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PubMed1999

Spatial correspondence between R-cadherin expression domains and retinal ganglion cell axons in developing zebrafish.

Mechanisms underlying axonal pathfinding have been investigated for decades, and numerous molecules have been shown to play roles in this process, including members of the cadherin family of cell adhesion molecules. We showed in the companion paper that a member of the cadherin family (zebrafish R-cadherin) is expressed in retinal ganglion cells, and in presumptive visual structures in zebrafish brain, during periods when the axons were actively extending toward their targets. The present study extends the earlier work by using 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) anterograde tracing techniques to label retinal ganglion cell axons combined with R-cadherin in situ hybridization to explicitly examine the association ofretinal axons and brain regions expressing R-cadherin message. We found that in zebrafish embryos at 46-54 hours postfertilization, DiI-labeled retinal axons were closely associated with cells expressing R-cadherin message in the hypothalamus, the pretectum, and the anterolateral optic tectum. These results demonstrate that R-cadherin is appropriately distributed to play a role in regulating development of the zebrafish visual system, and in particular, pathfinding and synaptogenesis of retinal ganglion cell axons.

Animals↗

PubMed1999

Cloning and expression of the zebrafish germ cell nuclear factor.

Nuclear orphan receptors are DNA-binding proteins that share the domain structure of the nuclear hormone receptor superfamily, although their ligands are unknown. Members of the nuclear receptor family are involved in the regulation of various developmental and reproductive processes. We have identified such a nuclear orphan receptor in the zebrafish and named it zebrafish germ cell nuclear factor (zfGCNF) based on its high sequence homology to previously described mouse, human, and Xenopus laevis GCNF. Detailed sequence comparison of zfGCNF with mouse, human, and frog GCNF revealed high homologies in the domains conserved in the nuclear receptor family. Homology in the DNA-binding domain is 97% for frog and even 98.5% for mouse and human when compared to the zebrafish sequence. Homology in the E III subdomain of the transactivation/ligand-binding E domain is 100% when compared to the mouse and human sequences. Transcripts of different size were detected by Northern blot analysis in the zebrafish ovary, whereas, in the testis only one transcript was present. In situ hybridization revealed that zfGCNF was predominantly expressed in previtellogenic oocytes in the ovary and in spermatocytes in the testis.

Amino Acid Sequence↗

PubMed1998

Zebrafish segmentation and pair-rule patterning.

Segmentation in the vertebrate embryo is evident within the paraxial mesoderm in the form of somites, which are repeated structures that give rise to the vertebrae and muscle of the trunk and tail. In the zebrafish, our genetic screen identified two groups of mutants that affect somite formation and pattern. Mutations of one class, the fss-type mutants, disrupt the formation of the anterior-posterior somite boundaries during somitogenesis. However, segmentation within the paraxial mesoderm is not completely eliminated in these mutants. Irregular somite boundaries form later during embryogenesis and, strikingly, the vertebrae are not fused. Here, we show that formation of the irregular somite boundaries in these mutants is dependent upon the activity of a second group of genes, the you-type genes, which include sonic you, the zebrafish homologue of the Drosophila segment polarity gene, sonic hedgehog. Further to characterize the defects caused by the fss-type mutations, we examined their effects on the expression of her1, a zebrafish homologue of the Drosophila pair-rule gene hairy. In wild-type embryos, her1 is expressed in a dynamic, repeating pattern, remarkably similar to that of its Drosophila and Tribolium counterparts, suggesting that a pair-rule mechanism also functions in the segmentation of the vertebrate paraxial mesoderm. We have found that the fss-type mutants have abnormal pair-rule patterning. Although a her1 mutant could not be identified, analysis of a double mutant that abolishes most her1 expression suggests that a her1 mutant may not display a pair-rule phenotype analogous to the hairy phenotype observed in Drosophila. Cumulatively, our data indicate that zebrafish homologues of both the Drosophila segment polarity genes and pair-rule genes are involved in segmenting the paraxial mesoderm. However, both the relationship between these two groups of genes within the genetic heirarchy governing segmentation and the precise roles that they play during segmentation likely differ significantly between the two organisms.

Animals↗

PubMed2000

Somite development in zebrafish.

A full understanding of somite development requires knowledge of the molecular genetic pathways for cell determination as well as the cellular behaviors that underlie segmentation, somite epithelialization, and somite patterning. The zebrafish has long been recognized as an ideal organism for cellular and histological studies of somite patterning. In recent years, genetics has proven to be a very powerful complementary approach to these embryological studies, as genetic screens for zebrafish mutants defective in somitogenesis have identified over 50 genes that are necessary for normal somite development. Zebrafish is thus an ideal system in which to analyze the role of specific gene products in regulating the cell behaviors that underlie somite development. We review what is currently known about zebrafish somite development and compare it where appropriate to somite development in chick and mouse. We discuss the processes of segmentation and somite epithelialization, and then review the patterning of cell types within the somite. We show directly, for the first time, that muscle cell and sclerotome migrations occur at the same time. We end with a look at the many questions about somitogenesis that are still unanswered.

Animals↗

PubMed2000

Structure and function of the developing zebrafish heart.

The combination of optical clarity and large scale of mutants makes the zebrafish vital for developmental biologists. However, there is no comprehensive reference of morphology and function for this animal. Since study of gene expression must be integrated with structure and function, we undertook a longitudinal study to define the cardiac morphology and physiology of the developing zebrafish. Our studies included 48-hr, 5-day, 2-week, 4-week, and 3-month post-fertilization zebrafish. We measured ventricular and body wet weights, and performed morphologic analysis on the heart with H&E and MF-20 antibody sections. Ventricular and dorsal aortic pressures were measured with a servonull system. Ventricular and body weight increased geometrically with development, but at different rates. Ventricle-to-body ratio decreased from 0.11 at 48-hr to 0.02 in adult. The heart is partitioned into sinus venosus, atrium, ventricle, and bulbus arteriosus as identified by the constriction between the segments at 48-hr. Valves were formed at 5-day post-fertilization. Until maturity, the atrium showed extensive pectinate muscles, and the atrial wall increased to two to three cell layers. The ventricular wall and the compact layer increased to three to four cell layers, while the extent and complexity in trabeculation continued. Further thickening of the heart wall was mainly by increase in cell size. The bulbus arteriosus had similar characteristics to the myocardium in early stages, but lost the MF-20 positive staining, and transitioned to smooth muscle layer. All pressures increased geometrically with development, and were linearly related to stage-specific values for body weight (P < 0.05). These data define the parameters of normal cardiac morphology and ventricular function in the developing zebrafish.

Animals↗

PubMed2001

Cardiac morphology and blood pressure in the adult zebrafish.

Zebrafish has become a popular model for the study of cardiovascular development. We performed morphologic analysis on 3 months postfertilization zebrafish hearts (n > or = 20) with scanning electron microscopy, hematoxylin and eosin staining and Masson's trichrome staining, and morphometric analysis on cell organelles with transmission electron photomicrographs. We measured atrial, ventricular, ventral, and dorsal aortic blood pressures (n > or = 5) with a servonull system. The atrioventricular orifice was positioned on the dorsomedial side of the anterior ventricle, surmounted by the single-chambered atrium. The atrioventricular valve was free of tension apparati but supported by papillary bands to prevent retrograde flow. The ventricle was spanned with fine trabeculae perpendicular to the compact layer and perforated with a subepicardial network of coronary arteries, which originated from the efferent branchial arteries by means of the main coronary vessel. Ventricular myocytes were larger than those in the atrium (P < 0.05) with abundant mitochondria close to the sarcolemmal. Sarcoplasmic reticulum was sparse in zebrafish ventricle. Bulbus arteriosus was located anterior to the ventricle, and functioned as an elastic reservoir to absorb the rapid rise of pressure during ventricular contraction. The dense matrix of collagen interspersed across the entire bulbus arteriosus exemplified the characteristics of vasculature smooth muscle. There were pressure gradients from atrium to ventricle, and from ventral to dorsal aorta, indicating that the valves and the branchial arteries, respectively, were points of resistance to blood flow. These data serve as a framework for structure-function investigations of the zebrafish cardiovascular system.

Animals↗

PubMed2001

Activity and localization of 3beta-hydroxysteroid dehydrogenase/ Delta5-Delta4-isomerase in the zebrafish central nervous system.

Little information is available for neurosteroidogenesis in the central nervous system (CNS) of lower vertebrates. Therefore, in the present study, we examined the enzymatic activity and localization of 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3betaHSD), a key steroidogenic enzyme, in the CNS of adult male zebrafish to clarify central progesterone biosynthesis. Biochemical studies together with HPLC analysis revealed that the zebrafish brain converted pregnenolone to progesterone, suggesting the enzymatic activity of 3betaHSD. This conversion was significantly reduced by trilostane, a specific inhibitor of 3betaHSD. By using Western immunoblotting with the polyclonal antiserum directed against purified bovine adrenal 3betaHSD, a 3betaHSD-like substance was found in homogenates of the zebrafish brain. Immunocytochemical analysis was then undertaken to investigate the localization of the 3betaHSD-like substance in the zebrafish brain and spinal cord. Clusters of immunoreactive cell bodies were localized in the dorsal telencephalic areas (D), central posterior thalamic nucleus (CP), preoptic nuclei (NPO), posterior tuberal nucleus (PTN), paraventricular organ (PVO), and nucleus of medial longitudinal fascicle (NMLF). 3betaHSD-like immunoreactivity was also observed in somata of cerebellar Purkinje neurons. A widespread distribution of immunoreactive fibers was found throughout the brain and spinal cord. In addition, positively stained cells were restricted to other organs, such as the pituitary and retina. Preabsorbing the antiserum with purified bovine adrenal microsome resulted in a complete absence of 3betaHSD-like immunoreactivity. These results suggest that the fish CNS possesses steroidogenic enzyme 3betaHSD and produces progesterone. The present study further provides the first immunocytochemical mapping of the site of 3betaHSD expression in the fish CNS.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

The development of GABA immunoreactivity in the retina of the zebrafish (Brachydanio rerio).

The goal of this study was to determine the pattern of gamma-aminobutyric acid (GABA) expression in the retina and optic nerve of the zebrafish (Brachydanio rerio) during embryonic development. Zebrafish embryos were fixed at intervals between 1 and 4 days postfertilization, and semithin plastic sections were prepared for postembedding immunocytochemistry with antisera against GABA. Sections were also prepared from several adult zebrafish eyes for comparison. GABA immunoreactivity first appeared in the optic nerve at 2 days postfertilization, and by 2.5 days the inner nuclear layer (INL), inner plexiform layer (IPL), retinal ganglion cell layer, and optic nerve were all positive for GABA. The GABA expression in the retinal ganglion cell layer and optic nerve was transient, however, and these structures were largely unlabeled by 4 days postfertilization. The pattern of GABA immunoreactivity at 4 days resembled that seen in the adult zebrafish: A large population of presumptive amacrine cells was labeled at the base of the INL, and the IPL was positive for GABA, as were occasional cells in the ganglion cell layer. Horizontal cells, particularly at the retinal margins, were also GABA positive beginning at about 3 days postfertilization. The transient expression of GABA in retinal ganglion cells and their axons during the period when synaptic contacts are being established both within the retina and between the retina and central targets suggests that GABA may have a role in the development of this system, in addition to serving as a classical neurotransmitter.

Animals↗

PubMed2002

Taste bud development in the zebrafish, Danio rerio.

Taste buds are chemosensory endorgans consisting of modified epithelial cells. Fish and other vertebrates use their taste bud cells to sample potential food, either selecting or rejecting substances according to their edibility. The adult gustatory system in fish has been studied thoroughly, including regeneration experiments. Taste buds occur in the epithelia of the lips, the mouth cavity, the oropharyngeal cavity, and also in the skin of the barbels, the head, and sometimes even all over the body surface. Despite its importance for feeding, little is known about the ontogeny of the fish taste system. We examined the development of taste buds in the zebrafish on the light microscopical and the scanning and transmission electron microscopical levels. Taste buds develop later than the olfactory organ and the solitary chemosensory cells, two other chemosensory systems in aquatic vertebrates. The first few taste bud primordia are visible within the epithelia of lips and gill arches 3 to 4 days after fertilization, and the first few taste buds with open receptor areas appear on the lips and simultaneously on the gill arches 4-5 days after fertilization, which coincides with the onset of feeding. Taste buds in the mouth cavity, on the head, and on the barbels are formed later in development. As seen in other fish, zebrafish taste buds contain elongate dark and light cells, termed according to their electron density. Dark cells with a cell apex of many short microvilli appear first, followed by the light cells with one large microvillus. In addition, the zebrafish has a third fusiform cell type, which appears last. This cell type is low in electron density and has a brush-like apical ending with several small microvilli. This cell type has not been described previously. Furthermore, in zebrafish, the ontogenetic processes of taste bud formation differ from regenerative processes described in the literature.

Animals↗

PubMed2003

Cardiac neural crest in zebrafish embryos contributes to myocardial cell lineage and early heart function.

Myocardial dysfunction is evident within hours after ablation of the cardiac neural crest in chick embryos, suggesting a role for neural crest in myocardial maturation that is separate from its role in outflow septation. This role could be conserved in an animal that does not have a divided systemic and pulmonary circulation, such as zebrafish. To test this hypothesis, we used cell marking to identify the axial level of neural crest that migrates to the heart in zebrafish embryos. Unlike the chick and mouse, the zebrafish cardiac neural crest does not originate from the axial level of the somites. The region of neural crest cranial to somite 1 was found to contribute cells to the heart. Cells from the cardiac neural crest migrated to the myocardial wall of the heart tube, where some of them expressed a myocardial phenotype. Laser ablation of the cardiac premigratory neural crest at the three- to four-somite stage resulted in loss of the neural crest cells migrating to the heart as shown by the absence of AP2- and HNK1-expressing cells and failure of the heart tube to undergo looping. Myocardial function was assessed 24 hr after the cardiac neural crest ablation in a subpopulation of embryos with normal heart rate. Decreased stroke volume, ejection fraction, and cardiac output were observed, indicating a more severe functional deficit in cardiac neural crest-ablated zebrafish embryos compared with neural crest-ablated chick embryos. These results suggest a new role for cardiac neural crest cells in vertebrate cardiac development and are the first report of a myocardial cell lineage for neural crest derivatives.

Animals↗

PubMed2003

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.

Animals↗

PubMed2003

Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics.

Negatively charged homo-oligomers of alternating trans-4-hydroxy-L-proline/phosphonate polyamides with DNA bases (HypNA-pPNA) display excellent hybridization properties toward DNA and RNA, while preserving the mismatch discrimination, nuclease resistance, and protease resistance of peptide nucleic acids (PNAs). Similar properties are associated with morpholino phosphorodiamidate (MO) DNA mimics, which have been used in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. We evaluated mixed sequence HypNA-pPNAs as an alternative to MOs, and found that even a single central DNA mismatch lowered the HypNA-pPNA melting temperature by 16 degrees C. We then observed that the melting temperatures of HypNA-pPNA 18-mers hybridized to RNA 25-mers were comparable to the melting temperatures of MO 25-mers, and that two HypNA-pPNA mismatches lowered the melting temperature with RNA by 18 degrees C. In zebrafish embryos we observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail, and uroD, with greater mismatch stringency. Finally we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. HypNA-pPNAs designed to inhibit translation of specific zebrafish RNA targets thus demonstrated stringent hybridization properties, relative to DNA and MO oligomers, and present a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes.

Animals↗

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↗

PubMed2001

Zebrafish mutants: behavioral genetic studies of visual system defects.

Zebrafish are a promising model for behavioral and genetic studies of vertebrate visual system development and retinal degeneration. In the past few years, numerous studies on zebrafish vision have been published. While most of the studies focus on the molecular and cellular characterization of mutations that disrupt zebrafish visual system structure in early development, others examine the mechanisms that underlie inherited visual system disorders in adults. Behavioral assays, along with morphologic and electrophysiological methods, are powerful tools for functional analyses of zebrafish visual development and performance.

Animals↗

PubMed2001

Rod contributions to the electroretinogram of the dark-adapted developing zebrafish.

Anatomical studies of the developing zebrafish retina have shown that rods approach maturity at about 15 days postfertilization (dpf). Past work has examined the photopic spectral sensitivity function of the developing zebrafish, but not spectral sensitivity under dark-adapted conditions. This study examined rod contributions to the dark-adapted spectral sensitivity function of the ERG b-wave component in developing zebrafish. ERG responses to stimuli of various wavelengths and irradiances were obtained from dark-adapted fish at 6-8, 13-15, 21-24, and 27-29 dpf. The results show that dark-adapted spectral sensitivity varied with age. Spectral sensitivity functions of the 6-8 and 13-15 dpf groups appeared to be cone dominated and contained little or no rod contributions. Spectral sensitivity functions of the 21-24 and 27-29 dpf groups appeared to have both rod and cone contributions. Even at the oldest age group tested, the dark-adapted spectral sensitivity function did not match the adult function. Thus, consistent with anatomical findings, the rod contributions to the ERG spectral sensitivity function appear to develop with age; however, these contributions are still not adult-like by 29 dpf, which is contrary to anatomical work. These results illustrate that the zebrafish is an excellent model for visual development.

Animals↗

PubMed2004

Activation of apoptosis and caspase-3 in zebrafish early gastrulae.

Nonmammalian vertebrate embryos do not manifest apoptosis before gastrulation, and it has been suggested that their cells are inhibited from undergoing apoptosis. To study this interesting possibility, the zebrafish (Danio rerio) embryo is an excellent model. However, the appearance of apoptosis varies among species, and many components of cell death are not highly conserved. To undertake the larger investigation, we first need to document by several criteria that cell death in the zebrafish embryo is apoptotic. Exposure of gastrulating germ-ring stage embryos to cycloheximide or staurosporine elicits an arrest in development and cell death within 8 hr. Caspase-3 activity increases, followed by translocation of phosphatidylserine, loss of cell-cell adhesion, cleavage of poly (ADP-ribose) polymerase, terminal deoxynucleotidyl transferase-mediated dNTP-fluorescein nick end labeling (TUNEL)-positive nuclei, internucleosomal DNA fragmentation, chromatin condensation and margination, and blebbing of the nuclear membrane. Thus, by many criteria, cell death in zebrafish is apoptotic; many of the markers of apoptosis found in mammals are conserved in zebrafish; and post-midblastula transition embryos have the capacity to activate a caspase-dependent apoptotic response well before naturally occurring programmed cell death is seen.

Adenosine Diphosphate Ribose↗

PubMed2004

Zebrafish intestinal fatty acid binding protein (I-FABP) gene promoter drives gut-specific expression in stable transgenic fish.

Mammalian intestinal fatty acid-binding protein (I-FABP) is a small cytosolic protein and is thought to play a crucial role of intracellular fatty acid trafficking and metabolism in gut. To establish an in vivo system for investigating its tissue-specific regulation during zebrafish intestinal development, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a transgenic strategy to generate gut-specific transgenic zebrafish with green/red fluorescent intestine. The 4.5-kb 5'-flanking sequence of zebrafish I-FABP gene was sufficient to direct fluorescent expression in intestinal tube, first observed in 3 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. In all five transgenic lines 45-52% of the F2 inheritance rates were consistent with the ratio of Mendelian segregation. These fish can also provide a valuable resource of labeled adult intestinal cells for in vivo or in vitro studies. Finally, it is possible to establish an in vivo system using these fish for screening genes required for gut development. genesis 38:26-31, 2004.

Animals↗

PubMed1991

Genomic structure and restricted neural expression of the zebrafish wnt-1 (int-1) gene.

The Wnt-1 (int-1) gene was originally identified as an oncogene, but its normal function is in embryogenesis. The gene is the vertebrate homologue of the Drosophila segment polarity gene wingless, and encodes a secretory protein. In mouse embryos, Wnt-1 expression is necessary for proper development of the midbrain and anterior hindbrain. Here we describe the molecular cloning and primary structure of the zebrafish Wnt-1 gene (denoted wnt-1). Comparison with its mouse homologue reveals that both the genomic organization of wnt-1 and the amino acid sequence of the corresponding gene product have been extensively conserved during vertebrate evolution. Moreover, there is probably at least one Wnt-1-related sequence in the zebrafish genome. In zebrafish embryos, wnt-1 is expressed during differentiation of the neural tube. In situ hybridization analysis reveals that the transcripts are confined to the dorsal surfaces of the midbrain, hindbrain and spinal cord, and to lateral cells at the midbrain-hindbrain junction. Thus, the pattern of wnt-1 expression in the developing central nervous system of zebrafish is virtually identical to that seen in mouse embryos. Unexpectedly, despite the striking similarities of Wnt-1 structure and expression in fish and higher vertebrates, we could not identify sequences of obvious homology outside the coding regions, neither in the promoter nor in the introns.

Amino Acid Sequence↗