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PubMed1998

Calcium transients accompany ooplasmic segregation in zebrafish embryos.

Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (KD = 1.5 micromol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: > or = 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (KD = 0.15 micromol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.

Aequorin↗
PubMed2001

Brain-derived neurotrophic factor and TrkB tyrosine kinase receptor gene expression in zebrafish embryo and larva.

The genes that encode the neurotrophin family of secreted polypeptides and the Trk family of high affinity neurotrophin transmembrane protein tyrosine kinase receptors are induced at the time of neurogenesis in mammals and are known to play critical roles in nervous system development. We show here that in contrast to mammals, the genes encoding the neurotrophin brain-derived neurotrophic factor (BDNF) and the neurotrophin receptor TrkB are expressed throughout embryonic development in the zebrafish. At the embryonic stages preceding transcription of endogenous genes all cells contain BDNF transcripts and immunoreactive BDNF and the trkB transcripts lack the region that encodes a kinase domain. As development proceeds, progressively fewer cells contain BDNF transcripts and by the time of neurogenesis the trkB transcripts encode a kinase-domain. In the 4-day-old larva, a small subset of specialized sensory cells on the surface and cells in deeper structures including the gill arches, fin, and cloaca express the BDNF gene at high levels in a promoter-specific fashion. This progressive restriction of BDNF gene expression must involve an extinction of BDNF gene transcription in some and induction of high levels of transcription in a promoter-specific fashion in other cells.

Aging↗
PubMed2005

Assessing water quality in a tropical lake using biomarkers in zebrafish embryos: developmental toxicity and stress protein responses.

In order to achieve a more substantial appraisal of lake water quality, the assessment must not be based only on chemical measurements and analyses of the water itself, but even more so on the impact of existing conditions on aquatic biota. This is possible with the use of biotests or biomarkers, e.g. investigations of the developmental parameters (96-h embryotoxicity evaluate) or of the induction of heat shock proteins (proteotoxicity evaluate). To evaluate the suitability of these tests for environmental screening, fertilized zebrafish eggs were exposed to water samples collected from five sites of varying levels of stress from Laguna Lake, Philippines. Reconstituted water was used as laboratory control while water samples from a highly polluted freshwater source was used as positive control. Developmental parameters were noted and described within 48 and 96 h of exposure. Dilution experiments of the positive control were also done to further assess and compare toxicity potentials of Laguna Lake waters with those originating from a polluted freshwater. After the 96-h exposure, the levels of stress proteins (hsp 70) were determined in embryos from all exposure groups. Results showed 100% mortality in embryos exposed to undiluted positive control (PC) within only 12 h. Increasing dilution levels, however, resulted in lower mortality and lower abnormality rates. No detectable developmental differences were noted among embryos exposed to either the laboratory control or Laguna Lake waters at the end of 96 h, regardless of the source. Very high survival rates and high hatching success rates were observed in embryos exposed to lake waters as well as laboratory control, and the data did not differ significantly among the groups. Likewise, no significant malformations were noted among all developing embryos throughout the exposure period. However, the levels of heat shock proteins in the two sites located closest to Manila, the Philippine capital (Northern West Bay and Central West Bay), showed a pronounced elevation relative to the control, indicating that these stress proteins protect the embryos from the detrimental effects of pollutants in the water. Based on the 96-h early life stage (ELS) test, the water quality of the lake is good for fishery propagation despite the current levels of pollutants in the water. This finding is in accordance with the Class C status (i.e. suitable for fish growth and propagation) as given to the lake by the local environmental agency. On the other hand, data on proteotoxicity showed that the fish are under stress, presumably deriving from pollutants. This calls for a continuous monitoring and improvement of the lake water. The present study indicates that the two biomarker methods are very easy to use, practical, rapid, and sensitive for assessing water quality in a tropical lake and recommends for their incorporation into the future monitoring program of Laguna Lake.

Animals↗
PubMed2006

Fluorescent protein expression driven by her4 regulatory elements reveals the spatiotemporal pattern of Notch signaling in the nervous system of zebrafish embryos.

Notch activation inhibits neuronal differentiation during development of the nervous system; however, the dynamic role of Notch signaling in individual cell lineages remains poorly understood. We have characterized 3.4 kb 5'-regulatory sequence of a Notch target gene, her4, and used it to drive fluorescent gene expression in transgenic lines where the spatiotemporal pattern of Notch activation can be examined in vivo. The 3.4 kb her4 promoter contains five predicted Su(H) binding sites of which two proximal sites were confirmed to be required for Notch-mediated transcriptional activation. Without Notch, Su(H) effectively represses transcription regulated by the promoter. Analyses of transgenic zebrafish showed that while the expression of proneural genes and Notch activation were both critical for endogenous her4 expression, reporter gene expression was primarily regulated by Notch activity. This study also showed that her4 may be differently regulated in sensory cranial ganglia, where Notch activity is not essential for her4 expression and where Su(H) may repress her4 expression. The establishment of a reporter line with Notch-Su(H)-dependent fluorescent gene expression provides a tool to explore the complex role of Notch signaling in the development of vertebrate nervous system.

Animals↗
PubMedDate not supplied

Cell lineages generating axial muscle in the zebrafish embryo.

Cell lineage may contribute to determining the numbers, positions and types of cells formed during embryogenesis. In vitro clonal analyses show that vertebrate cells can autonomously maintain lineage commitments to single fates and that terminal development may include an invariant sequence of cell divisions. In addition, in vivo studies with Xenopus led to the proposal that clonal restrictions to spatial 'compartmental' domains arise during early development, analogous to what is observed in insects. In the zebrafish, individual gastrula cells generate clones of progeny that are confined within single tissues, but spatial restrictions have not been described. We now have examined the in vivo terminal cell lineages of zebrafish axial muscles. We obtained no evidence either for strict developmental regulation of division pattern or for spatial compartmentation within muscle lineages.

Animals↗
PubMed2003

The expression of novel membrane-type matrix metalloproteinase isoforms is required for normal development of zebrafish embryos.

Matrix metalloproteinases (MMPs) play important roles in the turnover of components of extracellular matrix (ECM) and in the processing of active and latent-signaling molecules bound to the ECM or associated with the cell surface. Through such actions, MMPs regulate a variety of cellular and developmental processes. Membrane-type matrix metalloproteinases (MT-MMPs) are of particular importance because they function in the immediate pericellular environment that modulates both cell-cell and cell-ECM interactions. In this study, we utilized zebrafish as a developmental model to study the role of MT-MMPs during early embryogenesis. We successfully isolated two isoforms of a MT-MMP homologue that are structurally similar to MT1-MMP. They have been named zebrafish MT-MMPalpha and beta. Zebrafish MT-MMPbeta is unique among vertebrate MT-MMPs in that it contains an Arg-Glu-Asp (RED) multiple-repeat motif in its linker region. Whole mount in situ analysis, RT-PCR, immunofluorescence, reporter analysis, Western blot analysis, and zymography indicated that MT-MMPalpha and beta were expressed through at least the first 72 h of development and that this expression was targeted to the cell surface. Functional studies using injection of either mRNA or morpholino antisense oligonucleotides resulted in a truncation of the cranial to caudal axis as monitored through 72 h post fertilization, indicating that zebrafish MT-MMPalpha and beta had an important role in embryonic development. Axis markers indicated that these effects likely involved processes occurring later than 10 h of embryogenesis.

Amino Acid Sequence↗
PubMed2005

Loss of gata1 but not gata2 converts erythropoiesis to myelopoiesis in zebrafish embryos.

The differentiation of hematopoietic progenitors into erythroid or myeloid cell lineages is thought to depend upon relative levels of the transcription factors gata1 and pu.1. While loss-of-function analysis shows that gata1 is necessary for terminal erythroid differentiation, no study has demonstrated that loss of gata1 alters myeloid differentiation during ontogeny. Here we provide in vivo evidence that loss of Gata1, but not Gata2, transforms primitive blood precursors into myeloid cells, resulting in a massive expansion of granulocytic neutrophils and macrophages at the expense of red blood cells. In addition to this fate change, expression of many erythroid genes was found to be differentially dependent on Gata1 alone, on both Gata1 and Gata2, or independent of both Gata factors, suggesting that multiple pathways regulate erythroid gene expression. Our studies establish a transcriptional hierarchy of Gata factor dependence during hematopoiesis and demonstrate that gata1 plays an integral role in directing myelo-erythroid lineage fate decisions during embryogenesis.

Animals↗
PubMed1999

Gefiltin in zebrafish embryos: sequential gene expression of two neurofilament proteins in retinal ganglion cells.

Neurogenesis is correlated with the progressive synthesis of diverse neuronal intermediate filaments (IF) proteins. This apparent developmental regulation of IF protein gene expression suggests that specific neurofilament proteins impart unique structural attributes that support the staged growth of the neuron. In the teleost visual pathway, the sequential expression of two IF genes, plasticin and gefiltin, is linked to the age of retinal ganglion cells (RGCs) and to the regeneration of optic axons after nerve injury. Given this pattern of plasticin and gefiltin expression, we hypothesized that the two proteins would be sequentially expressed in zebrafish retina during development. We analyzed the pattern of gefiltin expression during zebrafish development and compared it to our previous determination of plasticin expression (Canger et al. 1998). Gefiltin is expressed after plasticin, during the later stages of retinal development when axons grow past the optic chiasm and innervate their targets. Thus, during RGC development, expression of plasticin and gefiltin resembles that with optic nerve regeneration. Outside of the visual pathway, gefiltin is predominantly expressed in the central nervous system whereas plasticin is primarily expressed in the peripheral nervous system. These results suggest that the expression of these genes is regulated in a neuron-specific manner. In addition, since plasticin and gefiltin are co-expressed during RGC development, these findings suggest a more complex mechanism of transcriptional regulation which orchestrates the sequential expression of these genes.

Animals↗
PubMed1997

Localized calcium transients accompany furrow positioning, propagation, and deepening during the early cleavage period of zebrafish embryos.

Through the injection of f-aequorin (a calcium-specific luminescent reporter) and the use of an imaging photon detector, we see a distinct localized elevation of intracellular calcium that accompanies the appearance of the first furrow arc at the blastodisc surface: the furrow positioning signal. As the leading edges of the arc progress outward toward the margins of the blastodisc, they are accompanied by two subsurface slow calcium waves moving at about 0.2 micron/s: the furrow propagation signal. As these wave fronts approach the edge of the blastodisc, another calcium signal arises in the central region where the positioning signal originally appeared. Like the propagation signal, it extends outward to the margins of the blastodisc, but in this case it also moves downward, accompanying the deepening process that separates the daughter cells: the furrow deepening signal. Both of these furrow deepening progressions move at around 0.1 to 0.2 micron/s. The deepening signal begins to diminish from the center outward, returning to precleavage resting levels on completion of cytokinesis. The signaling sequence is repeated during the second cell division cycle. These localized transients do not require external calcium and they can be dissipated after they have begun by introducing calcium shuttle buffers, resulting in furrow delocalization and regression. They also occur in parthenogenetically activated eggs in which, in an attenuated form, they accompany abortive cleavages.

Aequorin↗
PubMed1999

Conservation of intracellular Wnt signaling components in dorsal-ventral axis formation in zebrafish.

The mechanism of early dorso-ventral axis specification in zebrafish embryos is not well understood. While beta-catenin has been clearly implicated as a determinant of the axis, the factors upstream and downstream of beta-catenin in this system are not defined. Unlike in Xenopus, where a sperm-induced cortical rotation is used to localize beta-catenin on the future dorsal side of the embryo, zebrafish do not have an obviously similar morphogenetic movement. Recently, a GSK-3 (Glycogen Synthase Kinase-3) binding protein (GBP) was identified as a novel member of the Wnt pathway required for maternal dorsal axis formation in Xenopus. GBP stabilizes beta-catenin levels by inhibiting GSK-3 and potentially provides a link between cortical rotation and beta-catenin regulation. Since zebrafish may use a different mechanism for regulating beta-catenin, we asked whether zebrafish also express a maternal GBP. We report the isolation of the zebrafish GBP gene and show that it is maternally expressed and is present as mRNA ubiquitously throughout early embryonic development. Over-expression of zebrafish GBP in frogs and fish leads to hyper-dorsalized phenotypes, similar to the effects resulting from over-expression of beta-catenin, indicating that components upstream of beta-catenin are conserved between amphibians and teleosts. We also examined whether Tcf (T cell factor) functions in zebrafish embryos. As in frogs, ectopic expression of a dominant negative form of XTcf-3 ventralizes zebrafish embryos. In addition, ectopic beta-catenin expression activates the promoter of the Tcf-dependent gene siamois, indicating that the step immediately downstream of beta-catenin is also conserved between fish and frogs.

Amino Acid Sequence↗
PubMed2000

Uptake, toxicity, and effects on detoxication enzymes of atrazine and trifluoroacetate in embryos of zebrafish.

The uptake, toxicity, and elimination of atrazine and trifluoroacetate (TFA) were studied in early life stages of the zebrafish (Danio rerio). Furthermore, the effects of these xenobiotics on soluble (s) and microsomal (m) glutathione S-transferases (GST) of zebrafish embryos were investigated using 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), and [(14)C]atrazine. [(14)C]Atrazine was taken up by the embryos within seconds, unhindered by the chorions. It accumulated in the embryos by a factor of 19 after 24 h of exposure time. LC(50) (48 h) was determined at 36.8 mg/L. At a level of 5 mg/L atrazine, activities of s and m GSTs were elevated in most stages, especially in prim 6 and long pec stage (24, 48 h after fertilization, respectively). GST activity toward atrazine was detectable only in untreated D. rerio eggs, increasing with developmental time. [(14)C]Atrazine was eliminated from the embryos between 24 and 48 h, indicating a possible metabolism to a more hydrophilic GSH conjugate. [(14)C]TFA was taken up by embryos, reaching at maximum fivefold the concentration of the incubation medium after 10 h. The chorions served no physiological protection. TFA (1 g/L) caused low elevation of the GST activity. No acute toxic effects (48 h) were observed up to 4 g/L TFA.

Animals↗