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At least 415 records · Page 23Linked to original sources

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.

Animals↗

A moving wave patterns the cone photoreceptor mosaic array in the zebrafish retina.

In this paper, we describe the embryonic origin and patterning of the planar mosaic array of cone photoreceptor spectral subtypes in the zebrafish retina. A discussion of possible molecular mechanisms that might generate the cone mosaic array considers but discards a model that accounts for formation of neuronal mosaics in the inner retina and discusses limitations of mathematical simulations that reproduce the zebrafish cone mosaic pattern. The formation and organization of photoreceptors in the ommatidia of the compound eye of Drosophila is compared with similar features in the developing zebrafish cone mosaic, and a model is proposed that invokes spatiotemporally coordinated cell-cell interactions among cone progenitors to determine the identity and positioning of cone spectral subtypes.

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Laser-induced gene expression in specific cells of transgenic zebrafish.

Over the past few years, a number of studies have described the generation of transgenic lines of zebrafish in which expression of reporters was driven by a variety of promoters. These lines opened up the real possibility that transgenics could be used to complement the genetic analysis of zebrafish development. Transgenic lines in which the expression of genes can be regulated both in space and time would be especially useful. Therefore, we have cloned the zebrafish promoter for the inducible hsp70 gene and made stable transgenic lines of zebrafish that express the reporter green fluorescent protein gene under the control of a hsp70 promoter. At normal temperatures, green fluorescent protein is not detectable in transgenic embryos with the exception of the lens, but is robustly expressed throughout the embryo following an increase in ambient temperature. Furthermore, we have taken advantage of the accessibility and optical clarity of the embryos to express green fluorescent protein in individual cells by focussing a sublethal laser microbeam onto them. The targeted cells appear to develop normally: cells migrate normally, neurons project axons that follow normal pathways, and progenitor cells divide and give rise to normal progeny cells. By generating other transgenic lines in which the hsp70 promoter regulates genes of interest, it should be possible to examine the in vivo activity of the gene products by laser-inducing specific cells to express them in zebrafish embryos. As a first test, we laser-induced single muscle cells to make zebrafish Sema3A1, a semaphorin that is repulsive for specific growth cones, in a hsp70-sema3A1 transgenic line of zebrafish and found that extension by the motor axons was retarded by the induced muscle.

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Sequence, expression, and location of zebrafish frizzled 10.

Members of the frizzled gene family encode seven-pass transmembrane proteins that function in the interpretation and reception of Wnt-mediated cell-cell communication events. To investigate frizzled function in early zebrafish development, we isolated the maternally contributed frizzled 10 (fz10) gene and localized it to linkage group 8 using radiation hybrid mapping. The cloned zebrafish fz10 is closely related to the fz10 group from other organisms. Zygotic expression of fz10 is observed in the posterior tail mesenchyme, dorsal neural tube, and different parts of the brain.

Amino Acid Sequence↗

Position-independent expression of transgenes in zebrafish.

The variability in expression patterns of transgenes, caused by the influence of neighboring chromatin, is called 'position effect'. Border elements are DNA sequences, which have the ability to alleviate position effects. The abilities of two types of border elements, scs/scs' from the D. melanogaster 87A7 heat shock locus and the A-element from the chicken lysozyme gene, to protect transgenes from position effects were quantified in developing zebrafish embryos. The transgenic construct used was FV3CAT, which consists of the carp beta-actin transcriptional regulatory region, the chloramphenicol acetyltransferase (CAT) gene and the 3'-untranslated region from the Chinook salmon growth hormone gene. FV3CAT constructs flanked by either scs/scs'-elements or A-elements were introduced into zebrafish chromosomes and the spatial and temporal expression patterns of the transgenes were quantified in multiple generations of transgenic zebrafish. Levels of transgene expression were uniform in the pre-differentiated and fully differentiated populations of cells present during embryonic development. Levels of transgene expression were proportional to the numbers of integrated transgenes. Expression of transgenes per cell varied less than two-fold in different transgenic lines. Both types of border elements were able to prevent the influences of neighboring chromatin on transgene expression through three generations of fish. The results are consistent with the ability of border elements to function with equal efficiencies in the many cell types found in vertebrates. Thus, inclusion of border elements in genetic constructs can provide reliable and reproducible levels of gene expression in multiple lines of fish.

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Vertebrate model systems in the study of early heart development: Xenopus and zebrafish.

Xenopus and zebrafish serve as outstanding models in which to study vertebrate heart development. The embryos are transparent, allowing observation during organogenesis; they can be obtained in large numbers; and they are readily accessible to embryologic manipulation and microinjection of RNA, DNA, or protein. These embryos can live by diffusion for several days, allowing analysis of mutants or experimental treatments that perturb normal heart development. Xenopus embryos have been used to understand the induction of the cardiac field, the role of Nkx genes in cardiac development, and the role transforming growth factor beta molecules in the establishment and signaling of left-right axis information. Large-scale mutant screens in zebrafish and the development of transgenics in both Xenopus and zebrafish have accelerated the molecular identification of genes that regulate conserved steps in cardiovascular development.

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Cloning and embryonic expression patterns of the zebrafish Runt domain genes, runxa and runxb.

We isolated zebrafish homologues of the Runt-related transcription factor gene family (Runx family), runxa and runxb, and analyzed their developmental expression patterns. The deduced amino acid sequence of Runxa was highly homologous to that of AML1 (also called CBFA2, PEBP2alphaB or Runx1), a critical regulator of mammalian hematopoiesis expressed in cells of the hematopoietic lineage as well as other tissues. During zebrafish development, the runxa gene was not expressed in hematopoietic tissues but in the olfactory placodes and cells attached to the otic vesicles. We identified three kinds of runxb transcripts, which encoded two types of proteins with different N-terminal regions. The Runxb proteins were highly similar to AML2 (CBFA3, PEBP2alphaC or Runx3). The expression sites of the shared region of runxb mRNAs during development were the trigeminal ganglions, dorsal neurons of the neural tube and the lateral line primordia. These findings show that expression patterns of the zebrafish Runx genes are distinct from that of the mammalian genes.

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Zebrafish angiogenesis: a new model for drug screening.

Angiogenesis is necessary for tumor growth, making inhibition of vessel formation an excellent target for cancer therapy. Current assays for angiogenesis, however, are too complex to be practical for drug screening. Here, we demonstrate that the zebrafish is a viable whole animal model for screening small molecules that affect blood vessel formation. Blood vessel patterning is highly characteristic in the developing zebrafish embryo and the subintestinal vessels (SIVs) can be stained and visualized microscopically as a primary screen for compounds that affect angiogenesis. Small molecules added directly to the fish culture media diffuse into the embryo and induce observable, dose-dependent effects. To evaluate the zebrafish as a model, we used two angiogenesis inhibitors, SU5416 and TNP470, both of which have been tested in mammalian systems. Both compounds caused a reduction in vessel formation when introduced to zebrafish embryos prior to the onset of angiogenesis. Short duration (1 h) exposure of SU5416 was sufficient to block new angiogenic and vasculogenic vessel formation. In contrast, TNP470 required continuous exposure to block SIV formation and had no apparent effect on vasculogenic vessel formation. To ascertain whether blood vessels in the zebrafish embryo respond to angiogenic compounds, we introduced human VEGF into embryos. Injection of VEGF caused an observable increase in SIV formation.

Journal Article↗

Zebrafish cyclin D1 is differentially expressed during early embryogenesis.

We have isolated and determined the nucleotide sequence of a cDNA containing the complete coding region of cyclin D1 from embryonic zebrafish cDNA library. The cyclin D1 gene is a single copy gene within the zebrafish genome, which undergoes an alternative polyadenylation process. The initial expression of cyclin D1 transcript occurs at the presumed onset of G1 phase in the developing zebrafish embryo.

Amino Acid Sequence↗

Differential expression of the methyl-cytosine binding protein 2 gene in embryonic and adult brain of zebrafish.

Epigenetic gene repression occurs as the result of the interactions between DNA and a number of proteins, including methyl-cytosine binding protein 2 (MeCP2). We have isolated a 1680 bps MeCP2 cDNA from zebrafish that shows deduced amino acid identity with Xenopus and mammalian MeCP2alpha protein sequences. The zebrafish MeCP2 gene was mapped to linkage group 8 using the LN54 radiation hybrid cell panel. The genomic organizations of the zebrafish MeCP2 and mammalian MeCP2alpha are highly similar. Relatively high levels of expression of MeCP2 mRNA were found in embryos at 1 to 4 h postfertilization (hpf), after 24 hpf, and in adult brain and eyes. Whole mount in situ hybridization was performed on embryos and revealed ubiquitous MeCP2 expression during early zebrafish development. At 24 and 48 hpf, the highest levels of expression are found in the epiphysis, midbrain, ventricular zone, and the otic vesicles. In adult zebrafish, MeCP2-expressing cells were found throughout the brain. Regions that are enriched in MeCP2 mRNA include the pallium layer of the telencephalon, the preoptic area, the periventricular grey zone, lobus caudalis, and the vagus lobes. In the cerebellum, high expression is found in the Purkinje and Golgi type 2 cells.

Amino Acid Sequence↗

Key role played by RhoA in the balance between planar and apico-basal cell divisions in the chick neuroepithelium.

The cell division axis determines the position of daughter cells and is therefore critical for cell fate. During vertebrate neurogenesis, most cell divisions take place within the plane of the neuroepithelium (Das, T., Payer, B., Cayouette, M., and Harris, W.A. (2003). In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina. Neuron 37, 597-609. Haydar, T.F., Ang, E., Jr., and Rakic, P. (2003). Mitotic spindle rotation and mode of cell division in the developing telencephalon. Proc Natl Acad Sci U S A 100, 2890-5. Kosodo, Y., Roper, K., Haubensak, W., Marzesco, A. M., Corbeil, D., and Huttner, W. B. (2004). Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells. EMBO J. 23, 2314-24). The cellular constraints responsible for this preferential orientation are poorly understood. Combining electroporation and time-lapse confocal imaging of chick neural progenitors, the events responsible for positioning the mitotic spindle and their dependence on RhoA were investigated. The results indicate that the spindle forms with a random orientation. However, the final orientation of cell divisions is dependent on two main factors: (i) an early rotation of the spindle that aligns it within the plane of the neuroepithelium, and (ii) a specific limitation of spindle oscillations, despite free rotation around the apico-basal axis. Expressing a dominant-negative RhoA leads to apico-basal cell divisions after a correct initial rotation of the spindle. Our data reveal a specific role for RhoA in the maintenance of spindle orientation, prior to anaphase. Thus, RhoA could be a key player potentially regulated by the neurogenic program or by the neural stem cell environment to control the balance between planar and apico-basal divisions, during normal or pathological development.

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Growth cone and dendrite dynamics in zebrafish embryos: early events in synaptogenesis imaged in vivo.

We used time-lapse fluorescence microscopy to observe the growth of Mauthner cell axons and their postsynaptic targets, the primary motor neurons, in spinal cords of developing zebrafish embryos. Upon reaching successive motor neurons, the Mauthner growth cone paused briefly before continuing along its path. Varicosities formed at regular intervals and were preferentially associated with the target regions of the primary motor neurons. In addition, the postsynaptic motor neurons showed highly dynamic filopodia, which transiently interacted with both the growth cone and the axon. Both Mauthner cell and motor neurons were highly active, each showing motility sufficient to initiate synaptogenesis.

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The effects of endogenous and exogenous nitric oxide on gut motility in zebrafish Danio rerio embryos and larvae.

Using motion analysis, the ontogeny of the nitrergic control system in the gut was studied in vivo in zebrafish Danio rerio embryos and larvae. For the first time we show the presence of a nitrergic tonus, modulating both anterograde and retrograde contraction waves in the intestine of developing zebrafish. At 4 d.p.f. (days post fertilisation), the nitric oxide synthase (NOS) inhibitor L-NAME (three boluses of 50-100 nl, 10(-3) mol l(-1)) increased the anterograde contraction wave frequency by 0.50+/-0.10 cycles min(-1). Subsequent application of the NO donor sodium nitroprusside (SNP; three boluses of 50-100 nl, 10(-4) mol l(-1)) reduced the frequency of propagating anterograde waves (-0.71+/-0.20 cycles min(-1)). This coincided with the first appearance of an excitatory cholinergic tonus, observed in an earlier study. One day later, at 5 d.p.f., in addition to the effect on anterograde contraction waves, application of L-NAME increased (0.39+/-0.15 cycles min(-1)) and following SNP application reduced (-1.61+/-0.36 cycles min(-1)) the retrograde contraction wave frequency. In contrast, at 3 d.p.f., when no spontaneous motility is observed, application of L-NAME did not induce contraction waves in either part of the gut, indicating the lack of a functional inhibitory tonus at this early stage. Gut neurons expressing NOS-like immunoreactivity were present in the distal and middle intestine as early as 2 d.p.f., and at 1 day later in the proximal intestine. In conclusion, the present study suggests that a nitrergic inhibitory tonus develops shortly before or at the time for onset of exogenous feeding.

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Two Na,K-ATPase beta 2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis.

We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase beta 2 subunit. The beta 2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish beta 2a subunit. By using a zebrafish meiotic mapping panel, we determined that the beta 2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the beta 2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. beta 2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, beta 2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the beta 2a and beta 2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian beta 2 gene may be partitioned between the two zebrafish beta 2 orthologs.

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Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo.

The head skeleton and muscles of the zebrafish develop in a stereotyped pattern in the embryo, including seven pharyngeal arches and a basicranium underlying the brain and sense organs. To investigate how individual cartilages and muscles are specified and organized within each head segment, we have examined their early differentiation using Alcian labeling of cartilage and expression of several molecular markers of muscle cells. Zebrafish larvae begin feeding by four days after fertilization, but cartilage and muscle precursors develop in the pharyngeal arches up to 2 days earlier. These chondroblasts and myoblasts lie close together within each segment and differentiate in synchrony, perhaps reflecting the interdependent nature of their patterning. Initially, cells within a segment condense and gradually become subdivided into individual dorsal and ventral structures of the differentiated arch. Cartilages or muscles in one segment show similar patterns of condensation and differentiation as their homologues in another, but vary in size and shape in the most anterior (mandibular and hyoid) and posterior (tooth-bearing) arches, possibly as a consequence of changes in the timing of their development. Our results reveal a segmental scaffold of early cartilage and muscle precursors and suggest that interactions between them coordinate their patterning in the embryo. These data provide a descriptive basis for genetic analyses of craniofacial patterning.

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Molecular cloning of zebrafish (Danio rerio) MutS homolog 6(MSH6) and noncoordinate expression of MSH6 gene activity and G-T mismatch binding proteins in zebrafish larvae.

Eukaryotic MutS homolog 6(MSH6) is a DNA mismatch recognition protein associated with mismatch repair of simple base-base mispairs and small insertion-deletion loops. As replication or recombination errors generated during embryonic development of living organisms should be efficiently corrected to maintain the integrity of genetic materials, we attempted to study MSH6 gene expression in developing zebrafish (Danio rerio) and the influence of MSH6 expression on the production of mismatch binding factors. A full-length cDNA encoding zebrafish MSH6 (zMSH6) was first obtained by rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of zMSH6 shares 57% and 56% identity with human and mouse MSH6, respectively. The 190-kDa recombinant zMSH6 containing 1,369 amino acids bound preferentially to a heteroduplex than to a homoduplex DNA. Northern blot and semiquantitative RT-PCR analysis detected apparent levels of zMSH6 mRNA expression in 12 and 36-hr-old zebrafish embryos, while this expression in 84-hr-old larvae was dramatically reduced to 23% of that in 12-hr-old embryos when beta-actin mRNA was constitutively synthesized. Incubation of G-T and G-G heteroduplex probes with 12 to 60-hr-old zebrafish extracts produced predominantly high-shifting binding complexes with very similar band intensity. Although low in zMSH6 mRNA production, the extracts of 84-hr-old larvae interacted significantly stronger than the embryonic extracts with both G-T and G-G mispairs, producing high and low-shifting complexes. Heteroduplex-recognition proteins in 108-hr-old larvae gave a similar pattern of mismatch binding. The intensities of G-T complexes produced by 84 and 108-hr-old zebrafish extracts were 2.5 to 3-fold higher than those of G-G complexes. Our data indicate that the production of efficient MSH6-independent binding factors, particularly G-T-specific recognition proteins, is upregulated in zebrafish at the larval stage when MSH6 gene activity is downregulated.

Amino Acid Sequence↗

Postembryonic development of the cranial lateral line canals and neuromasts in zebrafish.

The development of the cranial lateral line canals and neuromast organs are described in postembryonic zebrafish (0-80 days postfertilization). Cranial canal development commences several weeks after hatch, is initiated in the vicinity of individual neuromasts, and occurs in four discrete stages that are described histologically. Neuromasts remain in open canal grooves for several weeks during which they dramatically change shape and increase in size by adding hair cells at a rate one-tenth that in the zebrafish inner ear. Scanning electron microscopy demonstrates that neuromasts elongate perpendicular to the canal axis and the axis of hair cell polarization and that they lack a prominent nonsensory cell population surrounding the hair cells-features that make zebrafish neuromasts unusual among fishes. These results demand a reassessment of neuromast and lateral line canal diversity among fishes and highlight the utility of the lateral line system of postembryonic zebrafish for experimental and genetic studies of the development and growth of hair cell epithelia.

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