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PubMed2002

Cloning of two tryptophan hydroxylase genes expressed in the diencephalon of the developing zebrafish brain.

The monoamine serotonin (5-HT) exerts key neuromodulatory activities in all animal phyla, but the development and function of the serotonergic system is still incompletely understood. The zebrafish Danio rerio is an excellent model to approach this question since it is amenable to a combination of genetic, molecular and embryological studies. In order to characterize the organization of serotonergic neurons in the zebrafish we cloned two cDNAs encoding distinct forms of tryptophan hydroxylase (Tph), the rate-limiting enzyme in serotonin synthesis. We report here the pattern of expression of these two genes in relation with immunoreactive TH and 5-HT nuclei in the developing zebrafish embryo and early larva. tphD1 expression starts at 22 h post-fertilization (hpf) in the epiphysis and in basal spinal cells. Expression persists in the epiphysis until at least 4 days (dpf). Between 48 hpf and 3 dpf, tphD1 expression is initiated in retinal amacrine cells and in restricted preoptic and posterior tubercular nuclei within the basal diencephalon. At 3 and 4 dpf, tphD1 expression is newly initiated in the caudal hypothalamus and in branchial arches-associated neurons. tphD2 mRNA is detected transiently (between 30 somites and 32 hpf) in a restricted preoptic nucleus. All sites of tphD1 or D2 expression within the anterior central nervous system are also immunoreactive for 5-HT, but are not positive for TH. However, neither tphD gene is expressed in raphe nuclei, suggesting that additional tph gene(s) exist in zebrafish to account for 5-HT synthesis in that location. The co-expression of tphD1, tphD2 and 5-HT in the zebrafish diencephalon appears in striking contrast to the situation in mammals, where diencephalic serotonin results from re-uptake rather than from local production.

Amino Acid Sequence↗

PubMed2002

Expression of zebrafish cyp11a1 as a maternal transcript and in yolk syncytial layer.

Cyp11a1 (P450scc, cholesterol side-chain cleavage enzyme) is the first enzyme for the synthesis of all steroid hormones. The regulation of steroid synthesis has been extensively investigated, except during embryogenesis. To study steroidogenesis in embryos, we have isolated the zebrafish cyp11a1 gene, which consists of 11 exons. Reverse transcription-polymerase chain reaction analysis indicates that zebrafish cyp11a1 is expressed temporally in two waves during embryonic stages and when sexual differentiation begins. It is expressed in adult brain, testicular Leydig cells, and the granulosa/theca layer of the ovary. In addition, zebrafish cyp11a1 is expressed in oocytes, and is inherited as a maternal transcript in early embryos. Throughout zebrafish epiboly and segmentation stages, cyp11a1 is expressed in the yolk syncytial layer. At 36 h post fertilization, cyp11a1 transcript is located ventral to the third somite, where the primordial interrenal gland is located. In summary, zebrafish cyp11a1 is expressed in the cytoplasm of oocytes, as a maternal transcript, and in yolk syncytial layer during early embryogenesis.

Amino Acid Sequence↗

PubMedDate not supplied

Mapping absorbance spectra, cone fractions, and neuronal mechanisms to photopic spectral sensitivity in the zebrafish.

The four spectral cone types in the zebrafish retina each contribute to photopic visual sensitivity as measured by the b-wave of the electroretinogram (ERG). The goal of the current study was to evaluate a model of photopic b-wave spectral sensitivity in the zebrafish that mapped first-order cellular and biophysical aspects of cone photoreceptors (visual pigment absorbance spectra and cone fractions) onto a second-order physiological aspect of cone-derived neural activity in the retina. Good correspondence between the model and photopic ERG data was attained using new visual pigment absorbance data for zebrafish cones (lambda(max) of the L, M, and S cones were 564, 473, and 407 nm, respectively), visual pigment templates, and linearly gained cone fractions. The model inferred four distinct cone processing channels that contribute to the photopic b-wave, two of which are antagonistic combinations of cone-derived signals (L-M and M-S), and two of which are noncombinatorial signals from S and U cones. The nature of the gains and the processing channels suggested general rules of cone-specific inputs to second-order neurons. The model further suggested that the zebrafish retina utilizes neuronal mechanisms for enhancing sensitivity to luminance contrast at short wavelengths and chromatic contrast at middle and long wavelengths. The results indicated that first-order cellular and biophysical aspects of cone photoreceptors can successfully explain physiological aspects of cone-derived neuronal activity in the zebrafish retina.

Animals↗

PubMedDate not supplied

Visual pigment composition in zebrafish: Evidence for a rhodopsin-porphyropsin interchange system.

Numerous reports have concluded that zebrafish (Danio rerio) possesses A1-based visual pigments in their rod and cone photoreceptors. In the present study, we investigated the possibility that zebrafish have a paired visual pigment system. We measured the spectral absorption characteristics of photoreceptors from zebrafish maintained in different temperature regimes and those treated with exogenous thyroid hormone using CCD-based microspectrophotometry. Rods from fish housed at 15 degrees C and 28 degrees C were not significantly different, having lambda max values of 503 +/- 5 nm (n = 106) and 504 +/- 6 nm (n = 88), respectively. Thyroid hormone treatment (held at 28 degrees C), however, significantly shifted the lambda max of rods from 503 +/- 5 nm (n = 194) to 527 +/- 8 nm (n = 212). Cone photoreceptors in fish housed at 28 degrees C (without thyroid hormone treatment) had lambda max values of 361 +/- 3 nm (n = 2) for ultraviolet-, 411 +/- 5 nm (n = 18) for short-, 482 +/- 6 nm (n = 9) for medium-, and 565 +/- 10 nm (n = 14) for long-wavelength sensitive cones. Thyroid hormone treatment of fish held at 28 degrees C significantly shifted the lambda max of long-wavelength sensitive cones to 613 +/- 11 nm (n = 20), substantially beyond that of the lambda max of the longest possible A1-based visual pigment (approximately 580 nm). Thyroid hormone treatment produced smaller shifts of lambda max in other cone types and increased the half-band width. All shifts in photoreceptor lambda max values resulting from thyroid hormone treatment matched predictions for an A1- to A2-based visual pigment system. We therefore conclude that zebrafish possess a rhodopsin-porphyropsin interchange system that functions to spectrally tune rod and cone photoreceptors. We believe that these observations should be carefully considered during analysis of zebrafish spectral sensitivity.

Animals↗

PubMedDate not supplied

Neurogenesis in the visual system of embryonic and adult zebrafish (Danio rerio). off.

The zebrafish has recently assumed a central position in the study of vertebrate development. Numerous studies of other fish have shown that their central nervous systems, and especially their visual systems, continue to add new neurons throughout life, which is probably related to their abilities to regenerate axons and whole nervous tissue. Retinal neurogenesis had not been examined in adult zebrafish, and two reports concluded that the optic tectum ceased neurogenesis early in life, so the question arose whether the zebrafish was anomalous in this regard. We labeled embryonic (24- and 48-h postfertilization) and adult zebrafish with the thymidine analog, bromo-deoxyuridine, and, after short and long survivals, examined the retina and brain for labeled cells. They were abundant in both the optic tectum and the retina. Although the rate of retinal growth slows considerably between embryonic and adult stages, the patterns of neurogenesis in both the embryo and the adult are similar to those described in other fish, so these "fish-specific" features of general interest can justifiably be studied in zebrafish.

Animals↗

PubMedDate not supplied

ERG assessment of zebrafish retinal development.

Research has shown that adult zebrafish have a complex visual system, with two possible opponent mechanisms. Anatomically, zebrafish retina develops in a sequential manner and is immature at hatching. The purpose of the present study was to assess zebrafish retinal development using the electroretinogram (ERG). ERG responses to visual stimuli were obtained from 4-5, 6-8, 13-15, and 21-24 days postfertilization (dpf) zebrafish. Individual waveforms were assessed and compared across the four age groups. Spectral-sensitivity functions were calculated for the a- and b-wave components of the ERG response. Results showed that the ERG waveforms and spectral-sensitivity functions varied with age. While the 21-24 dpf subjects had an ERG waveform that was similar to that of adults, the younger subjects did not. Although there were modest differences in the a-wave spectral sensitivity, substantial differences were found in the b-wave spectral sensitivities across the ages. There was a consistent strong response to ultraviolet wavelengths, while across the remaining parts of the spectrum, there was a gradual increase in sensitivity with age. Also, the 21-24 dpf subjects appear to have adult-like U- and S-cone functions, but were missing the L-M and the M-S opponent mechanisms found in the adult. These results support the findings of the anatomical studies and demonstrate that the zebrafish is a useful model for examining the development of retinal function.

Animals↗

PubMed2003

Chemical discovery and global gene expression analysis in zebrafish.

The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.

Animals↗

PubMed1998

A microsatellite genetic linkage map for zebrafish (Danio rerio).

We have constructed a zebrafish genetic linkage map consisting of 705 simple sequence-length polymorphism markers (SSLPs). The map covers 2350 centimorgans (cM) of the zebrafish genome with an average resolution of 3.3 cM. It is a complete map in genetic mapping terms (there is one linkage group for each of the 25 chromosomes), and it has been confirmed by somatic-cell hybrids and centromere-mapping using half-tetrad analysis. The markers are highly polymorphic in the zebrafish strains used for genetic crosses and provide a means to compare genetic segregation of developmental mutations between laboratories. These markers will provide an initial infrastructure for the positional cloning of the nearly 600 zebrafish genes identified as crucial to vertebrate development,and will become the anchor for the physical map of the zebrafish genome.

Animals↗

PubMed2005

Conserved structural, pharmacological and functional properties among the three human and five zebrafish alpha 2-adrenoceptors.

1. Zebrafish has five distinct alpha(2)-adrenoceptors. Two of these, alpha(2Da) and alpha(2Db), represent a duplicated, fourth alpha(2)-adrenoceptor subtype, while the others are orthologue of the human alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors. Here, we have compared the pharmacological properties of these receptors to infer structural determinants of ligand interactions. 2. The zebrafish alpha(2)-adrenoceptors were expressed in Chinese hamster ovary cells and tested in competitive ligand binding assays and in a functional assay (agonist-stimulated [(35)S]GTPgammaS binding). The affinity results were used to cluster the receptors and, separately, the ligands using both principal component analysis and binary trees. 3. The overall ligand binding characteristics, the order of potency and efficacy of the tested agonists and the G-protein coupling of the zebrafish and human alpha(2)-adrenoceptors, separated by approximately 350 million years of evolution, were found to be highly conserved. The binding affinities of the 20 tested ligands towards the zebrafish alpha(2)-adrenoceptors are generally comparable to those of their human counterparts, with a few compounds showing up to 40-fold affinity differences. 4. The alpha(2A) orthologues and the zebrafish alpha(2D) duplicates clustered as close pairs, but the relationships between the orthologues of alpha(2B) and alpha(2C) were not clearly defined. Applied to the ligands, our clustering methods segregated the ligands based on their chemical structures and functional properties. As the ligand binding pockets formed by the transmembrane helices show only minor differences among the alpha(2)-adrenoceptors, we suggest that the second extracellular loop--where significant sequence variability is located --might contribute significantly to the observed affinity differences.

Animals↗

PubMed1997

Scale development in zebrafish (Danio rerio).

In the course of an extensive comparative, structural and developmental study of the cranial and postcranial dermal skeleton (teeth and scales) in osteichthyan fishes, we have undertaken investigations on scale development in zebrafish (Danio (Brachydanio) rerio) using alizarin red staining, and light and transmission electron microscopy. The main goal was to know whether zebrafish scales can be used as a model for further research on the processes controlling the development of the dermal skeleton in general, especially epithelial-mesenchymal interactions. Growth series of laboratory bred specimens were used to study in detail: (1) the relationship of scale appearance with size and age; (2) the squamation pattern; and (3) the events taking place in the epidermis and in the dermis, before and during scale initiation and formation, with the aim of searching for morphological indications of epithelial-mesenchymal interactions. Scales form late in ontogeny, generally when zebrafish are more than 8.0 mm in standard length. Within a population of zebrafish of the same age scale appearance is related to standard length, but when comparing populations of different age the size of the fish at scale appearance is also related to age. Scales always appear first in the posterior region of the body and the squamation then extends anteriorly. Scales develop in the dermis but closely apposed to the epidermal-dermal boundary. Cellular modifications occurring in the basal layer of the epidermis and in the dermis before scale formation clearly indicate that the basal epidermal cells differentiate first, before any evidence of differentiation of the progenitors of the scale-forming cells in the dermis. This strongly suggests that scale differentiation could be initiated by the epidermal basal layer cells which probably produce a molecular signal towards the dermis below. Subsequently dermal cells accumulate close to the epidermis, and differentiate to form scale papillae. The late formation of the scales during ontogeny is due to a late colonisation of the dermis by the progenitors of the scale-forming cells. Because of their late formation during ontogeny and of their regular pattern of development, scales in zebrafish represent a good model for further investigations on the general mechanisms of epithelial-mesenchymal interactions during dermal skeleton development, and in particular for the study of the gene expression patterns.

Animals↗

PubMed1998

Cloning of zebrafish neurofilament cDNAs for plasticin and gefiltin: increased mRNA expression in ganglion cells after optic nerve injury.

During retinal growth and optic axon regeneration, the differential expression of the neuronal intermediate filament proteins, plasticin and gefiltin, in the goldfish visual pathway suggests that these proteins support programmed axonal growth. To investigate plasticin and gefiltin during axonogenesis, we turned to the zebrafish, a system that is more amenable to mutational analysis. As a first step, we demonstrated that the intermediate filament compositions of goldfish and zebrafish are similar. In addition, the cDNAs for zebrafish plasticin and gefiltin were cloned and characterized. Using in situ hybridization in retina, we show increased mRNA levels for these proteins following optic nerve crush. Zebrafish plasticin and gefiltin peak and return to baseline levels of expression more rapidly than in goldfish. Furthermore, in the unoperated eye of experimental fish, there was a moderate increase in the levels of plasticin and gefiltin mRNA, suggesting that soluble factors influence the expression of these proteins. The successive expression of plasticin and gefiltin suggests that these neuronal intermediate filament proteins are integral components of axonogenesis. The cloning and characterization of cDNAs for plasticin and gefiltin permit mutational analyses of these proteins during zebrafish axonogenesis.

Animals↗

PubMed2000

Zebrafish genes for neuropeptide Y and peptide YY reveal origin by chromosome duplication from an ancestral gene linked to the homeobox cluster.

Neuropeptide Y (NPY) and peptide YY (PYY) are related 36-amino acid peptides. NPY is widely distributed in the nervous system and has several physiological roles. PYY serves as an intestinal hormone as well as a neuropeptide. We report here cloning of the npy and pyy genes in zebrafish (Danio rerio). NPY differs at only one to four amino acid positions from NPY in other jawed vertebrates. Zebrafish PYY differs at three positions from PYY from other fishes and at 10 positions from mammals. In situ hybridization showed that neurons containing NPY mRNA have a widespread distribution in the brain, particularly in the telencephalon, optic tectum, and rhombencephalon. PYY mRNA was found mainly in brainstem neurons, as reported previously for vertebrates as divergent as the rat and the lamprey, suggesting an essential role for PYY in these neurons. PYY mRNA was observed also in the telencephalon. These results were confirmed by immunocytochemistry. As in the human, the npy gene is located adjacent to homeobox (hox) gene cluster A (copy a in zebrafish), whereas the pyy gene is located close to hoxBa. This suggests that npy and pyy arose from a common ancestral gene in a chromosomal duplication event that also involved the hox gene clusters. As zebrafish has seven hox clusters, it is possible that additional NPY family genes exist or have existed. Also, the NPY receptor system seems to be more complex in zebrafish than in mammals, with at least two receptor genes without known mammalian orthologues.

Amino Acid Sequence↗

PubMed2002

A call to fins! Zebrafish as a gerontological model.

Among the wide variety of model organisms commonly used for studies on aging, such as worms, flies and rodents, a wide research gap exists between the invertebrate and vertebrate model systems. In developmental biology, a similar gap has been filled by the zebrafish (Danio rerio). We propose that the zebrafish is uniquely suited to serve as a bridge model for gerontology. With high fecundity and economical husbandry requirements, large populations of zebrafish may be generated quickly and cheaply, facilitating large-scale approaches including demographic studies and mutagenesis screens. A variety of mutants identified in such screens have led to modelling of human disease, including cardiac disorders and cancer. While zebrafish longevity is at least 50% longer than in commonly used mouse strains, as an ectothermic fish species, its life span may be readily modulated by caloric intake, ambient temperature and reproductive activity. These features, coupled with a growing abundance of biological resources, including an ongoing genome sequencing project, make the zebrafish a compelling model organism for studies on aging.

Aging↗

PubMed2005

Mutation in sodium-calcium exchanger 1 (NCX1) causes cardiac fibrillation in zebrafish.

Cardiac fibrillation, a form of cardiac arrhythmia, is the most common cause of embolic stroke and death associated with heart failure. The molecular mechanisms underlying cardiac fibrillation are largely unknown. Here we report a zebrafish model for cardiac fibrillation. The hearts of zebrafish tremblor (tre) mutants exhibit chaotic movements and fail to develop synchronized contractions. Calcium imaging showed that normal calcium transients are absent in tre cardiomyocytes, and molecular cloning of the tre mutation revealed that the tre locus encodes the zebrafish cardiac-specific sodium-calcium exchanger (NCX) 1, NCX1h. Forced expression of NCX1h or other calcium-handling molecules restored synchronized heartbeats in tre mutant embryos in a dosage-dependent manner, demonstrating the critical role of calcium homeostasis in maintaining embryonic cardiac function. By creating mosaic zebrafish embryos, we showed that sporadic NCX1h-null cells were not sufficient to disrupt normal cardiac function, but clustered wild-type cardiomyocytes contract in unison in tre mutant hearts. These data signify the essential role of calcium homeostasis and NCX1h in establishing rhythmic contraction in the embryonic zebrafish heart.

Animals↗

PubMed2005

A zebrafish bmyb mutation causes genome instability and increased cancer susceptibility.

A major goal of cancer research has been to identify genes that contribute to cancer formation. The similar pathology between zebrafish and human tumors, as well as the past success of large-scale genetic screens in uncovering human disease genes, makes zebrafish an ideal system in which to find such new genes. Here, we show that a zebrafish forward genetic screen uncovered multiple cell proliferation mutants including one mutant, crash&burn (crb), that represents a loss-of-function mutation in bmyb, a transcriptional regulator and member of a putative proto-oncogene family. crb mutant embryos have defects in mitotic progression and spindle formation, and exhibit genome instability. Regulation of cyclin B levels by bmyb appears to be the mechanism of mitotic accumulation in crb. Carcinogenesis studies reveal increased cancer susceptibility in adult crb heterozygotes. Gene-expression signatures associated with loss of bmyb in zebrafish are also correlated with conserved signatures in human tumor samples, and down-regulation of the B-myb signature genes is associated with retention of p53 function. Our findings show that zebrafish screens can uncover cancer pathways, and demonstrate that loss of function of bmyb is associated with cancer.

Animals↗

PubMed1996

Active transposition in zebrafish.

We have identified a new family of Tc1-like transposons in the zebrafish, Danio rerio. The sequence of a candidate active transposon, deduced from sample Tzf elements, shows limited resemblance to the previously described Tdr1 elements of zebrafish. Both the Tzf and the Tdr elements are extremely abundant in zebrafish. We describe here a general strategy for detecting transposition events in a complex genome and demonstrate its utility by selectively monitoring hundreds of potentially active Tzf copies in the zebrafish genome against a background of other related elements. We have followed members of a zebrafish pedigree, using this two-dimensional transposon display strategy, to identify the first examples of active transposition of such elements in vertebrates.

Amino Acid Sequence↗

PubMed2003

Two zebrafish eIF4E family members are differentially expressed and functionally divergent.

Eukaryotic translation initiation factor 4E (eIF4E) is an essential component of the translational machinery that binds m(7)GTP and mediates the recruitment of capped mRNAs by the small ribosomal subunit. Recently, a number of proteins with homology to eIF4E have been reported in plants, invertebrates, and mammals. Together with the prototypical translation factor, these constitute a new family of structurally related proteins. To distinguish the prototypical translation factor eIF4E from other family members, it has been termed eIF4E-1 (Keiper, B. D., Lamphear, B. J., Deshpande, A. M., Jankowska-Anyszka, M., Aamodt, E. J., Blumenthal, T., and Rhoads, R. E. (2000) J. Biol. Chem. 275, 10590-10596). We describe the characterization of two eIF4E family members in the zebrafish Danio rerio. Based on their relative identities with human eIF4E-1, these zebrafish proteins are termed eIF4E-1A (82%) and eIF4E-1B (66%). eIF4E-1B, originally termed eIF4E(L), has been reported previously as the zebrafish eIF4E-1 counterpart (Fahrenkrug, S. C., Dahlquist, M. O., Clark, K., and Hackett, P. B. (1999) Differentiation 65, 191-201; Fahrenkrug, S. C., Joshi, B., Hackett, P. B., and Jagus, R. (2000) Differentiation 66, 15-22). Sequence comparisons suggest that the two genes probably evolved from a duplication event that occurred during vertebrate evolution. eIF4E-1A is expressed ubiquitously in zebrafish, whereas expression of eIF4E-1B is restricted to early embryonic development and to gonads and muscle of the tissues investigated. The ability of these two zebrafish proteins to bind m(7)GTP, eIF4G, and 4E-BP, as well as to complement yeast conditionally deficient in functional eIF4E, show that eIF4E-1A is a functional equivalent of human eIF4E-1. Surprisingly, although eIF4E-1B possesses all known residues thought to be required for interaction with the cap structure, eIF4G, and 4E-BPs, it fails to interact with any of these components, suggesting that this protein serves a role other than that assigned to eIF4E.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Proximal and distal sequences control UV cone pigment gene expression in transgenic zebrafish.

The molecular basis of cone photoreceptor-specific gene expression is largely unknown. In this study, we define cis-acting DNA sequences that control the cell type-specific expression of the zebrafish UV cone pigment gene by transient expression of green fluorescent protein transgenes following their injection into zebrafish embryos. These experiments show that 4.8 kb of 5'-flanking sequences from the zebrafish UV pigment gene direct expression specifically to UV cones and that this activity requires both distal and proximal sequences. In addition, we demonstrate that a proximal region located between -215 and -110 bp (with respect to the initiator methionine codon) can function in the context of a zebrafish rhodopsin promotor to convert its specificity from rod-only expression to rod and UV cone expression. These experiments demonstrate the power of transient transgenesis in zebrafish to efficiently define cis-acting regulatory sequences in an intact vertebrate.

5' Untranslated Regions↗