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PubMed2004

A conserved role for retinoid signaling in vertebrate pancreas development.

Retinoic acid (RA) signaling plays critical roles in the regionalization of the central nervous system and mesoderm of all vertebrates that have been examined. However, to date, a role for RA in pancreas and liver development has only been demonstrated for the teleost zebrafish. Here, we demonstrate that RA signaling is required for development of the pancreas but not the liver in the amphibian Xenopus laevis and the avian quail. We disrupted RA signaling in Xenopus tadpoles, using both a pharmacological and a dominant-negative strategy. RA-deficient quail embryos were obtained from hens with a dietary deficiency in vitamin A. In both species we found that pancreas development was dependent on RA signaling. Furthermore, treatment of Xenopus tadpoles with exogenous RA led to an expansion of the pancreatic field. By contrast, liver development was not perturbed by manipulation of RA signaling. Taken together with our previous finding that RA signaling is necessary and sufficient for zebrafish pancreas development, these data support the hypothesis that a critical role for RA signaling in pancreas development is a conserved feature of the vertebrates.

Animals↗
PubMed2004

Developmental toxicity of the dithiocarbamate pesticide sodium metam in zebrafish.

Sodium metam (NaM), a dithiocarbamate, is a general agricultural biocide applied prior to planting for the elimination of nematodes, soil pathogens, and weeds. There is a remarkable paucity of information about the mechanism of action and the risk that dithiocarbamates may pose to developing vertebrates. We have characterized NaM toxicity during early life stage exposure in zebrafish. Zebrafish embryos are most sensitive to NaM exposure during gastrulation and early segmentation (4-14 hours post fertilization, hpf). For mortality, the dose response curve is steep with an LC(50) estimate of 1.95 microM (248 ppb) at 48 hpf. The most notable malformation among surviving embryos was a severely twisted notochord, which became evident by 24 hpf. Surprisingly, this notochord defect was not immediately lethal and the animals continued to grow despite delays in hatching, apparent paralysis, and an inability to feed. We have characterized the notochord malformation using histological and in situ hybridization techniques. collagen 2a1 mRNA expression is normally localized to the notochord sheath cells at 24 hpf, whereas in NaM-exposed embryos it is misexpressed in the notochord cells. Histological staining and myoD expression indicate that the myotomes of the NaM-exposed embryos are less defined, compacted and block-shaped compared to controls. The degradation product of NaM, methyl isothiocyanate (MITC), causes similar malformations at similar concentrations as NaM, suggesting that MITC or another common product may be the active toxicant. Our results indicate that developing zebrafish are sensitive to NaM and MITC and we believe that this model is ideal to elucidate the molecular mechanism(s) and etiology of NaM toxicity in vertebrates.

Animals↗
PubMed1997

Zebrafish genetics and its implications for understanding vertebrate development.

The identification of genes via their mutant phenotypes is the most direct way of dissecting the molecular basis of developmental processes. While this approach has been extremely powerful in invertebrates such as Drosophila and Caenorhabditis elegans, it has until recently been lacking in vertebrates. Now two landmark studies by the groups of Nüsslein-Volhard and Driever have for the first time provided the basis for a comprehensive genetic dissection of vertebrate development using the teleost fish, Danio rerio. The mutations identified in these screens look set to revolutionise our understanding of vertebrate development.

Animals↗
PubMed2000

Regulation of gut and heart left-right asymmetry by context-dependent interactions between xenopus lefty and BMP4 signaling.

The Lefty subfamily of TGFbeta signaling molecules has been implicated in early development in mouse, zebrafish, and chick. Here, we show that Xenopus lefty (Xlefty) is expressed both bilaterally in symmetric midline domains and unilaterally in left lateral plate mesoderm and anterior dorsal endoderm. To examine the roles of Xlefty in left-right development, we created a system for scoring gut asymmetry and examined the effects of unilateral Xlefty misexpression on gut development, heart development, and Xnr-1 and XPitx2 expression. In contrast to the unilateral effects of Vg1, Activin, Nodal, or BMPs, targeted expression of Xlefty in either the left or the right side of Xenopus embryos randomized the direction of heart looping, gut coiling, and left-right positioning of the gut and downregulated the asymmetric expression of Xnr-1 and XPitx2. It is currently thought that Lefty proteins act as feedback inhibitors of Nodal signaling. However, this would not explain the effects of right-sided Xlefty misexpression. Here, we show that Xlefty interacts with the signaling pathways of other members of the TGFbeta family during left-right development. Results from coexpression of Xlefty and Vg1 indicate that Xlefty can nullify the effects of Vg1 ectopic expression and that Xlefty is downstream of left-sided Vg1 signaling. Results from coexpression of Xlefty and XBMP4 indicate that XLefty and XBMP4 interact both synergistically and antagonistically in a context-dependent manner. We propose a model in which interactions of Xlefty with multiple members of the TGFbeta family enhance the differences between the right-sided BMP/ALK2/Smad pathway and the left-sided Vg1/anti-BMP/Nodal pathway, leading to left-right morphogenesis of the gut and heart.

Amino Acid Sequence↗
PubMed2004

A requirement for kit in embryonic zebrafish melanocyte differentiation is revealed by melanoblast delay.

Exploring differences in gene requirements between species can allow us to delineate basic developmental mechanisms, provide insight into patterns of evolution, and explain heterochronic differences in developmental processes. One example of differences in gene requirements between zebrafish and mammals is the requirement of the kit receptor tyrosine kinase in melanocyte development. kit is required for migration, survival and differentiation of all neural crest-derived melanocytes in mammals. In contrast, zebrafish kit is not required for differentiation of embryonic melanocytes during normal development. When melanoblast development in zebrafish embryos is delayed by injecting morpholinos targeted to the mitfa gene, we show that these delayed melanoblasts fail to differentiate in kit mutants. Thus, we show that there is a kit requirement for melanocyte differentiation in zebrafish when melanoblast development is delayed. Furthermore, we show that kit is not involved in maintaining melanocyte precursors through the developmental delay, but instead is required for differentiation of melanocytes after the block on their development is removed. Finally, we suggest there is a heterochronic shift in the onset of melanocyte differentiation between fish and mouse, and developmental delay of melanoblast development in zebrafish removes this heterochronic difference.

Animals↗
PubMed2005

Zebrafish gonadotropins and their receptors: I. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors--evidence for their distinct functions in follicle development.

In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.

Amino Acid Sequence↗
PubMed1993

Regulation of expression of transgenes in developing fish.

The transcriptional regulatory elements of the beta-actin gene of carp (Cyprinus carpio) have been examined in zebrafish and goldfish harbouring transgenes. The high sequence conservation of the putative regulatory elements in the beta-actin genes of animals suggested that their function would be conserved, so that transgenic constructs with the same transcriptional control elements would promote similar levels of transgene expression in different species of transgenic animals. To test this assumption, we analysed the temporal expression of a reporter gene under the control of transcriptional control sequences from the carp beta-actin gene in zebrafish (Brachydanio rerio) and goldfish (Carrasius auratus). Our results indicated that, contrary to expectations, combinations of different transcriptional control elements affected the level, duration, and onset of gene expression differently in developing zebrafish and goldfish. The major differences in expression of beta-actin/CAT (chloramphenicol acetyltransferase) constructs in zebrafish and goldfish were: (1) overall expression was almost 100-fold higher in goldfish than in zebrafish embryos, (2) the first intron had an enhancing effect on gene expression in zebrafish but not in goldfish, and (3) the serum-responsive/CArG-containing regulatory element in the proximal promoter was not always required for maximal CAT activity in goldfish, but was required in zebrafish. These results suggest that in the zebrafish, but not in the goldfish, there may be interactions between motifs in the proximal promoter and the first intron which appear to be required for maximal enhancement of transcription.

Actins↗
PubMed2003

A periaxonal net in the zebrafish central nervous system.

We produced a monoclonal antibody, named A20, which specifically recognizes a 35 kDa protein and stains myelinated axons in zebrafish brain. The A20 antigen is located at the outside of the myelin layer of large axons, and comprises a fine meshwork composed of thin unit fibers about 1-2 microm in length and about 100-200 nm in thickness. The unit fibers form pentagonal and hexagonal structures, which further polymerize into an envelope structure on the axons. The A20 monoclonal antibody did not stain neuronal cell bodies nor synapses. Instead, the distribution of the A20 antigen was along axons, practically coincident with the distribution of myelin basic protein. The monoclonal antibody stained only axons in the central nervous system (CNS), and not the extracellular matrix surrounding Schwann cells. These results suggest that this antigenic meshwork (which we call the periaxonal net) is synthesized by oligodendrocytes. During the development of the zebrafish brain, the periaxonal net appeared after the formation of myelin on the axons. The periaxonal net developed first at the brain stem, then gradually appeared at the caudal end of the spinal cord. The thickness of the periaxonal net around the Mauthner axon changed during development. Although the thickness of the Mauthner axon continues to grow throughout life, the thickness of periaxonal net stopped growing at 6 months after fertilization.

Animals↗
PubMed1984

Epithelial fusion during early semicircular canal formation in the embryonic zebrafish, Brachydanio rerio.

The developing inner ear of the teleost, Brachydanio rerio, provides an opportunity for observing an epithelial fusion between the apical surfaces of apposed epithelia in a vertebrate embryo in vivo. The developing otocyst was filmed for periods up to 4 days in unanesthetized embryos, and specimens were fixed at intervals and processed for light microscopy, TEM, and SEM. The semicircular canals are formed as a consequence of the union between the tips of three cylindrical projections from the wall of the otocyst, which grow toward corresponding bulges of a projection from the lateral wall. The epithelial cells covering the projections contain extensive rough endopasmic reticulum, exhibit apical junctional complexes, and are not underlain by a basal lamina. The core of each projection contains large amounts of flocculent and fibrillar extracellular material. After a period of growth and elongation, the tip of each projection contacts, and adheres to, the appropriate bulge to create a circular, flattened, bilayered, epithelial plate. Small, focal junctions form between the apposed apical cell surfaces within the plate during this period, but they are not numerous. Junctional complexes do develop, however, between apposed cells at the periphery of the plate. After 1-2 hours, the basal surface of the plate exhibit considerable alteration in contour. Adjacent cells within the plate then separate to allow continuity of the connective tissue components of the two structures. The observations of this study indicate that following an initial period of contact and adhesion, cellular reorientation and changes in junctional contacts between adjacent cells within the epithelial plate, rather than cell degeneration, are responsible for perforation of the plate.

Animals↗
PubMed2005

Oligodendrocyte development and myelination in GFP-transgenic zebrafish.

Green fluorescent protein (GFP) transgenic zebrafish technology has been employed to directly visualize and analyze dynamic developmental processes, such as cell migration and morphogenesis. Stable transgenic zebrafish that express GFP in oligodendrocytes can be a valuable tool to visualize complex myelination processes in vivo, as well as to conduct rapid mutagenesis screens for defective myelination mutants. We investigated whether two myelin gene promoters, the zebrafish P0 promoter and the mouse proteolipid protein (PLP) promoter, drive GFP expression in zebrafish oligodendrocytes. Transiently, both promoters drive enhanced GFP (EGFP) expression in morphologically identifiable oligodendrocytes, premyelinating oligodendrocytes, and possible oligodendrocyte precursors. We have established a stable transgenic zebrafish line, tg(plp:EGFP) zebrafish, at the F1 generation, which expresses enhanced GFP (EGFP) driven by the mouse PLP promoter. In this transgenic line, EGFP-expressing cells are visually detectable around 24-hr postfertilization (hpf), and later at 54 hpf, these cells start exhibiting the clear morphologic characteristics of oligodendrocytes. Shortly afterward, EGFP-expressing oligodendrocytes establish a ventral dominant distribution pattern throughout the central nervous system. This transgenic zebrafish line is likely to serve as a useful tool, in which normal myelination as well as abnormal myelination can be recorded under time-lapse confocal microscopy. Furthermore, it has the potential to greatly facilitate mutagenesis screening for novel dysmyelinating mutants.

Animals↗
PubMed2000

Development of the pteridine pathway in the zebrafish, Danio rerio.

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.

Alcohol Oxidoreductases↗
PubMed2005

The zebrafish: a new model of T-cell and thymic development.

T-cell and thymic development are processes that have been highly conserved throughout vertebrate evolution. Mammals, birds, reptiles and fish share common molecular signalling pathways that regulate the development of the adaptive immune system. This Review article focuses on defining the similarities and differences between zebrafish and mammalian T-cell immunobiology, and it highlights the advantages of using the zebrafish as a genetic model to uncover mutations that affect T-cell and thymic development. Finally, we summarize the use of the zebrafish as a new model for assessing stem-cell function and for drug discovery.

Animals↗
PubMed2004

Zebrafish pitx3 is necessary for normal lens and retinal development.

The human PITX3 gene encodes a bicoid-like homeodomain transcription factor associated with a variety of congenital ocular conditions, including anterior segment dysgenesis, Peter's anomaly, and cataracts. We identified a zebrafish pitx3 gene encoding a protein (Pitx3) that possesses 63% amino acid identity with human PITX3. The zebrafish pitx3 gene encompasses approximately 16.5kb on chromosome 13 and consists of four exons, which is similar to the genomic organization of other pitx genes. Expression of the zebrafish pitx3 gene was studied by in situ mRNA hybridization and RT-PCR. The pitx3 transcripts were detected throughout development with the greatest level of expression occurring in the developing lens and brain at 24hpf. In adults, the highest expression was detected in the eye. Morpholinos were used to knockdown expression of the Pitx3 protein and a control morpholino that contains five mismatched bases was used to confirm the specificity of the phenotypes. The morphants had small eyes, misshapen heads and reduced jaws and fins relative to controls. The morphants exhibited abnormalities in lens development and their retinas contained pyknotic nuclei accompanied by a reduction in the number of cells in different neuronal classes. This suggests the lens is required for retinal development or Pitx3 has an unexpected role in retinal cell differentiation or survival. These results demonstrate zebrafish pitx3 represents a true ortholog of the human PITX3 gene and the general function of the Pitx3 protein in lens development is conserved between mammals and the teleost fish.

Actins↗
PubMedDate not supplied

Comparative expression analysis of Adh3 during arthropod, urochordate, cephalochordate, and vertebrate development challenges its predicted housekeeping role.

Gene and genome duplications in the vertebrate lineage explain the complexity of extant gene families. Among these, the medium-chain alcohol dehydrogenase (ADH), which expanded by tandem duplications after the cephalochordate-vertebrate split, is a good model with which to analyze the evolution of gene function. Although the ancestral member of this family, ADH3, has been strictly conserved throughout animal evolution, its physiological role is still controversial. Previous evidence indicates that it contributes to formaldehyde cytoprotection, retinoic acid metabolism, and nitric oxide homeostasis. We performed in situ hybridization during Drosophila, ascidian (Ciona intestinalis), and zebrafish (Danio rerio) development. We showed that Adh3 expression was restricted to the fat body in Drosophila embryos at stage 17 and to the anterior endoderm in C. intestinalis tail bud, whereas in the zebrafish 2.5-day larvae the signal appeared widespread. A more comprehensive expression analysis including amphioxus and mice revealed that ancestral Adh3 was tissue specific, whereas a widespread expression was later attained in vertebrates. These variations occurred concomitantly with the expansion of the ADH family and the acquisition of new functions but were unlinked to the genomic changes that led to the transition from fractional to global methylation in vertebrates. Our data challenge the housekeeping role of ADH3 and question its involvement in the prevertebrate retinoic acid pathway.

Alcohol Dehydrogenase↗
PubMed2004

Isoforms vatB1 and vatB2 of the vacuolar type ATPase subunit B are differentially expressed in embryos of the zebrafish (Danio rerio).

The v-type ATPase is a membrane anchored, multi-subunit proton pump, which in freshwater fish appears to play a major role in ionoregulative processes in the apical membrane of specialized gill cells. Very little is known about free-living fish embryos and larvae that are exposed to hypo-osmotic conditions with spawning but do not have their gills fully developed. By using reverse transcriptase-polymerase chain reaction and immunological methods, we could demonstrate the presence of two isoforms of the subunit B of this v-type ATPase in the early development of the zebrafish. Immunohistochemical analysis revealed the presence of one isoform (vatB1) in the apical membrane of embryonic skin cells, while vatB2 has been found ubiquitously. This differential localization of the two isoforms supports the hypothesis that vatB1 is preferentially involved in ionoregulative functions, while vatB2 may be preferentially responsible for acidification of intracellular vesicles.

Adenosine Triphosphatases↗
PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗
PubMed2003

Identifying axon guidance defects in the embryonic zebrafish brain.

The method described here outlines a simple protocol to investigate the in vivo function of axon guidance molecules during the development of the embryonic zebrafish brain. By 24 hours postfertilization, a simple scaffold of axon tracts and commissures can be visualised in the brain using acetylated alpha-tubulin, a panaxonal marker that stains all axons. The highly stereotypical trajectory of axons in the embryonic zebrafish brain provides an ideal system in which to study the molecular mechanisms of axon guidance, as defects in the axon scaffold can be clearly visualised. We describe here our approach to identify defects in the trajectory of axons that establish the initial template of tracts in the embryonic fore- and mid-brain. By combining immunohistochemical techniques and confocal microscopy on dissected wholemounts of embryonic brains we are able to observe at high resolution the complete scaffold of axon tracts. This approach provides a rapid and simple means of assessing axon guidance defects in the developing brain. Given the advantages of the zebrafish as a model system, and the range of molecular perturbation methods now available, this technique provides a valuable tool for assessing the phenotypic effects of gene perturbations in a biologically relevant context.

Animals↗