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PubMed2001

Expression of BMP signalling pathway members in the developing zebrafish inner ear and lateral line.

In this paper we describe the mRNA expression patterns of members of the bone morphogenetic protein (BMP) signalling pathway in the developing zebrafish ear. bmp2b, 4, and 7 are expressed in discrete areas of otic epithelium, some of which correspond to sensory patches. bmp2b and 4 mark the developing cristae before and during the appearance of differentiated hair cells. bmp4 is also expressed in a dorsal, non-sensory region of the ear. Expression of bmps in cristae is conserved between zebrafish, chick, and mouse, but there are also notable differences in ear expression patterns between these species. Of five zebrafish BMP antagonists, only one (follistatin) shows significant expression in the otic epithelium. The type I receptor bmpr-IB shows localised expression in the ear epithelium. Mediators of BMP signalling, smad1 and smad5, are expressed in statoacoustic and lateral line ganglia; smad5 is also expressed at low levels throughout the ear epithelium. An inhibitory smad, smad6, is expressed laterally in the ear epithelium. Lateral line primordia and neuromasts also express bmp2b, 4, follistatin, smad1, and smad5. The conservation of bmp expression in cristae among different species adds weight to the growing evidence that BMPs are required for the development of the vertebrate ear.

Animals↗

PubMed2000

Properties of miniature glutamatergic EPSCs in neurons of the locomotor regions of the developing zebrafish.

As a first step in understanding the development of synaptic activation in the locomotor network of the zebrafish, we examined the properties of spontaneous, glutamatergic miniature excitatory postsynaptic currents (mEPSCs). Whole cell patch-clamp recordings were obtained from visually identified hindbrain reticulospinal neurons and spinal motoneurons of curarized zebrafish 1-5 days postfertilization (larvae hatch after the 2nd day of embryogenesis). In the presence of tetrodotoxin (TTX) and blockers of inhibitory receptors (strychnine and picrotoxin), we detected fast glutamatergic mEPSCs that were blocked by the AMPA/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At positive voltages or in the absence of Mg(2+), a second, slower component of the mEPSCs was revealed that the N-methyl-D-aspartate (NMDA) receptor-selective antagonist DL-2-amino-5-phosphonovalerate (AP-5) abolished. In the presence of both CNQX and AP-5, all mEPSCs were eliminated. The NMDA component of reticulospinal mEPSCs had a large single-channel conductance estimated to be 48 pS. Larval AMPA/kainate and NMDA components of the mEPSCs decayed with biexponential time courses that changed little during development. At all stages examined, approximately one-half of synapses had only NMDA responses (lacking AMPA/kainate receptors), whereas the remainder of the synapses were composed of a mixture of AMPA/kainate and NMDA receptors. There was an overall increase in the frequency and amplitude of mEPSCs with an NMDA component in reticulospinal (but not motoneurons) during development. These results indicate that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.

2-Amino-5-phosphonovalerate↗

PubMed2003

Specific and conserved roles of TAp73 during zebrafish development.

p53, p63 and p73 are related transcription factors involved in the regulation of cell proliferation, survival and differentiation. Here, we report the isolation and characterization of p73 from zebrafish. While for zebrafish p63 only N-terminally truncated isoforms (DeltaNp63) have been reported, p73 appears to be predominantly or exclusively present in transactivating isoforms (TAp73). p73 shows a very restricted expression pattern during zebrafish development. Transcripts are found in a subset of cells of the olfactory system, the telencephalon, the dorsal diencephalon, and the pronephric ducts. In addition, p73 is expressed in differentiating slow muscle cells of the somites, and in the pharyngeal endoderm. We carried out TAp73 gain- and loss-of-function experiments, injecting either TAp73alpha mRNA, or antisense morpholino oligonucleotides to suppress translation of TAp73 transcripts. The overexpression studies indicate that in contrast to p53, TAp73alpha has no pro-apoptotic effect in zebrafish embryos. However, TAp73 appears to be required for specific processes during the development of the olfactory system, the telencephalon and the pharyngeal arches. Together, our data point to both conserved and class-specific roles of p73 during vertebrate development.

Amino Acid Sequence↗

PubMed2004

Cadherin-2 function in the cranial ganglia and lateral line system of developing zebrafish.

Cadherins are cell surface molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin-2 (also called N-cadherin), a member of classic cadherin subfamily, has been shown to play important roles in development of a variety of tissues and organs, including the nervous system. We recently reported that cadherin-2 was strongly expressed by the majority of cranial ganglia and lateral line system of developing zebrafish. To gain insight into cadherin-2 role in the formation of these structures, we have used several markers to analyze zebrafish embryos injected with a specific cadherin-2 antisense morpholino oligonucleotide (cdh2MO). We find that development of several cranial ganglia, including the trigeminal, facial, and vagal ganglia, and the lateral line ganglia and neuromasts of the cdh2MO-injected embryos are severely disrupted. These phenotypes were confirmed by analyzing a cadherin-2 mutant, glass onion. Our results suggest that cadherin-2 function is crucial for the normal formation of the zebrafish lateral line system and a subset of cranial ganglia.

Animals↗

PubMed2002

Gills are needed for ionoregulation before they are needed for O(2) uptake in developing zebrafish, Danio rerio.

A variation on the classic ablation method was used to determine whether O(2) uptake or ionoregulation is the first to shift from the skin to the gills in developing zebrafish, Danio rerio. Zebrafish larvae, ranging in age from 3 to 21 days postfertilization, were prevented from ventilating their gills and forced to rely on cutaneous processes by exposing them to one of two anaesthetics (tricaine methanesulphonate or phenoxyethanol) or by embedding their gills in agar. They were then placed in solutions designed to compensate selectively for impaired O(2) uptake (42% O(2)), impaired ionoregulatory capacity (50% physiological saline) or impairment of both functions (42% O(2)+50% physiological saline). Survival under these conditions was compared with that in normoxic (21% O(2)) fresh water. Neither hyperoxia nor 50% physiological saline had any significant effect on the survival of newly hatched larvae (3 days postfertilization), suggesting that at this stage cutaneous exchange was sufficient to satisfy both ionoregulatory and respiratory requirements. At 7 days postfertilization, the skin still appeared capable of satisfying the O(2) requirements of larvae but not their ionoregulatory requirements. Physiological saline significantly improved survival at 7 days postfertilization; hyperoxia did not. At 14 days postfertilization, both hyperoxia and 50% saline significantly improved survival, indicating that at this stage gills were required for O(2) uptake as well as for ionoregulation. At 21 days postfertilization, only hyperoxia significantly improved survival. By this stage, larvae apparently are so dependent on gills for O(2) uptake that they suffocate before the effects of ionoregulatory impairment become apparent. Thus, it would appear that in zebrafish it is the ionoregulatory capacity of the skin not its ability to take up O(2) that first becomes limiting. This raises the possibility that ionoregulatory pressures may play a more important role in gill development than is generally appreciated.

Animals↗

PubMed1997

Cross-interactions between two members of the Dlx family of homeobox-containing genes during zebrafish development.

The Dlx homeobox genes of vertebrates are transcribed in multiple cells of the embryo with overlapping patterns but often with different onsets of expression. Here we describe the interaction between two dlx genes, dlx3 and dlx4, during zebrafish development. The observation that dlx3 expression precedes that of dlx4 in the otic vesicle led us to investigate whether dlx3 had the ability to control expression of dlx4. Truncated versions of dlx3 were overexpressed in zebrafish embryos and the expression patterns of dlx4 were examined later in development. Overexpression of truncated forms of Dlx3 or of a Dlx3-Dlx2 chimera was found to result in perturbations in dlx4 expression. In addition, cotransfection experiments indicated the ability of Dlx3 to activate transcription through a 1.7-kb fragment of the 5' flanking region of dlx4. These results suggest that dlx4 is one of the target genes of dlx3 in embryos and that cross-regulatory interactions between Dlx genes may be one of the mechanisms responsible for their overlapping expression.

Animals↗

PubMed2002

Multiple functions of LIM domain-binding CLIM/NLI/Ldb cofactors during zebrafish development.

The crucial involvement of CLIM/NLI/Ldb cofactors for the exertion of the biological activity of LIM homeodomain transcription factors (LIM-HD) has been demonstrated. In this paper we show that CLIM cofactors are widely expressed during zebrafish development with high protein levels in specific neuronal cell types where LIM-HD proteins of the Isl class are synthesized. The overexpression of a dominant-negative CLIM molecule (DN-CLIM) that contains the LIM interaction domain (LID) during early developmental stages of zebrafish embryos results in an impairment of eye and midbrain-hindbrain boundary (MHB) development and disturbances in the formation of the anterior midline. On a cellular level we show that the outgrowth of peripheral but not central axons from Rohon Beard (RB) and trigeminal sensory neurons is inhibited by DN-CLIM overexpression. We demonstrate a further critical role of CLIM cofactors for axonal outgrowth of motor neurons. Additionally, DN-CLIM overexpression causes an increase of Isl-protein expression levels in specific neuronal cell types, likely due to a protection of the DN-CLIM/LIM-HD complex from proteasomal degradation. Our results demonstrate multiple roles of the CLIM cofactor family for the development of entire organs, axonal outgrowth of specific neurons and protein expression levels.

Animals↗

PubMed2005

Role of Yes kinase during early zebrafish development.

We have identified the Yes kinase in zebrafish eggs and investigated its role in development of the zebrafish embryo. In situ hybridization as well as immunofluorescence techniques demonstrated that Yes kinase is maternally expressed and is localized to the cortical region of the unfertilized egg. Fertilization resulted in concentration of Yes kinase to the blastodisc where it continued to be localized to the blastoderm cells through cleavage, gastrulation, and later development. Yes kinase activity was found to decrease abruptly at fertilization, then increase progressively during epiboly, and was maintained at high levels throughout gastrulation. The role of Yes kinase in development was tested by treating embryos with chemical protein tyrosine kinase (PTK) inhibitors such as 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2) and by injection of antisense morpholinos. Both treatments resulted in the arrest of development at the beginning of the epiboly. Co-immunoprecipitation studies demonstrated that Yes kinase participates in a stable complex with focal adhesion kinase (FAK), which is phosphorylated in vitro. These results demonstrate that Yes kinase plays an important role in epiboly and indicate that Yes kinase participates in signaling by focal adhesion kinase during early development.

Amino Acid Sequence↗

PubMed2004

Identification of 315 genes essential for early zebrafish development.

We completed a large insertional mutagenesis screen in zebrafish to identify genes essential for embryonic and early larval development. We isolated 525 mutants, representing lesions in approximately 390 different genes, and we cloned the majority of these. Here we describe 315 mutants and the corresponding genes. Our data suggest that there are roughly 1,400 embryonic-essential genes in the fish. Thus, we have mutations in approximately 25% of these genes and have cloned approximately 22% of them. Re-screens of our collection to identify mutants with specific developmental defects suggest that approximately 50 genes are essential for the development of some individual organs or cell types. Seventy-two percent of the embryonic-essential fish genes have homologues in yeast, 93% have homologues in invertebrates (fly or worm), and 99% have homologues in human. Yeast and worm orthologues of genes that are essential for early zebrafish development have a strong tendency to be essential for viability in yeast and for embryonic development in the worm. Thus, the trait of being a genetically essential gene is conserved in evolution. This mutant collection should be a valuable resource for diverse studies of cell and developmental biology.

Animals↗

PubMed2006

Selective labeling of central and peripheral sensory neurons in the developing zebrafish using P2X(3) receptor subunit transgenes.

The two paralogous P2X receptor subunit genes p2rx3.1 and p2rx3.2 are selectively expressed in overlapping, but unique, patterns of sensory neurons in the developing zebrafish. We constructed a series of transgenes derived from both genes using the recombineering technique. Transgenes utilizing either enhanced green fluorescent protein or monomeric red fluorescent protein-1 were shown to be expressed with the same spatial and temporal patterns as the native genes. The p2rx3.1-derived transgenes labeled the vast majority of the Rohon-Beard neurons in the spinal cord and neurons of the trigeminal ganglia. The p2rx3.2-derived transgene labeled fewer Rohon-Beard and trigeminal neurons than what was observed for the p2rx3.1-derived transgenes, but was also detected in neurons of the epibranchial ganglia. Three distinct populations of sensory neurons were detected: those expressing only one or the other paralog, and those expressing both paralogs. The fluorescent proteins encoded by the transgenes allowed for visualization of the neuronal somas as well as their peripheral and central projections. These reagents should prove extremely useful in providing the basis for future studies aimed at elucidating the developmental and physiological attributes of sensory neurons.

Animals↗

PubMed1996

Asynchronous onset of odorant receptor expression in the developing zebrafish olfactory system.

The functional identity of an olfactory neuron is determined in large part by the odorant receptors it expresses. As an approach toward understanding the events that underlie the specification of olfactory neurons, we have examined the patterns of odorant receptor gene expression in the developing zebrafish. Surprisingly, we find that the onset of specific odorant receptor expression occurs asynchronously in the developing olfactory placode. Our results suggest that odorant receptor expression is not strictly stochastic, but rather is governed by temporally regulated cues during development. Moreover, by restricting the number of receptor genes competent for transcription at different times of development, temporal waves of expression may provide a mechanism for simplifying the regulation of the large odorant receptor gene family.

Age Factors↗

PubMed2006

Impairment of lower jaw growth in developing zebrafish exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and reduced hedgehog expression.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause a multitude of detrimental effects to developing zebrafish (Danio rerio). Previously, we demonstrated that jaw growth was impaired by TCDD exposure, but the exact mechanism underlying these malformations remained unknown. In the present study, we investigated the involvement of hedgehog genes and their downstream signaling in TCDD-mediated jaw malformation. We demonstrate that the developing lower jaw expresses sonic hedgehog a (shha), sonic hedgehog b (shhb) and their receptors, patched1 (ptc1) and patched2 (ptc2), as well as the downstream transcription factors, gli1 and gli2a. Loss of Hh signaling in mutants (sonic you) and larvae treated with a Hh inhibitor (cyclopamine), resulted in similar effects as those caused by TCDD. Moreover, TCDD exposure caused downregulation of shha and shhb in a manner dependent on aryl hydrocarbon receptor 2 (ahr2). Although this suggested an involvement of Hh signaling in TCDD-mediated impairment of jaw growth, we did not observe downregulation of ptc1 and ptc2, receptors dependent on Hh signaling. Furthermore, while the overall occurrence of apoptosis in the developing jaw was minimal, it was significantly increased in larvae treated with cyclopamine. In contrast, both TCDD and cyclopamine markedly reduced immunoreactivity against phosphorylated histone 3, a cell proliferation marker in the developing jaw. Taken together, our data suggest that Ahr2-mediated downregulation of Hh signaling, leading to a failure of cell proliferation, contributes to TCDD induced inhibition of lower jaw growth. TCDD may impair jaw growth through other pathway(s) in addition to Hh signaling.

Animals↗

PubMed2004

Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway.

beta-arrestins are multifunctional proteins that act as scaffolds and transducers of intracellular signals from heptahelical transmembrane-spanning receptors (7TMR). Hedgehog (Hh) signaling, which uses the putative 7TMR, Smoothened, is established as a fundamental pathway in development, and unregulated Hh signaling is associated with certain malignancies. Here, we show that the functional knockdown of beta-arrestin 2 in zebrafish embryos recapitulates the many phenotypes of Hh pathway mutants. Expression of wild-type beta-arrestin 2, or constitutive activation of the Hh pathway downstream of Smoothened, rescues the phenotypes caused by beta-arrestin 2 deficiency. These results suggest that a functional interaction between beta-arrestin 2 and Smoothened may be critical to regulate Hh signaling in zebrafish development.

Animals↗

PubMed2005

Epigenetic variations in early cardiovascular performance and hematopoiesis can be explained by maternal and clutch effects in developing zebrafish (Danio rerio).

This is the first study to show maternal and clutch effects on early developmental parameters like blood cell concentration and cardiac performance (heart rate, stroke volume and cardiac output) in developing zebrafish larvae (2-8 days post fertilization, dpf). Ten individuals per parental pair and developmental stage were analyzed. A pronounced interclutch variation of heart rate has been found in younger stages (2-4 dpf), while interclutch variation of heart rate was small in later stages (8 dpf). This effect was more pronounced in offspring from parental fish nourished with living food. The opposite effect was observed in interclutch variation of blood cell concentration. Here only older stages showed significant interclutch variations. Stroke volume and cardiac output had very small interclutch variations throughout all stages. Heart rate was strongly dependent with developmental stage in all groups. Nutritional maternal effects on heart performance and also in blood cell concentration could be detected in the offspring of parent animals either fed with flake food or with living food. Red blood cell count, calculated as a product from red blood cell concentration, was not significantly different in both feeding groups. The number of spawned eggs was not different. In summary, these data indicate that "clutch effects" caused by maternal and/or genetic influences can affect the developmental pattern of cardiac performance and blood cell concentration.

Animals↗

PubMed2002

Differential regulation of spontaneous and heat-induced HSP 70 expression in developing zebrafish (Danio rerio).

A spontaneous high expression of heat shock protein 70 (HSP 70) was found to arise in zebrafish (Danio rerio) at the larval stage (84 hr after fertilization). The level of HSP 70 in 84-hr-old larvae was estimated to be six- to eightfold that of 12-hr-old embryos. As heat-induced HSP 70 synthesis in many eukaryotic organisms is known to be mediated by a transcriptional-dependent pathway activated by heat shock factor 1 (HSF-1), we then examined if the spontaneous and heat-induced HSP 70 synthesis in zebrafish were controlled by the same mechanism. Although the transient increase of a 62-kDa HSF-1-like polypeptide in 72- to 96-hr-old larvae seemed to correlate with the onset of the spontaneous HSP 70 production, an anti-HSF-1 antibody cocktail supershifted the heat shock element (HSE) binding complex induced by stressed but not by unstressed zebrafish extracts. Northern blot and quantitative RT-PCR analysis demonstrated the predominant presence of the cognate form of hsp 70 mRNA (hsc 70 mRNA) in developing zebrafish. The extent of heat-induced HSP 70 production in 84-hr-old larvae matched well with a dramatic increase in hsp 70 mRNA accumulation, while no apparent increase in total hsp 70 mRNA could be detected in 72- to 84-hr-old unstressed larvae by northern blot analysis. The stable expression of hsc 70 mRNA specific to beta-actin mRNA in normal zebrafish was confirmed by RT-PCR analysis. Hence, the spontaneous high expression of HSP 70 in zebrafish is believed to be controlled by a mechanism different from the HSF-1-dependent transcriptional activation of hsp 70 under heat stress. J. Exp. Zool. 293:349-359, 2002.

Animals↗

PubMed2003

Assessment of neuronal maturation and acquisition of functional competence in the developing zebrafish olfactory system.

Olfactory coding at the level of the olfactory bulb is thought to depend upon an ensemble response of mitral cells receiving input from chemotopically-organized projections of olfactory sensory neurons and regulated by lateral inhibitory circuits. Immunocytochemical methods are described to metabolically classify neurons in the developing zebrafish olfactory system based on the relative concentrations of taurine, glutamate, GABA (and potentially other small biogenic amines) and a small guanidium-based cation, agmatine, which labels NMDA-sensitive cells by permeating through active ionotropic glutamate receptor channels. Using metabolic profiling in conjunction with activity dependent labeling we demonstrate that neuronal differentiation in the developing olfactory bulb, as assessed by acquisition of a mature neurochemical profile, and sensitivity to an ionotropic glutamate receptor agonist, NMDA, occurs during the second day of development. This experimental approach is likely to be useful in studies concerned with the development of glutamatergic signaling pathways.

Agmatine↗

PubMed2006

Analysis and visualization of cell movement in the developing zebrafish brain.

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

Animals↗

PubMed2001

Differential distribution of the G protein gamma3 subunit in the developing zebrafish nervous system.

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein alpha subunit was the sole regulator of intracellular molecules. The G protein betagamma complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein gamma subunit. The 666 nucleotides of the zebrafish G protein gamma subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse gamma subunits. BLAST search analysis of GenBank revealed that the zebrafish gamma subunit is 93% identical and 97% similar to the mammalian gamma3 subunit. The gamma3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected gamma3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish gamma3 subunit to form a signaling heterodimeric complex with a beta subunit was tested using a human beta2 subunit. The gamma3 formed a heterodimer with beta2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the beta2gamma3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a betagamma heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.

Amino Acid Sequence↗