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Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter.

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

Amino Acid Sequence↗

Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone.

In a partial life-cycle test, the impact of 17alpha-ethinylestradiol (EE2) and 17alpha-methyltestosterone (MT) on juvenile zebrafish was evaluated by use of vitellogenin measurements and gonadal development. Exposure to EE2 (1-25 ng/l) resulted in a dose-dependent increase in vitellogenin production starting at 2 ng/l. Significant changes in sex ratios in female direction were detected at 1 ng/l, with complete sex reversal taking place after exposure to 2 ng/l. No intersex fish were observed after exposure to EE2. Exposure to MT resulted in decreased vitellogenin concentrations. Complete sex reversal was detected in all MT concentrations used (26-1000 ng/l). A large proportion of intersex fish was observed after exposure to 1000 ng MT/l. The period of gonadal sex reversal in non-exposed zebrafish was also studied. The main morphological features of the transformation of ovaries into testis were observed 4-5 weeks after hatching.

Animals↗

Genome structure and thymic expression of an endogenous retrovirus in zebrafish.

In a search for previously unknown genes that are required for lymphocyte development in zebrafish, a retroviral sequence was identified in a subtracted thymus cDNA library and in genomic DNA libraries. The provirus is 11.2 kb and contains intact open reading frames for the gag, pol, and env genes, as well as nearly identical flanking long terminal repeat sequences. As determined by in situ hybridization, the thymus appears to be a major tissue for retroviral expression in both larval and adult fish. Several viral transcripts were found by Northern blotting in the adult thymus. The provirus was found at the same genomic locus in sperm from four fish, suggesting that it is an endogenous retrovirus. Phylogenetic analysis indicates that it is closest to, yet distinct from, the cluster of murine leukemia virus-related retroviruses, suggesting that this virus represents a new group of retroviruses.

Animals↗

Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish.

Neuropilin-1 (NRP1) is a cell-surface receptor for both vascular endothelial growth factor(165) (VEGF(165)) and class 3 semaphorins that is expressed by neurons and endothelial cells. NRP1 is required for normal developmental angiogenesis in mice. The zebrafish is an excellent system for analyzing vascular development. Zebrafish intersegmental vessels correspond to mammalian capillary sprouts, whereas the axial vessels correspond to larger blood vessels, such as arteries. The zebrafish NRP1 gene (znrp1) was isolated and when overexpressed in cells, zNRP1 protein was a functional receptor for human VEGF(165). Whole-mount in situ hybridization showed that transcripts for znrp1 during embryonic and early larval development were detected mainly in neuronal and vascular tissues. Morpholino-mediated knockdown of zNRP1 in embryos resulted in vascular defects, most notably impaired circulation in the intersegmental vessels. Circulation via trunk axial vessels was not affected. Embryos treated with VEGF receptor-2 kinase inhibitor had a similar intersegmental vessel defect suggesting that knockdown of zNRP1 reduces VEGF activity. To determine whether NRP1 and VEGF activities were interdependent in vivo, zNRP1 and VEGF morpholinos were coinjected into embryos at concentrations that individually did not significantly inhibit blood vessel development. The result was a potent inhibition of blood cell circulation via both intersegmental and axial vessels demonstrating that VEGF and NRP1 act synergistically to promote a functional circulatory system. These results provide the first physiological demonstration that NRP1 regulates angiogenesis through a VEGF-dependent pathway.

Animals↗

Transient expression of a novel Six3-related zebrafish gene during gastrulation and eye formation.

Both the Drosophila homeobox gene sine oculis and its murine homologue Six3 have regulatory functions in eye development. In zebrafish, in addition to two previously reported homologues of murine Six3, we have identified a related gene (six7). Although the deduced Six7 protein shares less than 68% sequence identity with the other known zebrafish Six3-like proteins, the embryonic expression patterns have highly conserved features. The six7 transcripts are first detected in involuting axial mesendoderm and, subsequently, in the overlying neurectoderm from which the forebrain and optic primordia develop. Similar to the two other zebrafish Six3 homologues, the expression boundaries of six7 correspond quite closely with the edges of the optic vesicles. Hence, the partially overlapping expression domains of these three six genes probably contribute to anteroposterior specification and in defining the eye primordia.

Amino Acid Sequence↗

Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.

Defects in cardiac valve morphogenesis and septation of the heart chambers constitute some of the most common human congenital abnormalities. Some of these defects originate from errors in atrioventricular (AV) endocardial cushion development. Although this process is being extensively studied in mouse and chick, the zebrafish system presents several advantages over these models, including the ability to carry out forward genetic screens and study vertebrate gene function at the single cell level. In this paper, we analyze the cellular and subcellular architecture of the zebrafish heart during stages of AV cushion and valve development and gain an unprecedented level of resolution into this process. We find that endocardial cells in the AV canal differentiate morphologically before the onset of epithelial to mesenchymal transformation, thereby defining a previously unappreciated step during AV valve formation. We use a combination of novel transgenic lines and fluorescent immunohistochemistry to analyze further the role of various genetic (Notch and Calcineurin signaling) and epigenetic (heart function) pathways in this process. In addition, from a large-scale forward genetic screen we identified 55 mutants, defining 48 different genes, that exhibit defects in discrete stages of AV cushion development. This collection of mutants provides a unique set of tools to further our understanding of the genetic basis of cell behavior and differentiation during AV valve development.

Animals↗

Green fluorescent fusion proteins: powerful tools for monitoring protein expression in live zebrafish embryos.

The recent development of transgenic technology in zebrafish has opened an exciting new avenue in which to explore vertebrate development. However, as in other species, the inability to easily identify live transgenic fish severely limits the potential of this promising technology. To determine whether the recently described green fluorescent protein (GFP) might provide a convenient live staining method in zebrafish, we constructed a glutathione S-transferase/GFP fusion protein (GST-GFP). GST-GFP cRNA, when injected into individual blastomeres of early zebrafish embryos, resulted in the rapid development (3 hr) of easily detectable green fluorescence which persisted for up to 4 days. GFP fluorescence was restricted to progeny of the injected cell and appeared to have no adverse effects on embryonic development despite widespread expression. Our findings demonstrate that GFP fusion proteins will provide a simple yet powerful means of monitoring production of heterologous proteins in live zebrafish.

Animals↗

The bHLH transcription factor hand2 plays parallel roles in zebrafish heart and pectoral fin development.

The precursors of several organs reside within the lateral plate mesoderm of vertebrate embryos. Here, we demonstrate that the zebrafish hands off locus is essential for the development of two structures derived from the lateral plate mesoderm - the heart and the pectoral fin. hands off mutant embryos have defects in myocardial development from an early stage: they produce a reduced number of myocardial precursors, and the myocardial tissue that does form is improperly patterned and fails to maintain tbx5 expression. A similar array of defects is observed in the differentiation of the pectoral fin mesenchyme: small fin buds form in a delayed fashion, anteroposterior patterning of the fin mesenchyme is absent and tbx5 expression is poorly maintained. Defects in these mesodermal structures are preceded by the aberrant morphogenesis of both the cardiogenic and forelimb-forming regions of the lateral plate mesoderm. Molecular analysis of two hands off alleles indicates that the hands off locus encodes the bHLH transcription factor Hand2, which is expressed in the lateral plate mesoderm starting at the completion of gastrulation. Thus, these studies reveal early functions for Hand2 in several cellular processes and highlight a genetic parallel between heart and forelimb development.

Animals↗

Zebrafish cypher is important for somite formation and heart development.

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.

Alternative Splicing↗

Development of the retinotectal projection in zebrafish embryos under TTX-induced neural-impulse blockade.

The influence of neural activity on the morphology of retinal-axon-terminal arbors and the precision of the developing retinotectal projection in zebrafish embryos was explored. Terminal-arbor morphology and their distribution in the tectum was determined with anatomical fiber-tracing methods using the fluorescent dyes dil and diO. To allow development under activity-deprived conditions, TTX was injected into the eyes of 30-38-hr-old zebrafish embryos at concentrations that effectively blocked neural activity both in retinal ganglion cells and throughout the CNS. Much like axons with normal neural-activity patterns, activity-deprived axons from dorsal and ventral and from temporal and nasal regions in the retina terminated over retinotopically appropriate and nonoverlapping regions of the tectum. Even after ablation of 1 hemiretina at the time of axonal outgrowth, activity-deprived axons from the remaining hemiretina grew directed toward and arborized selectively within their retinotopically appropriate tectal half in the same way as would nondeprived axons. Besides being retinotopic, the area over which small populations of activity-deprived axons from neighboring ganglion cells arborize is as small as that of active axons. The size of terminal arbors of retinal ganglion cell axons was unaffected by blockade of neural activity. The mean terminal-arbor size was 27 x 18 microns for the TTX-injected and 31 x 22 microns for the control embryos. The tectal coverage of TTX-blocked and control axons was equally small, with values of 1.4% and 1.6%, respectively. These data show that a precisely organized retinotopic map in developing zebrafish forms independent of neural-impulse activity.

Animals↗

The zebrafish fth1, slc3a2, men1, pc, fgf3 and cycd1 genes define two regions of conserved synteny between linkage group 7 and human chromosome 11q13.

In addition to being an excellent model system for studying vertebrate development, the zebrafish has become a great tool for gene discovery by mutational analysis. The recent availability of the zebrafish EST database and radiation hybrid mapping panels has dramatically expanded the framework for genomic research in this species. Developing comparative maps of the zebrafish and human genomes is of particular importance for zebrafish mutagenesis studies in which human orthologs are sought for zebrafish genes. However, only partial cDNA sequences are determined routinely for mapped ESTs, leaving the identity of the EST in question. It previously had been reported that zebrafish linkage group 7 shares conserved synteny with human chromosome 11q13. In an effort to further define this relationship, five full-length zebrafish cDNAs, fth1, slc3a2, prkri, cd81, and pc, as well as one putative human gene, DBX were identified and their map positions ascertained. These six genes, along with men1, fgf3 and cycd1 define two regions of conserved synteny between linkage group 7 and 11q13.

Amino Acid Sequence↗

Cloning and developmental expression of the DEC1 ortholog gene in zebrafish.

DEC1/STRA13/SHARP2 is a transcription factor of the bHLH family that has been suggested to play key roles in mammalian cell differentiation, the cell cycle and circadian regulation. However, the function of the DEC1 gene during embryogenesis is not well understood. In the present study, we cloned a zebrafish ortholog of the human DEC1 gene and analyzed its expression during development of zebrafish. The predicted protein encoded by zebrafish DEC1 consists of 403 amino acids, and shares 59%, 60% and 59% identity in overall amino acid sequence with human DEC1, mouse STRA13 and rat SHARP2, respectively. Zebrafish DEC1 contains a bHLH domain exhibiting 97% identity with that of the mammalian ortholog. During zebrafish embryogenesis, DEC1 is expressed in a strong ubiquitous manner before early segmentation. At 15-72 hpf, DEC1 shows a specific and dynamic expression in the developing eyes, somites, pineal gland (epiphysis), heart, brain, spinal cord, notochord, pronephric duct, common cardinal vein and blood cells. In older zebrafish, DEC1 also is expressed in multiple tissues including the brain, eye, gut, liver and pancreas. Our data provide evidence that expression of DEC1 is evolutionally conserved in zebrafish.

Amino Acid Sequence↗

Intraocular pressure in zebrafish: comparison of inbred strains and identification of a reduced melanin mutant with raised IOP.

PURPOSE: The goal was to establish an accurate method for measuring intraocular pressure (IOP) in living adult zebrafish and to determine the average IOPs of genetically different strains and mutants. The overall purpose was to develop the zebrafish system for studies of IOP and glaucoma. Elevated IOP and anterior segment dysgenesis are two known risk factors for glaucoma. Because albino mammals have increased IOP and are prone to anterior segment anomalies, the study was focused on a reduced melanin zebrafish mutant, brass. METHODS: Servo-null electrophysiology was used to measure IOP in anesthetized adult zebrafish. A pulled-glass microelectrode was used to penetrate the cornea into the anterior chamber, and pressure transduction was recorded. IOP, histology, and visual behavioral assays were used to assess the brass mutant. RESULTS: Significant differences in IOP were detected between genetically distinct zebrafish strains. IOP averages were highest in the long fin (LF) strain (20.5 +/- 1.2 mm Hg) and lowest in the Oregon AB (AB) strain (10.8 +/- 0.3 mm Hg). In brass mutants, which show a reduced melanin phenotype, IOPs were elevated and mild iris hypoplasia was noted. No defects were observed within the retina or in visual function. CONCLUSIONS: In the present study, an accurate method was developed for measuring IOP in adult zebrafish. Both the ability to measure IOP in the genetically powerful zebrafish model and the conservation of elevated IOP and anterior segment anomalies between albino mammals and hypopigmented fish are important. They strongly support the use of zebrafish for studying the complex genetics of elevated IOP and glaucoma.

Animals↗

Development of a circadian melatonin rhythm in embryonic zebrafish.

We investigated the time course of circadian system development in zebrafish and the role of environmental light cycles in this process, using a rhythm in melatonin content of embryos and larvae as a marker of circadian function. When zebrafish were raised in a cycle of 14 h light and 10 h dark at 28.5 degrees C, nocturnal increases in melatonin content were detectable beginning on the second night post-fertilization (PF). When embryos were transferred to constant darkness (DD) at the end of the second light period, a circadian rhythm of melatonin content persisted for at least three cycles. However, when embryos were transferred from light to DD at 14 h PF, no rhythm was detectable in the population. Phase-locked circadian melatonin rhythms were measurable after embryos were exposed to a transition from constant light (LL) to darkness at 26 or 32 h PF, but not at 20 h. These data indicate that a circadian oscillator that regulates melatonin synthesis becomes functional and responsive by light between 20 and 26 h PF. At this stage, pineal photoreceptors have begun to differentiate, but retinal photoreceptors have not, suggesting that the first circadian melatonin rhythms are of pineal origin. The absence of melatonin rhythms in populations of embryos exposed to DD beginning at earlier stages indicates that there is no timed developmental event that sets the circadian clock in the absence of environmental input. Exposure to DD starting at 14 or 20 h PF did not retard overall development as determined by gross morphological staging criteria, and did not prevent later synchronization of melatonin rhythms by light-dark (LD) cycles.

Animals↗

Targeted "knockdown" of channel expression in vivo with an antisense morpholino oligonucleotide.

We have examined whether antisense morpholino oligonucleotides (morpholinos) can be used as a tool to suppress or "knockdown" the expression of ion channels during development of the zebrafish. Because the acetylcholine receptor channel is well characterized in zebrafish and is abundant as skeletal muscle is found throughout the body, we sought to knock down its expression as a general test of the feasibility of this approach. A 25-mer morpholino was designed to target the 5' region of the cloned alpha-subunit and was injected into early stage blastulae in order to trap it in all developing cells. From the time of hatching (early on the third day of development) and for a few days after, a fraction of the injected embryos were immobile, i.e. were "morphant". Injection of blastulae without the morpholino or with a control morpholino containing four mispaired bases did not affect the embryos. Although the morphant embryos were generally normal in appearance, they lacked staining with alpha-bungarotoxin or an alpha-subunit-specific monoclonal antibody. In whole muscle cell recordings from morphant embryos, miniature end-plate potentials were undetectable in many of the cells and in most they had a slower, immature time course. These results are consistent with a greatly reduced, dysfunctional level of expression of acetylcholine receptors in morphant embryos. Because of their stability and specificity, morpholinos should prove useful for targeted deletion of transmitter receptors and channels in developing zebrafish and possibly in other preparations.

Animals↗

The development of the paired fins in the zebrafish (Danio rerio).

In the present study, we describe the structure and normal development of the zebrafish (Danio rerio) paired fins. Particularly, we focus on the structure of the apical epidermis and on endoskeletal morphogenesis. Endoskeletal development proceeds differently in the pectoral and pelvic fins. Whereas in both fins major parts of the endoskeletal girdle develop within the fin bud mesenchyme, the pattern of chondrogenic condensations observed in the pelvic fins directly reflects the adult endoskeletal pattern. In the pectoral fin, a morphogenetic detour is taken via a functional larval endoskeleton, the endoskeletal disc. It arises in the fin bud mesenchyme from a chondrogenic anlage common with the girdle. The disc chondrifies and represents the functional endoskeleton of the larval pectoral fin. The pectoral fin endoskeleton is expanded as well as restructured during larval stages in a process which involves decomposition of cartilage matrix in the endoskeletal disc. Our comparisons of apical fold morphology with reports on other teleosts and tetrapod apical ridges show them to be homologous on the structural level. Comparisons of endoskeletal development of the zebrafish with reports on teleosts, actinopterygians and chondrichthyans show that endoskeletal morphogenesis in the zebrafish pectoral fin follows a morphogenetic process which is wide-spread among actinopterygians.

Animals↗

Identification of a promoter element within the zebrafish colXalpha1 gene responsive to runx2 isoforms Osf2/Cbfa1 and til-1 but not to pebp2alphaA2.

Type X collagen is a short chain collagen specifically expressed by hypertrophic chondrocytes during endochondral ossification. We report here the functional analysis of the zebrafish (Danio rerio) collagen Xalpha1 gene (colXalpha1) promoter with the identification of a region responsive to two isoforms of the runt domain transcription factor runx2. Furthermore, we provide evidence for the presence of dual promoter usage in zebrafish, a finding that should be important to further understanding of the regulation of its restricted tissue distribution and spatial-temporal expression during early development. The zebrafish colXalpha1 gene structure is comparable to that recently identified by comparative genomics in takifugu and shows homology with corresponding mammalian genes, indicating that its general architecture has been maintained throughout vertebrate evolution. Our data suggest that, as in mammals, runx2 plays a role in the development of the osteogenic lineage, supporting zebrafish as a model for studies of bone and cartilage development.

Amino Acid Sequence↗