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PubMed2003

Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening.

An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approach enabled the rapid isolation of the target proteins by facile affinity matrix preparation and elucidated the first small-molecule inhibitors for several ribosomal accessory proteins or their complex as the target.

Animals↗

PubMed1999

Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo.

Adult pancreatic islets comprise four cell types, alpha, beta, delta and PP, expressing glucagon, insulin, somatostatin and pancreatic-polypeptide, respectively, arising from cell lineages whose relationships during endocrine pancreas differentiation are still uncertain [Edlund, 1998. Diabetes 47, 1817-1823]. As zebrafish (Danio rerio) represents an attractive vertebrate model to study mutants affecting pancreatic organogenesis [Pack et al., 1996. Development 123, 321-328], we have investigated the expression patterns of islet hormones in zebrafish embryos, from the 16-somite (17 h) to 48-h stages, by whole-mount in situ hybridization and immunofluorescence. Results showed that in the zebrafish pancreatic primordium (a) insulin is the first hormone gene to be expressed, and (b) somatostatin colocalizes with insulin while glucagon-expressing cells, since their appearance, are distinct from insulin- or insulin/somatostatin-expressing cells. Notably, both somatostatin and glucagon, but not insulin, are first expressed in extrapancreatic regions.

Animals↗

PubMed2002

Cadmium-induced ectopic apoptosis in zebrafish embryos.

In this study, we tested the hypothesis that cadmium-induced developmental toxicity was mediated via ectopic occurrence of apoptosis during embryonic development. We employed confocal microscopy to acquire images of whole-mount staining of apoptotic cells in zebrafish embryo exposed to 100 micro M cadmium from 5 hours post fertilisation (hpf) to 28 hpf. Three-dimensional reconstruction of the images was performed and the spatial and temporal distributions of apoptotic cells in the embryos were compared. In cadmium-treated embryos with varying degrees of gross developmental malformations, significantly higher numbers of apoptotic cells were detected with this method. In order to detect the precise locations of apoptotic cells, we performed terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay in sectioned embryos. In the degenerating neural tube of cadmium-treated embryos apoptotic cells were detected, while in the healthy neural tube of the untreated controls no apoptotic cells were found. We then employed flow cytometry to investigate whether cadmium exposure would affect the dynamics of apoptosis or induce any abnormalities in cell-cycle progression. It appeared that cadmium did not induce cell-cycle arrest. The percentages of apoptotic cells did not differ in the two groups at 13, 16 or 19 hpf. At 28 hpf, however, a significantly higher percentage of apoptotic cells were found in the cadmium-treated group. Exposure to cadmium, therefore, induced ectopic apoptosis at 28 hpf without affecting the dynamics of apoptosis at earlier developmental stages.

Animals↗

PubMed2004

A role for MKP3 in axial patterning of the zebrafish embryo.

Fibroblast growth factors (FGFs) are secreted molecules that can activate the RAS/mitogen-activated protein kinase (MAPK) pathway to serve crucial functions during embryogenesis. Through an in situ hybridization screen for genes with restricted expression patterns during early zebrafish development, we identified a group of genes that exhibit similar expression patterns to FGF genes. We report the characterization of zebrafish MAP kinase phosphatase 3 (MKP3; DUSP6 - Zebrafish Information Network), a member of the FGF synexpression group, showing that it has a crucial role in the specification of axial polarity in the early zebrafish embryo. MKP3 dephosphorylates the activated form of MAPK, inhibiting the RAS/MAPK arm of the FGF signaling pathway. Gain- and loss-of-function studies reveal that MKP3 is required to limit the extent of FGF/RAS/MAPK signaling in the early embryo, and that disturbing this inhibitory pathway disrupts dorsoventral patterning at the onset of gastrulation. The earliest mkp3 expression is restricted to the future dorsal region of the embryo where it is initiated by a maternal beta-catenin signal, but soon after its initiation, mkp3 expression comes under the control of FGF signaling. Thus, mkp3 encodes a feedback attenuator of the FGF pathway, the expression of which is initiated at an early stage so as to ensure correct FGF signaling levels at the time of axial patterning.

Amino Acid Sequence↗

PubMed1998

Ethanol impairs migration of the prechordal plate in the zebrafish embryo.

Exposure of vertebrate embryos to ethanol causes cyclopia, but little is known about the underlying mechanisms of this effect. Here we show that cyclopia can be induced in the zebrafish by a short ethanol treatment during early gastrula stages and is accompanied by loss of gene expression characteristic of the ventral aspects of the fore- and midbrain. Interestingly, defects in the expression of ventral brain markers are linked to impaired migration of the prechordal plate mesoderm indicating that the correct position of the prechordal plate mesoderm under the anterior neural plate in the zebrafish embryo is required for specification of the anterior neural midline. Ethanol-induced cyclopia does not, however, impair the induction of anterior neuroectodermal structures in general. Finally, as genes like goosecoid and islet-1 are expressed in prechordal plate cells in a temporal pattern similar to control embryos despite the ectopic position of expressing cells, it appears that regulation of prechordal plate-specific gene expression is largely independent of the final position of the prechordal plate.

Animals↗

PubMed2004

Targeted knockdown of insulin-like growth factor binding protein-2 disrupts cardiovascular development in zebrafish embryos.

IGF binding protein-2 (IGFBP-2) is an evolutionarily conserved protein that binds IGFs and modulates their biological activities. Although the actions of IGFBP-2 have been well studied in vitro, we have a poor understanding of its in vivo functions, particularly during early development. Using the transparent zebrafish embryo as a model, we show that IGFBP-2 mRNA is expressed in lens epithelium and cranial boundary regions during early embryonic development and becomes localized to the liver by the completion of embryogenesis. Targeted knock-down of IGFBP-2 by antisense morpholino-modified oligonucleotides resulted in delayed development, reduced body growth, reduced IGF-I mRNA levels, and disruptions to cardiovascular development, including reduced blood cell number, reduced blood circulation, cardiac dysfunction, and brain ventricle edema. Detailed examination of vascular tissues, using a stable transgenic line of zebrafish expressing green fluorescent protein in vascular endothelial cells, revealed specific angiogenic (vessel sprouting) defects in IGFBP-2 knockdown embryos, with effects being localized in regions associated with IGFBP-2 mRNA expression. These findings suggest that IGFBP-2 is required for general embryonic development and growth and plays a local role in regulating vascular development in a model vertebrate organism.

Animals↗

PubMed1991

Retinoic acid modifies development of the midbrain-hindbrain border and affects cranial ganglion formation in zebrafish embryos.

Considerable evidence now suggests that retinoic acid (RA) is an important modulator of patterning events in early neuronal development in vertebrates. In this paper, we describe the effects of exogenously applied RA on early neural development in the zebrafish embryo. Neural anatomy is assessed by immunocytochemical and histochemical analysis of the developing embryo in whole mounts at 24 h post-fertilization. RA was applied for one hour at concentrations ranging from 10(-9) to 10(-6) M to embryos at 50% epiboly, the midgastrula stage, and at 10(-7) M to embryos at early and late gastrula stages. The neuroanatomical analysis shows that 10(-7) M RA causes a defined lesion to the developing central nervous system which corresponds to a loss of a region of the brain in the caudal midbrain-rostral hindbrain area, the precursor of the cerebellum and associated neural structures. The region that fails to develop corresponds to the cranial expression domain of the engrailed protein as assessed by the monoclonal antibody 4D9 (Patel et al. 1989: Expression of engrailed proteins in arthropods, annelids and chordates. Cell 58, 955-968). Structures caudal to rhombomere 4 are unaffected by 10(-7) M RA, as are the cranial midbrain and forebrain: 10(-7) M RA also affects the development of cranial ganglia, principally the Vth, anterior lateral line and VIIIth ganglia, suggesting that RA affects normal development of the cranial neural crest. Effects of RA at stages immediately prior to and after gastrulation show some similar and some distinct features. Results are discussed in terms of the possible role of RA as an endogenous moderator of normal head development.

Animals↗

PubMed2005

Hedgehog signaling is required for adult blood stem cell formation in zebrafish embryos.

Studies with embryonic explants and embryonic stem cells have suggested a role for Hedgehog (Hh) signaling in hematopoiesis. However, targeted deletion of Hh pathway components in the mouse has so far failed to provide in vivo evidence. Here we show that zebrafish embryos mutant in the Hh pathway or treated with the Hh signaling inhibitor cyclopamine display defects in adult hematopoietic stem cell (HSC) formation but not in primitive hematopoiesis. Hh is required in the trunk at three consecutive stages during vascular development: for the medial migration of endothelial progenitors of the dorsal aorta (DA), for arterial gene expression, and for the formation of intersomitic vessel sprouts. Interference with Hh signaling during the first two stages also interferes with HSC formation. Furthermore, HSC and DA formation also share Vegf and Notch requirements, which further distinguishes them from primitive hematopoiesis and underlines their close relationship during vertebrate development.

Animals↗

PubMed2003

Gap junctional coupling in the olfactory organ of zebrafish embryos.

Intercellular communication through gap junctions is vital for many developmental processes, including cell division and synaptogenesis. This study is the first demonstration that olfactory organ cells are functionally coupled by gap junctions. Cell coupling was examined during development in the olfactory organ using gap junction permeable dyes in live zebrafish embryos. At 1 day post-fertilization (dpf), cells of the olfactory organ were not coupled by gap junctions. At 2 and 3 dpf, olfactory organ cells passed dye from one cell to another, indicating functional coupling via gap junctions. Coupled cell cohorts included combinations of all three olfactory cell types: basal cells, support cells, and olfactory receptor cells. As the olfactory organ matured, the number of cells per coupled cell cohort increased. Gap junctional coupling corresponded with maturation of the olfactory organ and indicates that functional gap junctions may be involved in proper development of the olfactory organ.

Animals↗

PubMed1995

Tubulin dynamics in neuronal axons of living zebrafish embryos.

The mechanism of cytoskeletal protein transport, especially the question of what kind of form the cytoskeletal proteins assume during transport in neurons in situ, has been an important, as yet unsettled issue. To clear up this matter, we adopted the embryonic zebrafish as a living animal model and applied the fluorescence recovery after photobleaching (FRAP) method. The zebrafish embryo is appropriate for this kind of study because of its transparency during the early developmental stage, allowing the observation of neurons that incorporate the microinjected fluorescent tubulin directly under fluorescence microscopy. FRAP revealed no movement of the bleached zone proximodistally, where fluorescence recovered gradually (recovery half-time, 44.2 +/- 11.2 min; n = 36), suggesting that the polymers are stationary but dynamic and that the true moving form could be small oligomers or heterodimers.

Animals↗

PubMed2002

Development of pigment cells in the zebrafish embryo.

In recent years, the zebrafish Danio rerio has emerged as a powerful model organism for the study of vertebrate embryogenesis. Zebrafish, like other vertebrates, possess pigment cells that arise from two distinct embryonic sources: those of the dermis and epidermis originate from the neural crest, while those that comprise the outermost layer of the retina, the retinal pigment epithelium or RPE, derive from the optic cup. A better understanding of processes behind the specification and differentiation of these cells will provide insight to the evolutionary diversification of all classes of vertebrates and will have clinical relevance to human disorders of pigmentation and certain retinopathies. In the first part of this review, the present knowledge of the ontogeny of both of these populations of pigment cells in the embryonic zebrafish is summarized, in terms of both genetics and molecular markers. The final part of the review focuses on duplicate zebrafish genes encoding orthologs of the basic helix-loop-helix/leucine zipper protein Mitf (Microphthalmia-associated transcription factor), and presents a hypothesis concerning their divergent roles in neural crest and retinal pigment cells.

Animals↗

PubMed2001

Buffering intracellular calcium disrupts motoneuron development in intact zebrafish embryos.

Numerous studies, performed mainly on dissociated cells, have shown that calcium signals have a role during different stages of neuronal development. However, the actions of calcium during neuronal development in vivo remain to be established. The present study has investigated the role of intracellular calcium signals during development of motoneurons in the spinal cord of intact zebrafish embryos. Loading blastomeres of early embryos with either the calcium buffer BAPTA or the calcium reporter dye Calcium Green, was shown to disrupt motoneuron development in the spinal cord of embryos at 24 h postfertilisation. Loading the calcium buffer BAPTA, at an intracellular concentration of 1 mM, into the blastomeres of early embryos did not alter the resting levels of intracellular calcium, but significantly dampened transient rises in intracellular calcium in the cells of later stage embryos. Loading cells with 1 mM BAPTA significantly decreased the number of motoneurons present in the spinal cord at 24 h, indicating that calcium signals are important for normal motoneuron differentiation. Furthermore, in those BAPTA-filled cells that did adopt a motoneuron cell fate, axogenesis was found to be inhibited, suggestive of a role for calcium signalling in neurite initiation. This work provides evidence that calcium signals are necessary at several stages of motoneuron development in vivo.

Animals↗

PubMed2003

Semaphorin3a1 regulates angioblast migration and vascular development in zebrafish embryos.

Semaphorins are a large family of secreted and cell surface molecules that guide neural growth cones to their targets during development. Some semaphorins are expressed in cells and tissues beyond the nervous system suggesting the possibility that they function in the development of non-neural tissues as well. In the trunk of zebrafish embryos endothelial precursors (angioblasts) are located ventral and lateral to the somites. The angioblasts migrate medially and dorsally along the medial surface of the somites to form the dorsal aorta just ventral to the notochord. Here we show that in zebrafish Sema3a1 is involved in angioblast migration in vivo. Expression of sema3a1 in somites and neuropilin 1, which encodes for a component of the Sema3a receptor, in angioblasts suggested that Sema3a1 regulates the pathway of the dorsally migrating angioblasts. Antisense knockdown of Sema3a1 inhibited the formation of the dorsal aorta. Induced ubiquitous expression of sema3a1 in hsp70:(gfp)sema3a1(myc) transgenic embryos inhibited migration of angioblasts ventral and lateral to the somites and retarded development of the dorsal aorta, resulting in severely reduced blood circulation. Furthermore, analysis of cells that express angioblast markers following induced expression of sema3a1 or in a mutant that changes the expression of sema3a1 in the somites confirmed these results. These data implicate Sema3a1, a guidance factor for neural growth cones, in the development of the vascular system.

Animals↗

PubMed1997

Axon tracts correlate with netrin-1a expression in the zebrafish embryo.

Netrins are secreted molecules that can attract or repel growth cones from a variety of organisms. In order to clarify the extent and scope of the effects of netrins for guiding growth cones, we have analyzed netrin-1a within the relatively simple and well-characterized nervous system of zebrafish embryos. netrin-1a is expressed in dynamic patterns that suggest that it guides the growth cones of a wide variety of neurons. The spatiotemporal relationship of netrin-1a expression and extending growth cones further suggests that netrins may act to delineate specific pathways and stimulate axonal outgrowth in addition to attracting and repelling growth cones. Furthermore, aberrant outgrowth by commissural growth cones in the spinal cords of floating head mutants, in which netrin-1a expression is altered, is consistent with an in vivo, chemoattractive action of netrin-1a. These data suggest that netrins act on many growth cones and influence their behavior in a variety of ways.

Amino Acid Sequence↗

PubMed1998

Pathfinding of olfactory neuron axons to stereotyped glomerular targets revealed by dynamic imaging in living zebrafish embryos.

In the vertebrate olfactory system, sensory neurons with common odorant specificities project to specific glomeruli in the olfactory bulb. How do olfactory sensory neurons find their glomerular targets? To address this question, we have visualized the genesis of the peripheral olfactory system in living zebrafish embryos. Dye labelings reveal that a primordial yet stereotyped map of glomeruli is apparent during embryogenesis. By labeling a small number of cells with an ectopically expressed green fluorescent protein reporter, we can observe the dynamic growth behaviors of individual olfactory neuron growth cones as they project to their glomeruli. We find that olfactory axons extend directly to their partner glomeruli, suggesting that these cells' growth cones rely upon pathfinding cues to reach their targets.

Afferent Pathways↗

PubMed2004

A relational-tubular (ReTu) deformable model for vasculature quantification of zebrafish embryo from microangiography image series.

Embryonic cardiovascular system plays a vital role in embryonic development of human and animal. In this work, we introduce a novel deformable model, which we called Relational-tubular (ReTu) deformable model for segmenting and quantifying the embryonic vasculature of zebrafish embryo from microangiography image series. Particularly, to incorporate additional constraints on the spatial relationships among vessel branches, we introduce a new energy term called relation energy into the model energy function. This energy item acts as a repulsion force between neighboring vessels during the deformation to encourage them to move towards their respective volume data. Using the ReTu deformable model, the deformation process is an iterative two-stage procedure: vascular axis deformation and vascular surface deformation. The efficiency and robustness of this approach are demonstrated by experiments which show that satisfactory quantifications of the vasculature can be obtained after 3-4 iterations.

Angiography↗

PubMed2005

Quantum dots are powerful multipurpose vital labeling agents in zebrafish embryos.

Recently, inorganic fluorescent contrast agents composed of semiconductor materials have been introduced to biological imaging approaches. These so-called quantum dots provide unique and promising properties unreached by organic fluorophores, but their use as contrast agents within live organisms has been limited, probably due in part to concerns about their in vivo tolerance. Using transparent zebrafish embryos, we challenged quantum dots with a series of intravital imaging problems. We show that quantum dots provide a high fluorescent yield within targeted tissues, possess immense photostability, can be targeted to specific subcellular compartments, remain within targeted cells as lineage tracers, are easily separable from conventional organic fluorescent dyes, and are fixable, allowing them to be used in combination with immunohistochemistry after live recordings. Thus, quantum dots combine the specific advantages of different organic fluorescent contrast agents and promise to become the first fluorophore feasible for long-lasting intravital time-lapse studies. Finally, we show by co-labeling blood vessels of the vasculature and major axon tracts of the nervous system that, for establishing these networks, the same guidance cues might be used in some body parts, whereas in others, both networks appear to develop independently from one another. Thus, the bright fluorescence of quantum dots will help to unravel many open questions in the fields of embryology, cell biology, as well as phenotyping and disease diagnosis.

Animals↗

PubMed1995

A localized elevation of cytosolic free calcium is associated with cytokinesis in the zebrafish embryo.

Cytokinesis, a key step in cell division, is known to be precisely regulated both in its timing and location. At present, the regulatory mechanism of cytokinesis is not well understood, although it has been suggested that calcium signaling may play an important role in this process. To test this notion, we introduced a sensitive fluorescent Ca2+ indicator into the zebrafish embryo and used confocal microscopy to measure the spatiotemporal variation of intracellular free Ca2+ concentration ([Ca2+]i) during cell cleavage. It was evident that a localized elevation of [Ca2+]i is closely associated with cytokinesis. First, we found that during cytokinesis, the level of free Ca2+ was elevated locally precisely at the cleavage site. Second, the rise of free Ca2+ was very rapid and occurred just preceding the initiation of furrow contraction. These observations strongly suggest that cytokinesis may be triggered by a calcium signal. In addition, we found that this cytokinesis-associated calcium signal arose mainly from internal stores of Ca2+ rather than from external free Ca2+; it could be blocked by the antagonist of inositol trisphosphate (InsP3) receptors. These findings suggest that the localized elevation of [Ca2+]i is caused by the release of free Ca2+ from the endoplasmic reticulum through the InsP3-regulated calcium channels.

Animals↗