebrafish Groucho family genes, grg3, and demonstrated an interaction between Six3 and Grg3 using yeast two-hybrid analysis. Point-mutations in the eh1-related motifs in Six3 reduced both its eye and forebrain enlarging activities and its interaction with Grg3. These results strongly argue that Six3 functions as a Groucho-dependent repressor in eye and forebrain formation. Furthermore, zebrafish Six2 and Six4 also interacted with Grg3, implying a conserved function among the Six family proteins as transcriptional repressors.

Amino Acid Motifs↗

PubMed1989

Genetics and early development of zebrafish.

Zebrafish genes and development are being studied in a growing number of laboratories. Given that many other organisms are already being exploited by large numbers of investigators, and that our general knowledge about the zebrafish embryo and genome is at present rather sketchy, why should we now concern ourselves with how this tropical fish develops? Whereas the zebrafish embryo is similar in important ways to other vertebrate embryos, it is relatively simple and unusually accessible for both cellular and genetic analyses.

Animals↗

PubMed2004

Transcription factor Ap-2alpha is necessary for development of embryonic melanophores, autonomic neurons and pharyngeal skeleton in zebrafish.

The genes that control development of embryonic melanocytes are poorly defined. Although transcription factor Ap-2alpha is expressed in neural crest (NC) cells, its role in development of embryonic melanocytes and other neural crest derivatives is unclear because mouse Ap-2alpha mutants die before melanogenesis. We show that zebrafish embryos injected with morpholino antisense oligonucleotides complementary to ap-2alpha (ap-2alpha MO) complete early morphogenesis normally and have neural crest cells. Expression of c-kit, which encodes the receptor for the Steel ligand, is reduced in these embryos, and, similar to zebrafish c-kit mutant embryos, embryonic melanophores are reduced in number and migration. The effects of ap-2alpha MO injected into heterozygous and homozygous c-kit mutants support the notion that Ap-2alpha works through C-kit and additional target genes to mediate melanophore cell number and migration. In contrast to c-kit mutant embryos, in ap-2alpha MO-injected embryos, melanophores are small and under-pigmented, and unexpectedly, analysis of mosaic embryos suggests Ap-2alpha regulates melanophore differentiation through cell non-autonomous targets. In addition to melanophore phenotypes, we document reduction of other neural crest derivatives in ap-2alpha MO-injected embryos, including jaw cartilage, enteric neurons, and sympathetic neurons. These results reveal that Ap-2alpha regulates multiple steps of melanophore development, and is required for development of other neuronal and non-neuronal neural crest derivatives.

Amino Acid Sequence↗

PubMed1994

Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish.

The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector, originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.

Animals↗

PubMed2001

Delta-Notch signaling and lateral inhibition in zebrafish spinal cord development.

BACKGROUND: Vertebrate neural development requires precise coordination of cell proliferation and cell specification to guide orderly transition of mitotically active precursor cells into different types of post-mitotic neurons and glia. Lateral inhibition, mediated by the Delta-Notch signaling pathway, may provide a mechanism to regulate proliferation and specification in the vertebrate nervous system. We examined delta and notch gene expression in zebrafish embryos and tested the role of lateral inhibition in spinal cord patterning by ablating cells and genetically disrupting Delta-Notch signaling. RESULTS: Zebrafish embryos express multiple delta and notch genes throughout the developing nervous system. All or most proliferative precursors appeared to express notch genes whereas subsets of precursors and post-mitotic neurons expressed delta genes. When we ablated identified primary motor neurons soon after they were born, they were replaced, indicating that specified neurons laterally inhibit neighboring precursors. Mutation of a delta gene caused precursor cells of the trunk neural tube to cease dividing prematurely and develop as neurons. Additionally, mutant embryos had excess early specified neurons, with fates appropriate for their normal positions within the neural tube, and a concomitant deficit of late specified cells. CONCLUSIONS: Our results are consistent with the idea that zebrafish Delta proteins, expressed by newly specified neurons, promote Notch activity in neighboring precursors. This signaling is required to maintain a proliferative precursor population and generate late-born neurons and glia. Thus, Delta-Notch signaling may diversify vertebrate neural cell fates by coordinating cell cycle control and cell specification.

Animals↗

PubMed2006

Knockdown of nuclear Ca2+/calmodulin-dependent protein kinase phosphatase causes developmental abnormalities in zebrafish.

Nuclear Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP-N) is an enzyme that dephosphorylates and concomitantly downregulates multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) in vitro. However, the functional roles of this enzyme in vivo are not well understood. To investigate the biological significance of CaMKP-N during zebrafish embryogenesis, we cloned and characterized zebrafish CaMKP-N (zCaMKP-N). Based on the nucleotide sequences in the zebrafish whole genome shotgun database, we isolated a cDNA clone for zCaMKP-N, which encoded a protein of 633 amino acid residues. Transiently expressed full-length zCaMKP-N in mouse neuroblastoma, Neuro2a cells, was found to be localized in the nucleus. In contrast, the C-terminal truncated mutant lacking RKKRRLDVLPLRR (residues 575-587) had cytoplasmic staining, suggesting that the nuclear localization signal of zCaMKP-N exists in the C-terminal region. Ionomycin treatment of CaMKIV-transfected Neuro2a cells resulted in a marked increase in the phosphorylated form of CaMKIV. However, cotransfection with zCaMKP-N significantly decreased phospho-CaMKIV in ionomycin-stimulated cells. Whole mount in situ hybridization analysis of zebrafish embryos showed that zCaMKP-N is exclusively expressed in the head and neural tube regions. Gene knockdown of zCaMKP-N using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. A number of apoptotic cells were observed in brain and spinal cord of the abnormal embryos. These results suggest that zCaMKP-N plays a crucial role in the early development of zebrafish.

Amino Acid Sequence↗

PubMed2001

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.

Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.

Actins↗

PubMed2006

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio.

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K(m) value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Amino Acid Sequence↗

PubMedDate not supplied

Developmental chlorpyrifos effects on hatchling zebrafish swimming behavior.

Chlorpyrifos (CPF), a widely used organophosphate insecticide and potent acetylcholinesterase inhibitor, interferes with neurobehavioral development. Rat models have been key in demonstrating that developmental CPF exposure causes learning deficits and locomotor activity alterations, which persist into adulthood. Complementary nonmammalian models can be useful in determining the neurodevelopmental mechanisms underlying these persisting behavioral effects. Zebrafish (Danio rerio) with their clear chorion and extensive developmental information base provide an excellent model for assessment of molecular processes of toxicant-impacted neurodevelopment. We have developed methods for assessing spatial discrimination learning in adult zebrafish and have documented persisting effects of developmental CPF exposure on swimming activity and learning after low and high doses of CPF (10 and 100 ng/ml) administered to zebrafish embryos on Days 1-5 postfertilization (pf). In the current study, we developed methods for behavioral assessment of CPF exposure on swimming activity in newly hatched zebrafish. An equal area segmented annular grid (concentric circles divided into quadrants through the diameter) was made in a 16-mm diameter cylinder. The test area was placed on a heating device secured to an Olympus SZH10 dissecting scope stage. Zebrafish embryos were exposed to 10 ng/ml CPF, 100 ng/ml CPF, or vehicle control (25 microl/ml DMSO) (n=8-10/treatment group). Each treatment group was kept in a total volume of 25 ml of egg water (60 mg/ml Instant Ocean) including DMSO with or without CPF mixed to above dilutions in an incubator set at 28.5 degrees C. CPF dilutions or vehicle were changed daily with exposure ending on Day 5 pf. Testing of larval zebrafish was performed on Days 6 and 9 pf. The fish were placed in the test cylinder with 1.5 ml of egg H(2)O (28.5 degrees C). After a 2-min acclimation period, the swimming activity of the fish was measured for a 3-min testing session. The 100 ng/ml CPF dose caused significant slowing of swimming activity on Days 6 and 9 pf and had persisting effects of impairing spatial discrimination and decreasing response latency in adulthood. Developmental exposure to 10 ng/ml of CPF did not cause a significant change in locomotor activity during the period soon after hatching. CPF exposure during early development caused clear behavioral impairments detectable during the posthatching period. In a previous study, we found that early developmental CPF exposure caused behavioral alterations in zebrafish, which lasted throughout adulthood. The molecular mechanisms by which early developmental CPF exposure produces these behavioral impairments expressed in adulthood can now be studied in the zebrafish model.

Animals↗

PubMed2004

Sequence, linkage mapping and early developmental expression of the intestinal-type fatty acid-binding protein gene (fabp2) from zebrafish (Danio rerio).

The intestinal fatty acid-binding protein (I-FABP) shows binding specificity for long-chain fatty acids and is proposed to be involved in uptake of dietary fatty acids and their intracellular transport. We have determined the sequence of the gene encoding I-FABP in zebrafish. The zebrafish I-FABP gene contains four exons interrupted by three introns. Radiation hybrid mapping assigned the I-FABP gene to linkage group 1. A 924 bp sequence 5' upstream of the initiation codon in the I-FABP gene contained several putative cis-acting regulatory elements. In adult zebrafish, reverse transcription-polymerase chain reaction (RT-PCR) detected I-FABP mRNA in intestine, brain, liver, muscle and testis. Quantitative RT-PCR demonstrated that I-FABP mRNA was most abundant in intestine, followed by brain. I-FABP mRNA levels were very low in muscle, testis, heart, liver, skin and ovary. RT-PCR using total RNA extracted from zebrafish embryos detected I-FABP mRNA as early as 12 h post-fertilization. Whole-mount in situ hybridization to zebrafish embryos detected I-FABP mRNA in the yolk syncytial layer (YSL) at early somitogenesis. Later during embryonic development the I-FABP mRNA was detected in the intestinal bulb, liver and pancreas primordium. Expression in YSL, liver or pancreas has not been previously reported for fish or mammalian I-FABP genes and may be related to specific physiological differences between fishes and mammals.

Amino Acid Sequence↗

PubMed2003

Cloning and expression of zebrafish neuronal nicotinic acetylcholine receptors.

We propose to use the zebrafish (Danio rerio) as a vertebrate model to study the role of neuronal nicotinic acetylcholine receptors (nAChR) in development. As a first step toward using zebrafish as a model, we cloned three zebrafish cDNAs with a high degree of sequence similarity to nAChR beta3, alpha2 and alpha7 subunits expressed in other species. RT-PCR was used to show that the beta3 and alpha2 subunit RNAs were present in zebrafish embryos only 2-5hours post-fertilization (hpf) while alpha7 subunit RNA was not detected until 8hpf, supporting the differential regulation of nAChRs during development. In situ hybridization was used to localize zebrafish beta3, alpha2, and alpha7 RNA expression. nAChR binding techniques were used to detect the early expression of two high-affinity [3H]-epibatidine binding sites in 2 days post-fertilization (dpf) zebrafish embryos with IC(50) values of 28.6pM and 29.7nM and in 5dpf embryos with IC(50) values of 28.4pM and 8.9nM. These studies are consistent with the involvement of neuronal nAChRs in early zebrafish development.

Animals↗

PubMedDate not supplied

Chemical genetics: Drug screens in Zebrafish.

High throughput chemical genetic screens for compounds with specific biological activity in a whole organism are feasible using zebrafish embryos. At least two medium to large scale drug screens have been carried out to date, leading to the identification of compounds that disturb zebrafish development. Chemical genetics using zebrafish embryos may become an important step in the discovery of drugs and their targets.

Animals↗

PubMed2003

Suppression of lens growth by alphaA-crystallin promoter-driven expression of diphtheria toxin results in disruption of retinal cell organization in zebrafish.

In order to study lens-retina relationships during development, we cloned the zebrafish alphaA-crystallin cDNA and its promoter region. Using a 2.8-kb fragment of the zebrafish alphaA-crystallin promoter (z(alpha)Acry), we expressed the diphtheria toxin A fragment (DTA) in zebrafish embryos in a lens-specific manner. Injection of the z(alpha)Acry-DTA plasmid into eggs at the one-or two-cell stage resulted in the formation of small eyes, in which both lens and retina were reduced in size. In the DTA-expressing lenses, their fiber structure was disorganized, indicating that normal lens development had been abrogated. The neural retina also showed abnormal development, although this tissue did not express DTA. Lamination in the retina did not develop well, and molecular markers for the outer and inner plexiform layers were either abnormally expressed or absent. However, cell type-specific markers of ganglion and bipolar cells, as well as photoreceptors, were expressed in appropriate positions, indicating that initial differentiation of these retinal subpopulations occurred in the DTA-expressing embryos. Cell proliferation also proceeded normally in these embryos, although apoptosis was enhanced. These results suggest that the differentiated lens plays a critical role in the morphogenetic organization of retinal cells during eye development in zebrafish embryos.

Animals↗

PubMedDate not supplied

RecA-mediated, targeted mutagenesis in zebrafish.

We have evaluated the efficacy of RecA, a prokaryotic protein involved with homologous recombination, to direct site-specific mutagenesis in zebrafish embryos. For this we coinjected a vector containing a mutated enhanced green fluorescent protein (EGFP) gene plus 236-nucleotide corrective single-stranded DNAs coated with RecA into 1-cell zebrafish embryos. Twenty-hours after fertilization, about 5% to 20% of injected embryos showed EGFP expression in 1 or more cells when RecA-coated corrective DNAs were used, but not when RecA was omitted. Mutated EGFP genes with 1-bp insertions or deletions were inefficiently activated, whereas those with 7-bp insertions were activated about 4-fold more efficiently. RecA-coated template strand had a higher efficiency than its complementary strand in activation of EGFP expression. Prior irradiation of the embryos with UV light enhanced RecA-mediated restoration of gene activity, suggesting that the effects we observed were augmented by one or more factors of zebrafish DNA repair systems.

Animals↗

PubMed2006

Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival.

The Mycobacterium tuberculosis exported repetitive protein (Erp) is a virulence determinant required for growth in cultured macrophages and in vivo. To better understand the role of Erp in Mycobacterium pathogenesis, we generated a mutation in the erp homologue of Mycobacterium marinum, a close genetic relative of M. tuberculosis. erp-deficient M. marinum was growth attenuated in cultured macrophage monolayers and during chronic granulomatous infection of leopard frogs, suggesting that Erp function is similarly required for the virulence of both M. tuberculosis and M. marinum. To pinpoint the step in infection at which Erp is required, we utilized a zebrafish embryo infection model that allows M. marinum infections to be visualized in real-time, comparing the erp-deficient strain to a DeltaRD1 mutant whose stage of attenuation was previously characterized in zebrafish embryos. A detailed microscopic examination of infected embryos revealed that bacteria lacking Erp were compromised very early in infection, failing to grow and/or survive upon phagocytosis by host macrophages. In contrast, DeltaRD1 mutant bacteria grow normally in macrophages but fail to induce host macrophage aggregation and subsequent cell-to-cell spread. Consistent with these in vivo findings, erp-deficient but not RD1-deficient bacteria exhibited permeability defects in vitro, which may be responsible for their specific failure to survive in host macrophages.

Animals↗

PubMed2005

Identification and characterization of a second fibronectin gene in zebrafish.

Fibronectin (FN) is a highly conserved extracellular matrix protein that plays crucial roles in vertebrate embryogenesis. Previously, it was reported that zebrafish possess a single FN gene (fn1a). Here we report the presence of a second zebrafish FN gene (fn1b) that encodes a protein with a predicted primary structure that is similar to FNs identified in other vertebrates possessing 12 type I, 2 type II and 17 type III repeats including two alternative splice sites (EIIIA and EIIIB) and a variable region (V). Zebrafish FN1b exhibits 62.0% amino acid identity with zebrafish FN1a, 54% with human and 55% with Xenopus laevis FNs respectively. Employing RT-PCR analysis, we demonstrate that EIIIB- and V- isoforms are produced by alternative splicing of a single fn1b transcript. The FN1b EIIIA- isoform was not detected in zebrafish embryos or adult tissues nor were EIIIA, EIIIB or V region splice variants of fn1a found. FN1b mRNA was detected by RT-PCR in embryos at the gastrula-stage (8hpf) through 72hpf and in various adult tissues. EIIIB- and V+ are the predominant forms of FN1b present in the zebrafish embryo. Unlike FN1a, which is present at a relatively high amount at the embryonic stages before gastrulation, the FN1b isoforms are present at very low amounts at the early cleavage stage. The presence of multiple isoforms of FN1b along with a different pattern of expression compared to FN1a indicates that the two fn genes have separate roles in zebrafish development.

Alternative Splicing↗

PubMed2006

The transcription factor ZBP-89 controls generation of the hematopoietic lineage in zebrafish and mouse embryonic stem cells.

Hematopoietic development is closely linked to that of blood vessels and the two processes are regulated in large part by transcription factors that control cell fate decisions and cellular differentiation. Both blood and blood vessels derive from a common progenitor, termed the hemangioblast, but the factor(s) specifying the development and differentiation of this stem cell population into the hematopoietic and vascular lineages remain ill defined. Here, we report that knockdown of the Krüppel-like transcription factor ZBP-89 in zebrafish embryos results in a bloodless phenotype, caused by disruption of both primitive and definitive hematopoiesis, while leaving primary blood vessel formation intact. Injection of ZBP-89 mRNA into cloche zebrafish embryos, which lack both the hematopoietic and endothelial lineages, rescues hematopoiesis but not vasculogenesis. Injection of mRNA for Stem Cell Leukemia (SCL), a transcription factor that directs hemangioblast development into blood cell precursors, rescues the bloodless phenotype in ZBP-89 zebrafish morphants. Forced expression of ZBP-89 induces the expansion of hematopoietic progenitors in wild-type zebrafish and in mouse embryonic stem cell cultures but inhibits angiogenesis in vivo and in vitro. These findings establish a unique regulatory role for ZBP-89, positioned at the interface between early blood and blood vessel development.

Animals↗