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PubMed2004

Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism.

The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.

Animals↗

PubMed2026

Development of the zebrafish foveal analogue: a quantitative atlas of high-acuity zone growth and retinal regionalisation.

The vertebrate retina contains specialised regions for high-acuity vision, exemplified by the human fovea and its zebrafish analogue, the high-acuity zone (HAZ). Despite the widespread use of zebrafish to model retinal disease, a stage-resolved quantitative reference describing normal eye, photoreceptor layer (PRL) and lens growth has been lacking. Here, we apply contrast-enhanced micro-computed tomography (micro-CT) to construct the first three-dimensional micro-CT normative atlas of wild-type zebrafish eye development across five larval stages [3, 5, 7, 10 and 18 days post-fertilisation (dpf)], mapping circumferential PRL thickness, eye and lens morphology, and compartment growth rates. Regional PRL thickening within the temporo-ventral region of the expected HAZ emerged by 5 dpf and was sustained by a localised redistribution of growth, persisting and extending towards the optic nerve through 18 dpf. The PRL, lens and eye grew through four phases, alternating between disproportionate PRL expansion and coordinated growth, while the eye remodelled from a nasal-dominant to a temporo-ventral-dominant form. This regional specialisation was protracted relative to gross ocular growth and could proceed independently of it, paralleling the extended postnatal maturation of the human fovea. This atlas provides a quantitative baseline for distinguishing disease-induced changes from normal variation, supporting zebrafish models of foveal hypoplasia and related disorders.

Animals↗

PubMed2004

A role for MKP3 in axial patterning of the zebrafish embryo.

Fibroblast growth factors (FGFs) are secreted molecules that can activate the RAS/mitogen-activated protein kinase (MAPK) pathway to serve crucial functions during embryogenesis. Through an in situ hybridization screen for genes with restricted expression patterns during early zebrafish development, we identified a group of genes that exhibit similar expression patterns to FGF genes. We report the characterization of zebrafish MAP kinase phosphatase 3 (MKP3; DUSP6 - Zebrafish Information Network), a member of the FGF synexpression group, showing that it has a crucial role in the specification of axial polarity in the early zebrafish embryo. MKP3 dephosphorylates the activated form of MAPK, inhibiting the RAS/MAPK arm of the FGF signaling pathway. Gain- and loss-of-function studies reveal that MKP3 is required to limit the extent of FGF/RAS/MAPK signaling in the early embryo, and that disturbing this inhibitory pathway disrupts dorsoventral patterning at the onset of gastrulation. The earliest mkp3 expression is restricted to the future dorsal region of the embryo where it is initiated by a maternal beta-catenin signal, but soon after its initiation, mkp3 expression comes under the control of FGF signaling. Thus, mkp3 encodes a feedback attenuator of the FGF pathway, the expression of which is initiated at an early stage so as to ensure correct FGF signaling levels at the time of axial patterning.

Amino Acid Sequence↗

PubMed2004

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

Adenoviridae↗

PubMed2004

The chianti zebrafish mutant provides a model for erythroid-specific disruption of transferrin receptor 1.

Iron is a crucial metal for normal development, being required for the production of heme, which is incorporated into cytochromes and hemoglobin. The zebrafish chianti (cia) mutant manifests a hypochromic, microcytic anemia after the onset of embryonic circulation, indicative of a perturbation in red blood cell hemoglobin production. We show that cia encodes tfr1a, which is specifically expressed in the developing blood and requisite only for iron uptake in erythroid precursors. In the process of isolating zebrafish tfr1, we discovered two tfr1-like genes (tfr1a and tfr1b) and a single tfr2 ortholog. Abrogation of tfr1b function using antisense morpholinos revealed that this paralog was dispensable for hemoglobin production in red cells. tfr1b morphants exhibited growth retardation and brain necrosis, similar to the central nervous system defects observed in the Tfr1 null mouse, indicating that tfr1b is probably used by non-erythroid tissues for iron acquisition. Overexpression of mouse Tfr1, mouse Tfr2, and zebrafish tfr1b partially rescued hypochromia in cia embryos, establishing that each of these transferrin receptors are capable of supporting iron uptake for hemoglobin production in vivo. Taken together, these data show that zebrafish tfr1a and tfr1b share biochemical function but have restricted domains of tissue expression, and establish a genetic model to study the specific function of Tfr1 in erythroid cells.

Amino Acid Sequence↗

PubMed2005

Slow muscle regulates the pattern of trunk neural crest migration in zebrafish.

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.

Animals↗

PubMed1992

Expression of four zebrafish wnt-related genes during embryogenesis.

The wnt gene family codes for a group of cysteine-rich, secreted proteins, which are differentially expressed in the developing embryo and are possibly involved in cellular communication. Here, we describe the polymerase chain reaction based cloning and embryonic expression patterns of four zebrafish wnt-related sequences; wnt[a], wnt[b], wnt[c] and wnt[d]. One of these genes, wnt[a], is a potential homologue of murine Wnt-3, while the other three genes most likely represent new members of the vertebrate wnt gene family. In zebrafish embryos, transcripts of wnt[a] are confined to the dorsal diencephalon, the dorsal midbrain, the rhombic lips and the dorsal portions of the spinal cord. wnt[b] is expressed in the tail bud and at considerably lower levels in the mesoderm of the head. wnt[c] transcripts are present within the diencephalon and the posterior midbrain whereas wnt[d] shows a surprisingly similar expression pattern to zebrafish wnt-1. By analogy to wnt-1, it is likely that the members of the zebrafish wnt gene family play an important role in cell-to-cell signalling during pattern formation in the neural tube and the tail bud.

Amino Acid Sequence↗

PubMed1994

A zebrafish retinoic acid receptor expressed in the regenerating caudal fin.

Retinoic acid (RA) is an important signalling molecule in vertebrate pattern formation both in developing and regenerating tissues. The effects of RA are due largely to regulation of gene transcription, mediated by retinoic acid receptors (RAR-alpha, RAR-beta, RAR-gamma) and retinoid X receptors (RXR-alpha, RXR-beta, RXR-gamma). We have been using zebrafish as a model of regeneration to study the role of retinoic acid and its receptors in vertebrate pattern formation. In this report, we describe the molecular cloning and characterization of one of the zebrafish RARs that is the predominant receptor in the regenerating caudal fin and corresponds most closely to the RAR-gamma subtype isolated from mouse and human and to RAR-delta from newt. Zebrafish RAR-gamma (zfRAR-gamma) exhibits both structural and functional conservation with its mammalian counterparts. Studies utilizing both normal and regenerating caudal fins of the zebrafish have indicated that it is the RAR-gamma subtype, compared to RAR-alpha or RAR-beta, which is expressed at the highest levels in the tail fin. To localize the expression pattern of RAR-gamma during fin regeneration, we have carried out whole-mount in situ hybridization. ZfRAR-gamma transcripts, during fin regeneration, are localized in the blastemal tissue formed at the distal ends of the bony rays following amputation. Treatment of fish with RA during fin regeneration induces a number of striking morphological effects on the regenerate. When amputations are performed distal to the branch points or dichotomies, where a single ray bifurcates to extend two individual 'daughter' rays, RA treatment causes a dichotomy reduction where the two 'daughter' rays fuse to once again form a single ray. The single ray subsequently bifurcates in a comparatively normal manner. Our data suggest that exogenous RA can respecify pattern in the regenerating caudal fin and identifies the blastemae as possible RA target tissues.

Animals↗

PubMed1995

Distribution of tissue progenitors within the shield region of the zebrafish gastrula.

The zebrafish has emerged as an important model system for the experimental analysis of vertebrate development because it is amenable to genetic analysis and because its optical clarity allows the movements and the differentiation of individual cells to be followed in vivo. In this paper, we have sought to characterize the spatial distribution of tissue progenitors within the outer cell layers of the embryonic shield region of the early gastrula. Single cells were labeled by iontophoretic injection of fluorescent dextrans. Subsequently, we documented their position with respect to the embryonic shield and their eventual fates. Our data show that progenitor cells of the neural, notochordal, somitic and endodermal lineages were all present within the embryonic shield region, and that these progenitors were arranged as intermingled populations. Moreover, close to the midline, there was evidence for significant biases in the distribution of neural and notochord progenitors between the layers, suggesting some degree of radial organization within the zebrafish embryonic shield region. The distributions of tissue progenitors in the zebrafish gastrula differ significantly from those in amphibians; this bears not only on interpretations of mutant phenotypes and in situ staining patterns, but also on our understanding of morphogenetic movements during gastrulation and of neural induction in the zebrafish.

Animals↗

PubMed1999

Intronic enhancers control expression of zebrafish sonic hedgehog in floor plate and notochord.

The signalling molecule Sonic hedgehog (Shh) controls a wide range of differentiation processes during vertebrate development. Numerous studies have suggested that the absolute levels as well as correct spatial and temporal expression of shh are critical for its function. To investigate the regulation of shh expression, we have studied the mechanism controlling its spatial expression in the zebrafish. We employed an enhancer screening strategy in zebrafish embryos based on co-injection of putative enhancer sequences with a reporter construct and analysis of mosaic expression in accumulated expression maps. Enhancers were identified in intron 1 and 2 that mediate floor plate and notochord expression. These enhancers also drive notochord and floor plate expression in the mouse embryo strongly suggesting that the mechanisms controlling shh expression in the midline are conserved between zebrafish and mouse. Functional analysis in the zebrafish embryo revealed that the intronic enhancers have a complex organisation. Two activator regions, ar-A and ar-C, were identified in intron 1 and 2, respectively, which mediate mostly notochord and floor plate expression. In contrast, another activating region, ar-B, in intron 1 drives expression in the floor plate. Deletion fine mapping of ar-C delineated three regions of 40 bp to be essential for activity. These regions do not contain binding sites for HNF3beta, the winged helix transcription factor previously implicated in the regulation of shh expression, indicating the presence of novel regulatory mechanisms. A T-box transcription factor-binding site was found in a functionally important region that forms specific complexes with protein extracts from wild-type but not from notochord-deficient mutant embryos.

Animals↗

PubMed1999

Sequential PKC- and Cdc2-mediated phosphorylation events elicit zebrafish nuclear envelope disassembly.

Molecular markers of the zebrafish inner nuclear membrane (NEP55) and nuclear lamina (L68) were identified, partially characterized and used to demonstrate that disassembly of the zebrafish nuclear envelope requires sequential phosphorylation events by first PKC, then Cdc2 kinase. NEP55 and L68 are immunologically and functionally related to human LAP2beta and lamin B, respectively. Exposure of zebrafish nuclei to meiotic cytosol elicits rapid phosphorylation of NEP55 and L68, and disassembly of both proteins. L68 phosphorylation is completely inhibited by simultaneous inhibition of Cdc2 and PKC and only partially blocked by inhibition of either kinase. NEP55 phosphorylation is completely prevented by inhibition or immunodepletion of cytosolic Cdc2. Inhibition of cAMP-dependent kinase, MEK or CaM kinase II does not affect NEP55 or L68 phosphorylation. In vitro, nuclear envelope disassembly requires phosphorylation of NEP55 and L68 by both mammalian PKC and Cdc2. Inhibition of either kinase is sufficient to abolish NE disassembly. Furthermore, novel two-step phosphorylation assays in cytosol and in vitro indicate that PKC-mediated phosphorylation of L68 prior to Cdc2-mediated phosphorylation of L68 and NEP55 is essential to elicit nuclear envelope breakdown. Phosphorylation elicited by Cdc2 prior to PKC prevents nuclear envelope disassembly even though NEP55 is phosphorylated. The results indicate that sequential phosphorylation events elicited by PKC, followed by Cdc2, are required for zebrafish nuclear disassembly. They also argue that phosphorylation of inner nuclear membrane integral proteins is not sufficient to promote nuclear envelope breakdown, and suggest a multiple-level regulation of disassembly of nuclear envelope components during meiosis and at mitosis.

Animals↗

PubMed2003

Olfactory input increases visual sensitivity in zebrafish: a possible function for the terminal nerve and dopaminergic interplexiform cells.

Centrifugal innervation of the neural retina has been documented in many species. In zebrafish Danio rerio, the only so-far described centrifugal pathway originates from terminal nerve (TN) cell bodies that are located in the olfactory bulb. Most of the TN axons terminate in the forebrain and midbrain, but some project via the optic nerve to the neural retina, where they synapse onto dopaminergic interplexiform cells (DA-IPCs). While the anatomical pathway between the olfactory and visual organs has been described, it is unknown if and how olfactory signals influence visual system functions. We demonstrate here that olfactory input is involved in the modulation of visual sensitivity in zebrafish. As determined by a behavioral assay and by electroretinographic (ERG) recording, zebrafish visual sensitivity was increased upon presentation of amino acids as olfactory stimuli. This effect, however, was observed only in the early morning hours when zebrafish are least sensitive to light. The effect of olfactory input on vision was eliminated after lesion of the olfactory bulbs or after the destruction of DA-IPCs. Intraocular injections of a dopamine D(2) but not a D(1) receptor antagonist blocked the effect of olfactory input on visual sensitivity. Although we cannot exclude the involvement of other anatomical pathways, our data suggest that the TN and DA-IPCs are the prime candidates for olfactory modulation of visual sensitivity.

Amino Acids↗

PubMed2003

Waterborne iron acquisition by a freshwater teleost fish, zebrafish Danio rerio.

Waterborne iron accumulation by the gills of the zebrafish Danio rerio was assessed in ion-poor water. Branchial iron uptake, which comprises both the iron that has entered the gill cells and iron that is strongly bound to the epithelia, has high- and low-affinity components. At low nominal [Fe] (<40 nmol l(-1)) the high-affinity component demonstrated saturation kinetics, with an apparent K(m) of 5.9 nmol l(-1) Fe and V(max) of 2.1 pmol g(-1) h(-1). Over a range of higher nominal [Fe] (40-200 nmol l(-1)), branchial uptake was linear. In the presence of 2 micro mol l(-1) of the reducing agent dithiothreitol (DTT), branchial iron accumulation was significantly enhanced at [Fe]>15 nmol l(-1). The proton pump inhibitor bafilomycin A significantly reduced iron uptake in the presence of DTT. On the basis of these observations we conclude that branchial iron uptake at low [Fe] shows characteristics similar to those of other iron-transporting epithelia, coupling an apical membrane ferric reductase to a Fe(2+)/H(+) symporter. Zebrafish branchial iron transport at 18.6 nmol l(-1) was inhibited by 200 nmol l(-1) Cd(2+). But, unlike other Fe(2+)/H(+) symporters, iron uptake was not affected by other divalent metals (Co(2+), Ni(2+), Pb(2+), Cu(2+), Zn(2+) and Mn(2+)). Zebrafish loaded with (59)Fe from the water showed a loss of 7.9 pmol Fe g(-1) body mass over the first day and a further loss of 5.7 pmol Fe g(-1) body mass over the following 28 days. The depuration kinetics followed a two-component exponential model; for the short-lived component, t(1/2)=0.31 days, and for the long-lived component, t(1/2)=13.2 days. The daily iron loss by zebrafish can be compensated by iron uptake at exceedingly low water iron concentrations (uptake rate at 1.625 nmol l(-1) Fe=0.425 pmol g(-1) h(-1)), demonstrating that uptake of iron from the water is potentially an important source of this nutritive metal in freshwater teleost fish.

Animals↗

PubMed2004

Swimming of larval zebrafish: fin-axis coordination and implications for function and neural control.

Adult actinopterygian fishes typically perform steady forward swimming using either their pectoral fins or their body axis as the primary propulsor. In most species, when axial undulation is employed for swimming, the pectoral fins are tucked (i.e. adducted) against the body; conversely, when pectoral fins are beating, the body axis is held straight. In contrast to adults, larval fishes can combine their pectoral fin and body-axis movements during locomotion; however, little is known about how these locomotor modes are coordinated. With this study we provide a detailed analysis of the coordinated fin and axial movements during slow and fast swimming by examining forward locomotion in larval zebrafish (Danio rerio L.). In addition, we describe the musculature that powers pectoral fin movement in larval zebrafish and discuss its functional implications. As larvae, zebrafish alternate their pectoral fins during slow swimming (0.011+/-0.001 mm ms(-1)) in conjunction with axial undulations of the same frequency (18-28 Hz). During fast swimming (0.109+/-0.030 mm ms(-1); 36-67 Hz), the fins are tucked against the body and propulsion occurs by axial undulation alone. We show that during swimming, larval fishes can use a similar limb-axis coordination pattern to that of walking and running salamanders. We suggest that the fin-axis coordination observed in larval zebrafish may be attributed to a primitive neural circuit and that early terrestrial vertebrates may have gained the ability to coordinate limbs and lateral bending by retaining a larval central pattern generator for limb-axis coordination in the adult life history stage.

Animals↗

PubMed2005

Development of oxygen sensing in the gills of zebrafish.

Previous studies have described the morphology, innervation and O(2)-chemoreceptive properties of neuroepithelial cells (NECs) of the zebrafish gill filaments. The present work describes the ontogenesis of these cells, and the formation of functional O(2)-sensing pathways in developing zebrafish. Confocal immunofluorescence was performed on whole-mount gill preparations using antibodies against serotonin (5-HT) and a zebrafish-derived neuronal marker (zn-12) to identify the appearance and innervation of gill NECs during larval stages. NECs were first expressed in gill filament primordia of larvae at 5 days postfertilization (d.p.f.) and were fully innervated by 7 d.p.f. In vivo ventilation frequency analysis revealed that a behavioural response to hypoxia (11.2+/-2.8 min(-1)) developed in embryos as early as 2 d.p.f., and a significant increase (P<0.05) in the ventilatory response to hypoxia (200.8+/-23.0 min(-1)) coincided with innervation of NECs of the filaments. In addition, exogenous application of quinidine, a blocker of O(2)-sensitive background K(+) channels in NECs, induced hyperventilation in adults in a dose-dependent manner and revealed the development of a quinidine-sensitive ventilatory response in 7 d.p.f. larvae. This study shows that NEC innervation in the gill filaments may account for the development of a functional O(2)-sensing pathway and the hyperventilatory response to hypoxia in zebrafish larvae. At earlier stages, however, O(2)-sensing must occur through another pathway. The possibility that a new type of 5-HT-positive NEC of the gill arches may account for this earlier hypoxic response is discussed.

Analysis of Variance↗

PubMed2005

Analysis of cell movements in zebrafish embryos: morphometrics and measuring movement of labeled cell populations in vivo.

Cell movements occur in all phases of animal life from embryogenesis, to maintaining adult organs, to comprising a critical component of pathology. During gastrulation, cells demonstrate a repertoire of morphogenetic movements coordinated with fate inductions to sculpt the embryonic body. The morphogenetic behaviors, underlying mechanisms, and their control, are the subject of much current study. External development of the transparent zebrafish embryo, the abundance of mutations influencing cell movements, as well as a range of observation and manipulation methods, make the zebrafish valuable for cell movement studies. This chapter offers a conceptual background for analysis of gastrulation cell movements by reviewing how region specific cell movements shape the wild-type zebrafish embryo, and how defective morphogenetic movements alone or in combination with altered cell fate specification distort the body plans of known zebrafish mutants. We furnish methods for the morphometric analysis of embryonic shape and organ rudiments in live and fixed embryos, and present data collected from live wild-type, dorsoventral patterning (somitabun and chordino) and convergence and extension (knypek and trilobite) classes of mutants. We provide a method for quantitative assessment of the movements of cell populations in vivo, and a method for determining whether cell fate and/or movement are disturbed.

Animals↗

PubMed2006

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development.

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca(2+) signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP -mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca(2+). Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca(2+ )signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca(2+ )signaling during the 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.

Aequorin↗

PubMed2002

Characterization of oestrogen receptors in zebrafish (Danio rerio).

We cloned the cDNAs corresponding to three oestrogen receptors (ERs) in zebrafish (Danio rerio). Sequence analysis and phylogenetic studies demonstrated that two of these genes, ER beta.1 and ER beta.2, arose from duplication of the original ER beta in many species of the fish phylum, whereas ER alpha is unique. Zebrafish ERs behaved as oestrogen-dependent transcription factors in transactivation assays. However, their reactivity to various oestrogen modulators was different compared with that of mouse ERs. ER mRNA expression during zebrafish development is restricted to distinct time periods, as observed by RNase protection assays. ER beta.2 is initially expressed as maternally transmitted RNA, until 6 h after fertilization, when expression disappears. Between 6 and 48 h after fertilization, no ER expression could be observed. After 48 h after fertilization, all ERs, but predominantly ER alpha, began to be expressed. We conclude that oestrogen signal transduction can operate during zebrafish development only within discrete time windows.

Amino Acid Sequence↗