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PubMed2004

The early ontogeny of neuronal nitric oxide synthase systems in the zebrafish.

To examine a putative role for neuronal nitric oxide synthase (nNOS) in early vertebrate development we investigated nNOS mRNA expression and cGMP production during development of the zebrafish Danio rerio. The nNOS mRNA expression in the central nervous system (CNS) and periphery showed a distinct spatio-temporal pattern in developing zebrafish embryo and young larvae. nNOS mRNA expression was first detected at 19 h postfertilisation (h.p.f.), in a bilateral subpopulation of the embryonic ventrorostral cell cluster in the forebrain. The number of nNOS mRNA-expressing cells in the brain slowly increased, also appearing in the ventrocaudal cell cluster from about 26 h.p.f., and in the dorsorostral and hindbrain cell cluster and in the medulla at 30 h.p.f. A major increase in nNOS mRNA expression started at about 40 h.p.f., and by 55 h.p.f. the expression constituted cell populations in differentiated central nuclei and in association with the proliferation zones of the brain, and in the medulla and retina. In parts of the skin, nNOS mRNA expression started at 20 h.p.f. and ended at 55 h.p.f. Between 40 and 55 h.p.f., nNOS mRNA expression started in peripheral organs, forming distinct populations after hatching within or in the vicinity of the presumptive swim bladder, enteric ganglia, and along the alimentary tract and nephritic ducts. Expression of nNOS mRNA correlated with the neuronal differentiation pattern and with the timing and degree of cGMP production. These studies indicate spatio-temporal actions by NO during embryogenesis in the formation of the central and peripheral nervous system, with possible involvement in processes such as neurogenesis, organogenesis and early physiology.

Animals↗

PubMed2004

Differential expression of duplicated genes for brain-type fatty acid-binding proteins (fabp7a and fabp7b) during early development of the CNS in zebrafish (Danio rerio).

A gene for the zebrafish brain-type fatty acid-binding protein (fabp7b) was identified and its structure defined. The zebrafish fabp7b gene spans 1479 bp and consists of four exons encoding 24, 58, 34 and 16 amino acids, respectively, which is identical to the structure of the fabp7a gene previously described. The complete fabp7b cDNA was isolated by 5' and 3' RACE and its nucleotide sequence determined. The deduced amino acid sequence of FABP7B encoded by the zebrafish fabp7b gene shares 82% identity with that of FABP7A encoded by the zebrafish fabp7a gene. A single transcription start site for the fabp7b gene was mapped by 5' RNA ligase-mediated RACE. Phylogenetic analysis indicated that the duplication of the fabp7 genes occurred in the fish lineage after their divergence from mammals. The zebrafish fabp7b gene was assigned to linkage group 20 by radiation hybrid mapping. Reverse transcription-polymerase chain reaction detected fabp7b transcripts in the same adult tissues as fabp7a transcripts. In the brain, levels of fabp7b transcripts were lower than fabp7a transcripts. Whole-mount in situ hybridization showed that the zebrafish fabp7a transcripts were distributed in the early developing central nervous system. In addition to being expressed in the developing brain and retina, zebrafish fabp7b mRNA was also detected in the swim bladder and pharynx during the embryonic to larval transitory phase.

Animals↗

PubMed2003

Behavioral genetic approaches to visual system development and function in zebrafish.

The zebrafish is a recent vertebrate model system that shows great potential for a genetic analysis of behavior. Early development is extraordinarily rapid, so that larvae already display a range of behaviors 5 days after fertilization. In particular the visual system develops precociously, supporting a number of visually mediated behaviors in the larva. This provides the opportunity to use these visually mediated behaviors to screen chemically mutagenized strains for defects in vision. Larval optokinetic and optomotor responses have already been successfully employed to screen for mutant strains with defects in the visual system. In the adult zebrafish a visually mediated escape response has proved useful for screening for dominant mutations of the visual system. Here, I summarize visually mediated behaviors of both larval and adult zebrafish and their applicability for genetic screens, and present, the approaches and results of visual behavior carried out to date.

Animals↗

PubMed2006

The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish.

The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl(-/-) embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2-binding form of Scl but can be rescued by non-DNA-binding forms, suggesting tethering to target genes through DNA-binding partners linked via Lmo2. Interestingly, unlike other hematopoietic regulators, the Scl/Lmo2 complex does not appear to autoregulate, as neither gene's expression is affected by depletion of the other. Thus, expression of these critical regulators is dependent on continued expression of upstream regulators, which may include cell-extrinsic signals.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Formation of the digestive system in zebrafish. II. Pancreas morphogenesis.

Recent studies have suggested that the zebrafish pancreas develops from a single pancreatic anlage, located on the dorsal aspect of the developing gut. However, using a transgenic zebrafish line that expresses GFP throughout the endoderm, we report that, in fact, two pancreatic anlagen join to form the pancreas. One anlage is located on the dorsal aspect of the developing gut and is present by 24 h postfertilization (hpf), the second anlage is located on the ventral aspect of the developing gut in a position anterior to the dorsal anlage and is present by 40 hpf. These two buds merge by 52 hpf to form the pancreas. Using heart and soul mutant embryos, in which the pancreatic anlagen most often do not fuse, we show that the posterior bud generates only endocrine tissue, while the anterior bud gives rise to the pancreatic duct and exocrine cells. Interestingly, at later stages, the anterior bud also gives rise to a small number of endocrine cells usually present near the pancreatic duct. Altogether, these studies show that in zebrafish, as in the other model systems analyzed to date, the pancreas arises from multiple buds. To analyze whether other features of pancreas development are conserved and investigate the influence of surrounding tissues on pancreas development, we examined the role of the vasculature in this process. Contrary to reports in other model systems, we find that, although vascular endothelium is in contact with the posterior bud throughout pancreas development, its absence in cloche mutant embryos does not appear to affect the early morphogenesis or differentiation of the pancreas.

Animals↗

PubMed2004

Sexual dimorphic expression pattern of a splice variant of zebrafish vasa during gonadal development.

In Drosophila, the RNA helicase VASA (VAS) is required for both germ line formation and oocyte differentiation. While the murine VAS homologue is required for spermatogenesis, it is dispensable for germ line formation. The molecular basis for this apparently dual role of VAS in germ line ontogeny is, however, unclear. Recent evidence indicates that fish, like flies, employs VAS both in early and late stages of the germ line development and that there is a sex-linked differential expression of splice variants. We show here that the longer of two splice variants of zebrafish vas is transiently downregulated in the germ line around the time when the germ cells reach the developing gonad. Using transgenic vas::EGFP fish lines, which allow us to distinguish between male and female individuals, we show that the long splice variant reappears in both sexes at around day 25 and is subsequently downregulated during male gonadal development. Our data further suggest that there is a switch from maternal to zygotic expression of the long splice variant of vas as sexual dimorphic development commences.

Alternative Splicing↗

PubMed1991

Motoneuronal development in the embryonic zebrafish.

To learn how neurons find their appropriate targets, we have studied two populations of motoneurons in the embryonic zebrafish: primary motoneurons, individually identified cells whose growth cones pioneer the first nerve pathways in the muscle, and secondary motoneurons, cells which develop later and whose growth cones apparently extend along the axons of the primary motoneurons. Transplantation studies of single, identified primary motoneurons suggest that commitment of these cells to innervate their cell-specific muscle territories may be a multistep process in which they are first committed to be motoneurons and are later committed to extend axons along specific pathways. Ablation studies suggest that interactions among the primary motoneurons are unlikely to be necessary for proper pathfinding or commitment. However, interactions with the primary motoneurons may be important for proper development of the secondary motoneurons.

Animals↗

PubMed2002

Runx1 is required for zebrafish blood and vessel development and expression of a human RUNX1-CBF2T1 transgene advances a model for studies of leukemogenesis.

RUNX1/AML1/CBFA2 is essential for definitive hematopoiesis, and chromosomal translocations affecting RUNX1 are frequently involved in human leukemias. Consequently, the normal function of RUNX1 and its involvement in leukemogenesis remain subject to intensive research. To further elucidate the role of RUNX1 in hematopoiesis, we cloned the zebrafish ortholog (runx1) and analyzed its function using this model system. Zebrafish runx1 is expressed in hematopoietic and neuronal cells during early embryogenesis. runx1 expression in the lateral plate mesoderm co-localizes with the hematopoietic transcription factor scl, and expression of runx1 is markedly reduced in the zebrafish mutants spadetail and cloche. Transient expression of runx1 in cloche embryos resulted in partial rescue of the hematopoietic defect. Depletion of Runx1 with antisense morpholino oligonucleotides abrogated the development of both blood and vessels, as demonstrated by loss of circulation, incomplete development of vasculature and the accumulation of immature hematopoietic precursors. The block in definitive hematopoiesis is similar to that observed in Runx1 knockout mice, implying that zebrafish Runx1 has a function equivalent to that in mammals. Our data suggest that zebrafish Runx1 functions in both blood and vessel development at the hemangioblast level, and contributes to both primitive and definitive hematopoiesis. Depletion of Runx1 also caused aberrant axonogenesis and abnormal distribution of Rohon-Beard cells, providing the first functional evidence of a role for vertebrate Runx1 in neuropoiesis. To provide a base for examining the role of Runx1 in leukemogenesis, we investigated the effects of transient expression of a human RUNX1-CBF2T1 transgene [product of the t(8;21) translocation in acute myeloid leukemia] in zebrafish embryos. Expression of RUNX1-CBF2T1 caused disruption of normal hematopoiesis, aberrant circulation, internal hemorrhages and cellular dysplasia. These defects reproduce those observed in Runx1-depleted zebrafish embryos and RUNX1-CBF2T1 knock-in mice. The phenotype obtained with transient expression of RUNX1-CBF2T1 validates the zebrafish as a model system to study t(8;21)-mediated leukemogenesis.

Acute Disease↗

PubMed2005

Zebrafish R-cadherin (Cdh4) controls visual system development and differentiation.

In zebrafish, R-cadherin (cadherin-4 or Cdh4) is expressed in the retina and in retinorecipient brain regions, suggesting that Cdh4 functions during visual system development. Cdh4 function was examined during retinogenesis and retinal axon outgrowth using antisense morpholino oligonucleotides and mutant Cdh4 construct expression. In knockdowns, Cdh4 was reduced or absent, eyes were small, and retinae lacked discrete laminae. Increased cell death produced the small eye phenotype. Zn5-, Pax6-, and zpr-1-positive cells were reduced or absent in knockdown retinas but, when present, were in the correct laminae. Cdh4 knockdowns had sparse or absent retinal ganglion cell axons. When present, axons projected contralaterally but lacked fine branching and failed to reach the tectum or arborize the entire tectum. Mutant Cdh4 construct expression during retinal ganglion cell differentiation reduced or ablated neurite formation. Cdh4 is necessary for neural retina survival and differentiation, and required for normal retinotectal projection formation and tectal arborization.

Animals↗

PubMed2003

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.

Amino Acid Sequence↗

PubMed1999

Epidermal expression of apolipoprotein E gene during fin and scale development and fin regeneration in zebrafish.

Apolipoprotein E (apoE) plays an important role in systemic and local lipid homeostasis. We have examined the expression of apoE during morphogenesis and regeneration of paired and unpaired fins and during scale development in zebrafish (Danio rerio). In situ hybridization analysis revealed that, during embryogenesis, apoE is expressed in the epithelial cells of the median fin fold and of the pectoral fin buds. ApoE remains expressed in the elongating fin folds throughout development of the fins. During the larval to juvenile transition, apoE transcripts were present in the distal, interray and lateral epidermis of developing fins. Furthermore, as scale buds started to form, apoE was expressed in large scale domains which later, became restricted to the external posterior epidermal part of scales. A low level of transcripts could be observed at later developmental stages at these locations probably because fins and scales continue to grow throughout the animal's life. During regeneration of both pectoral and caudal fins, a marked increase in apoE expression is observed as early as 12 hours after amputation in the wound epidermis. High levels of apoE transcripts are then localized primarily in the basal cell layer of the apical epidermis. The levels of apoE expression were maximum between the second to fourth days and then progressively declined to basal level by day 14. ApoE transcripts were also observed in putative macrophages infiltrated in the mesenchymal compartment of regenerating fins a few hours after amputation. In conclusion, apoE is highly expressed in the epidermis of developing fins and scales and during fin regeneration while no expression can be detected in the skin of the trunk. ApoE may play a specific role in fin and scale differentiation at sites where important epidermo-dermal interactions occur for the elaboration of the dermal skeleton and/or for lipid uptake and redistribution within these rapidly growing structures.

Animals↗

PubMed2004

Melanophore sublineage-specific requirement for zebrafish touchtone during neural crest development.

The specification, differentiation and maintenance of diverse cell types are of central importance to the development of multicellular organisms. The neural crest of vertebrate animals gives rise to many derivatives, including pigment cells, peripheral neurons, glia and elements of the craniofacial skeleton. The development of neural crest-derived pigment cells has been studied extensively to elucidate mechanisms involved in cell fate specification, differentiation, migration and survival. This analysis has been advanced considerably by the availability of large numbers of mouse and, more recently, zebrafish mutants with defects in pigment cell development. We have identified the zebrafish mutant touchtone (tct), which is characterized by the selective absence of most neural crest-derived melanophores. We find that although wild-type numbers of melanophore precursors are generated in the first day of development and migrate normally in tct mutants, most differentiated melanophores subsequently fail to appear. We demonstrate that the failure in melanophore differentiation in tct mutant embryos is due at least in part to the death of melanoblasts and that tct function is required cell autonomously by melanoblasts. The tct locus is located on chromosome 18 in a genomic region apparently devoid of genes known to be involved in melanophore development. Thus, zebrafish tct may represent a novel as well as selective regulator of melanoblast development within the neural crest lineage. Further, our results suggest that, like other neural crest-derived sublineages, melanogenic precursors constitute a heterogeneous population with respect to genetic requirements for development.

Alleles↗

PubMed2005

Zebrafish: a tool to study hemostasis and thrombosis.

PURPOSE OF REVIEW: In the past eight years our laboratory has developed the zebrafish model to study hemostasis and thrombosis. The purpose of this review is to explore current developments involving the zebrafish model in the study of hemostasis and thrombosis because the time is now ripe to apply this model to identify novel players that participate in hemostasis and thrombosis. RECENT FINDINGS: In the past twelve months, three papers appeared in the hemostasis and thrombosis area using the zebrafish model. The first one is a review article that summarizes establishment of the zebrafish model to study hemostasis and thrombosis. The second study is a methodological paper describing assays for measuring hemostasis and thrombosis by inducing vascular occlusion in zebrafish larvae. The third paper describes a knockdown of prothrombin in zebrafish, which recapitulates knockout studies in mouse, and marks the beginning of studies in the hemostasis and thrombosis area by this new knockdown technology. In addition to the above papers, there is one abstract that describes kinetics of thrombocyte and thrombocyte-microparticle recruitment in laser-induced arterial thrombus formation in zebrafish. SUMMARY: With the above advances, the zebrafish model has now matured to the point that it can address more important questions in the hemostasis and thrombosis area using genetic approaches. This review therefore summarizes the issues described in the above papers along with thoughts about future progress of the zebrafish model as a tool to study hemostasis and thrombosis.

Animals↗

PubMed1998

Left-right development in Xenopus and zebrafish.

One of the most striking features of the vertebrate body plan is that most exterior structures are bilaterally symmetric while many interior structures are left-right asymmetric. Left-right asymmetries are displayed in the heart, the circulatory, digestive and respiratory systems and in the central nervous system. A fundamental question in the study of all patterning events, including left-right axis formation, is how does asymmetry arise from apparent symmetry. A second important question that is perhaps unique to the study of left-right development, is how does the left-right axis align with the asymmetries that develop along the orthogonal axes; dorsal-ventral and anterior-posterior. Recent experiments in Xenopus laevis and zebrafish address both of these questions and have identified signaling molecules and interactions with midline cells that regulate left-right development.

Animals↗

PubMed2002

Role of hlx1 in zebrafish brain morphogenesis.

hlx1 is a related homeobox gene expressed in a dynamic spatiotemporal expression pattern during development of the zebrafish brain. The homologues of hlx1, mouse dbx1 and Xenopus Xdbx, are known to play a role in the specification of neurons in the spinal cord. However, the role of these molecules in the brain is less well known. We have used two different approaches to elucidate a putative function for hlx1 in the developing zebrafish brain. Blastomeres were injected with either synthetic hlx1 mRNA in gain-of-function experiments or with antisense morpholino oligonucleotides directed against hlx1 in loss-of-function experiments. Mis-expression of hlx1 produced severe defects in brain morphogenesis as a result of abnormal ventricle formation, a phenotype we referred to as "fused-brain". These animals also showed a reduction in the size of forebrain neuronal clusters as well as abnormal axon pathfinding. hlx1 antisense morpholinos specifically perturbed hindbrain morphogenesis leading to defects in the integrity of the neuroepithelium. While hindbrain patterning was in the most part unaffected there were select disruptions to the expression pattern of the neurogenic gene Zash1B in specific rhombomeres. Our results indicate multiple roles for hlx1 during zebrafish brain morphogenesis.

Animals↗

PubMed2005

A three-dimensional functional assessment of heart and vessel development in the larva of the zebrafish (Danio rerio).

There has been considerable recent interest in the development of the circulation in the zebrafish. Optical techniques typically used to visualize changes in heart size allow measurement of stroke volume during early vertebrate development, but this approach is complicated in zebrafish larvae because of the heart's irregular shape and its significant change in morphology during the first 6 d of development. By use of a three-dimensional integration of the early zebrafish heart and vessels, we have greatly reduced measurement error of stroke volume and cardiac output and have determined the cross-sectional growth of major vessels in the developing zebrafish larvae. A dramatic 500%-600% increase in cardiac output (from 10 to 50-60 nL min(-1)) occurs on days 5 and 6 postfertilization in Danio rerio. Cross-sectional area of key vessels (dorsal artery, caudal artery, dorsal vein) as well as between-individual variation significantly decreased over the first 6 d of development. Associated with the decrease in cross-sectional area is a significant increase in red blood cell velocity on days 5 and 6 postfertilization. Together, the three-dimensional data of the cardiac and vascular systems have shown that the most profound physiological and developmental changes occur in days 5 and 6, which corresponds with the appearance of the adult form of the heart and the transition from diffusive to convective O2 supply to internal tissues.

Animals↗

PubMed2003

Myostatin expression during development and chronic stress in zebrafish (Danio rerio).

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle mass in mammals. We have studied myostatin expression during embryonic and post-hatching development in zebrafish by semiquantitative RT-PCR. The transcript is present in just-fertilized eggs and declines at 8 h post-fertilization (hpf), suggesting a maternal origin. A secondary rise occurs at 16 hpf, indicating the onset of embryonic transcription at the time of muscle cell differentiation. The level of myostatin mRNA decreases slightly at 24 hpf, when somitogenesis is almost concluded, and rises again at and after hatching, during the period of limited muscle hyperplastic growth that is typical of slow-growing, small fish. In the adult muscle, we found the highest expression of myostatin mRNA and protein, which were detectable by Northern and Western blot analyses respectively. Although only the precursor protein form was revealed in the adult lateral muscle, we demonstrated that zebrafish myostatin is proteolytically processed and secreted in cultured cells, as is its mammalian counterpart. These results suggest that myostatin may play an important regulatory role during myogenesis and muscle growth in fish, as it does in mammals. In chronically stressed fish, grown from 16 days post-fertilization to adulthood in an overcrowded environment, we observed both depression of body growth and a diminished level of myostatin mRNA in the adult muscle, as compared with controls. We propose that chronic stunting in fish brings about a general depression of muscle protein synthesis which does not spare myostatin.

Animals↗

PubMed1999

Boron is required for zebrafish embryogenesis

Boron is the only element known to be essential for plants, but only circumstantial evidence for essentiality exists in animals. We report here that boron is essential for the embryonic development of zebrafish (Danio rerio). Zebrafish were maintained in water with a boron concentration of 0.1 micromol l-1 or supplemented to a concentration of 45 micromol l-1 using ultrapure boric acid. Both groups were fed boron-depleted brine shrimp. This procedure depleted the boron content of the blastulas from low-boron parents to only 5 % of the boron content of the blastulas from boron-supplemented parents. Sperm from low-boron males successfully fertilized eggs from low-boron females, but 92 % of the embryos died within 10 days. The early cleavage stage of development was the most sensitive to boron deficiency. Of the fertilized embryos, 46% did not live to complete the blastula stage. Repletion of low-boron embryos during the first hour after fertilization rescued them from death. These observations provide strong evidence that boron is essential for zebrafish development.

Journal Article↗