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PubMed1991

The spt-1 mutation alters segmental arrangement and axonal development of identified neurons in the spinal cord of the embryonic zebrafish.

We examined the arrangement and development of identified neurons in zebrafish embryos homozygous for the mutation spt-1, which acts autonomously and specifically to alter the development of precursors of trunk segmented mesoderm, resulting in muscle-deficient myotomes. We found that the mutation alters the morphology, number, and arrangement of identified motoneurons. By transplanting identified motoneurons between wild-type and mutant embryos, we found that the effect of the mutation was nonautonomous. We suggest that the segmental arrangement and proper axonal development of motoneurons may result from interactions with segmented mesoderm.

Animals↗

PubMed2005

Sub-lethal concentrations of waterborne copper are toxic to lateral line neuromasts in zebrafish (Danio rerio).

In teleosts, the lateral line system is composed of neuromasts containing hair cells that are analogous to those present in the inner ear of all vertebrates. In the zebrafish embryo and early larva, this system is composed of the anterior lateral line (ALL), which covers the head, and the posterior lateral line (PLL), present in the trunk and tail. The mechanosensory hair cells found in neuromasts can be labeled in vivo using fluorescent dyes such as 4-di-2-Asp (DiAsp) or FM1-43. We have studied the effects of water-borne copper exposure on the function of the lateral line system in zebrafish larvae. Our results show that transient incubation of post-hatching larvae for 2h with non-lethal concentrations of copper (1-50 microM CuSO4) induces cellular damage localized to neuromasts, apoptosis, and loss of hair cell markers. This effect is specific to copper, as other metals did not show these effects. Since hair cells in fish can regenerate, we followed the reappearance of viable hair cells in neuromasts after copper removal. In the PLL, we determined that there is a threshold concentration of copper above which regeneration does not occur, whereas, at lower concentrations, the length of time it takes for viable hair cells to reappear is dependent on the amount of copper used during the treatment. The ALL behaves differently though, as regeneration can occur even after treatments with concentrations of copper an order of magnitude higher than the one that irreversibly affects the PLL. Regeneration of hair cells is dependent on cell division within the neuromasts as damage that precludes proliferation prevents reappearance of this cell type.

Animals↗

PubMed2005

Characterization of zebrafish Rad52 and replication protein A for oligonucleotide-mediated mutagenesis.

Zebrafish has become a favorite model organism not only in genetics and developmental biology, but also for the study of cancer, neuroscience and metabolism. However, strategies for reverse genetics in zebrafish are mostly limited to the use of antisense oligonucleotides, and therefore the development of other targeting methods is highly desirable. Here, we report an approach to gene targeting in this system in which single-stranded oligonucleotides and zebrafish Rad52 protein are employed. It has been proposed that a single-stranded oligonucleotide containing a mutation can be incorporated into the genome by annealing to the single-stranded region of the lagging strand of the replication fork. Rad52 is expected to accelerate the annealing step. In vitro experiments using purified truncated Rad52 proteins and replication protein A (RPA) showed that annealing of oligonucleotides is accelerated by Rad52 in the presence of RPA. We developed a simple and sensitive PCR-based method to detect point mutations in the genome. In exploratory experiments, we found that microinjection of single-stranded oligonucleotide targeted to a specific gene together with truncated Rad52 into zebrafish embryos resulted in a low level of recombinant copies in 3 of the 80 embryos tested under these conditions.

Amino Acid Sequence↗

PubMed2000

The zebrafish colourless gene regulates development of non-ectomesenchymal neural crest derivatives.

Neural crest forms four major categories of derivatives: pigment cells, peripheral neurons, peripheral glia, and ectomesenchymal cells. Some early neural crest cells generate progeny of several fates. How specific cell fates become specified is still poorly understood. Here we show that zebrafish embryos with mutations in the colourless gene have severe defects in most crest-derived cell types, including pigment cells, neurons and specific glia. In contrast, craniofacial skeleton and medial fin mesenchyme are normal. These observations suggest that colourless has a key role in development of non-ectomesenchymal neural crest fates, but not in development of ectomesenchymal fates. Thus, the cls mutant phenotype reveals a segregation of ectomesenchymal and non-ectomesenchymal fates during zebrafish neural crest development. The combination of pigmentation and enteric nervous system defects makes colourless mutations a model for two human neurocristopathies, Waardenburg-Shah syndrome and Hirschsprung's disease.

Animals↗

PubMed2004

accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1.

When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.

Amino Acid Sequence↗

PubMed2005

Synergistic activity of Sef and Sprouty proteins in regulating the expression of Gbx2 in the mid-hindbrain region.

Sef and Sprouty proteins function as feedback antagonists of fibroblast growth factor (Fgf) signaling in zebrafish embryos. To study the role of Sef in mice, we generated Sef homozygous mutant animals. These animals are viable and show normal expression of mid-hindbrain genes at embryonic days 8.5 and 9.5. To investigate the possibility of functional synergism between Sef and Sprouty proteins, we electroporated Sprouty2(Y55A), which functions in a dominant-negative manner in tissue culture cells into the mid-hindbrain region of wildtype and Sef mutant embryos. The expression pattern of Gbx2, a downstream target of Fgf signaling, was expanded or shifted in electroporated embryos, and this effect was significantly enhanced in the Sef mutant background. Altogether, our results demonstrate that Sef and Sproutys function synergistically to regulate Gbx2 expression in the anterior hindbrain.

Adaptor Proteins, Signal Transducing↗

PubMed2003

JLK isocoumarin inhibitors: selective gamma-secretase inhibitors that do not interfere with notch pathway in vitro or in vivo.

gamma-Secretase activity is involved in the generation of Abeta and therefore likely contributes to the pathology of Alzheimer's disease. Blocking this activity was seen as a major therapeutic target to slow down or arrest Abeta-related AD progression. This strategy seemed more doubtful when it was established that gamma-secretase also targets other substrates including Notch, a particularly important transmembrane protein involved in vital functions, at both embryonic and adulthood stages. We have described previously new non-peptidic inhibitors able to selectively inhibit Abeta cellular production in vitro without altering Notch pathway. We show here that in vivo, these inhibitors do not alter the Notch pathway responsible for somitogenesis in the zebrafish embryo. In addition, we document further the selectivity of JLK inhibitors by showing that, unlike other described gamma-secretase inhibitors, these agents do not affect E-cadherin processing. Finally, we establish that JLKs do not inhibit beta-site APP cleaving enzymes (BACE) 1 and BACE2, alpha-secretase, the proteasome, and GSK3beta kinase. Altogether, JLK inhibitors are the sole agents to date that are able to prevent Abeta production without triggering unwanted cleavages of other proteins.

Amyloid Precursor Protein Secretases↗

PubMed2005

Roles for fgf8 signaling in left-right patterning of the visceral organs and craniofacial skeleton.

Laterality is fundamental to the vertebrate body plan. Here, we investigate the roles of fgf8 signaling in LR patterning of the zebrafish embryo. We find that fgf8 is required for proper asymmetric development of the brain, heart and gut. When fgf8 is absent, nodal signaling is randomized in the lateral plate mesoderm, leading to aberrant LR orientation of the brain and visceral organs. We also show that fgf8 is necessary for proper symmetric development of the pharyngeal skeleton. Attenuated fgf8 signaling results in consistently biased LR asymmetric development of the pharyngeal arches and craniofacial skeleton. Approximately 1/3 of zebrafish ace/fgf8 mutants are missing Kupffer's vesicle (KV), a ciliated structure similar to Hensen's node. We correlate fgf8 deficient laterality defects in the brain and viscera with the absence of KV, supporting a role for KV in proper LR patterning of these structures. Strikingly, we also correlate asymmetric craniofacial development in ace/fgf8 mutants with the presence of KV, suggesting roles for KV in lateralization of the pharyngeal skeleton when fgf8 is absent. These data provide new insights into vertebrate laterality and offer the zebrafish ace/fgf8 mutant as a novel molecular tool to investigate tissue-specific molecular laterality mechanisms.

Animals↗

PubMed2002

A zebrafish orthologue (whnb) of the mouse nude gene is expressed in the epithelial compartment of the embryonic thymic rudiment.

The cloning and characterization of the zebrafish orthologue of the mouse nude (Whn/Foxn1) gene, whnb are reported. A previously described Whn-like gene from zebrafish, now designated as whna, is shown to be the orthologue of the mouse Foxn4 gene. The whnb gene is specifically expressed in the thymic rudiment of zebrafish embryos at day 3 after fertilization, whereas the whna gene is expressed in eye and brain structures. Whnb expression is maintained in cloche mutants, where endothelial and haematopoietic cell differentiation is defective, but absent in casanova mutants where endoderm formation is impaired. In adult thymi, whnb is expressed throughout cortical and medullary areas, whereas whna expression is observed in rare cell clusters only. Our results provide the first specific marker for the epithelial compartment of the zebrafish thymus.

Amino Acid Sequence↗

PubMed2004

2,3,7,8-Tetrachlorodibenzo-p-dioxin inhibits regression of the common cardinal vein in developing zebrafish.

A role for the aryl hydrocarbon receptor (AHR) pathway in vascular maturation has been implicated by studies in Ahr-null mice. In this study the hypothesis that activation of AHR signaling by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters common cardinal vein (CCV) development in the zebrafish embryo was investigated. The CCV is a paired vessel that grows across the yolk, connecting to the heart. It is extensively remodeled and regresses as the heart migrates dorsally within the pericardium. TCDD significantly reduced CCV growth as early as 44 h post fertilization (hpf), and CCV area was reduced to 63% of control at 62 hpf. This vascular response to TCDD was at least as sensitive as previously defined endpoints of TCDD developmental toxicity in zebrafish. TCDD also blocked regression of the CCV (by 80 hpf), possibly contributing to the "string-like" heart phenotype seen in TCDD-exposed zebrafish larvae. Dependence of the block in CCV regression on zebrafish (zf) AHR2 was investigated using a zfahr2 specific morpholino to knock down expression of AHR2. The zfahr2 morpholino had no effect on CCV regression in the absence of TCDD, but did protect against the TCDD-induced block of CCV regression. This demonstrates that the TCDD-induced block in CCV regression is AHR2 dependent. It is significant that decreased CCV growth occurs before and inhibition of CCV regression occurs concurrent with overt signs of TCDD developmental toxicity. This suggests that alterations of vascular growth and remodeling may play a role in TCDD developmental toxicity in zebrafish.

Animals↗

PubMed2004

Screen for genes differentially expressed during regeneration of the zebrafish caudal fin.

The zebrafish caudal fin constitutes an important model for studying the molecular basis of tissue regeneration. The cascade of genes induced after amputation or injury, leading to restoration of the lost fin structures, include those responsible for wound healing, blastema formation, tissue outgrowth, and patterning. We carried out a systematic study to identify genes that are up-regulated during "initiation" (1 day) and "outgrowth and differentiation" (4 days) of fin regeneration by using two complementary methods, suppression subtraction hybridization (SSH) and differential display reverse transcriptase polymerase chain reaction (DDRT-PCR). We obtained 298 distinct genes/sequences from SSH libraries and 24 distinct genes/sequences by DDRT-PCR. We determined the expression of 54 of these genes using in situ hybridization. In parallel, gene expression analyses were done in zebrafish embryos and early larvae. The information gathered from the present study provides resources for further investigations into the molecular mechanisms of fin development and regeneration.

Amputation, Surgical↗

PubMed2005

Structural and functional evidence for a singular repertoire of BMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests a shared ancestral BMP/activin pathway.

The transforming growth factor beta (TGF-beta) superfamily includes bone morphogenetic proteins, activins and TGF-betasensu stricto (s.s). These ligands, which transduce their signal through a heteromeric complex of type I and type II receptors, have been shown to play a key role in numerous biological processes including early embryonic development in both deuterostomes and ecdyzozoans. Lophochotrozoans, the third major group of bilaterian animals, have remained in the background of the molecular survey of metazoan development. We report the cloning and functional study of the central part of the BMP pathway machinery in the bivalve mollusc Crassostrea gigas (Cg-BMPR1 type I receptor and Cg-TGFbetasfR2 type II receptor), showing an unusual functional mode of signal transduction for this superfamily. The use of the zebrafish embryo as a reporter organism revealed that Cg-BMPR1, Cg-TGFbetasfR2, Cg-ALR I, an activin Type I receptor or their dominant negative acting truncated forms, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning, due to strong modulations of ventrolateral mesoderm patterning. The results suggest that Cg-BMPR1, and to a certain degree Cg-TGFbetasfR2 proteins, function in C. gigas in a similar way to their zebrafish orthologues. Finally, based on phylogenetic analyses, we propose an evolutionary model within the complete TGF-beta superfamily. Thus, evidence provided by this study argues for a possible conserved endomesoderm/ectomesoderm inductive mechanism in spiralians through an ancestral BMP/activin pathway in which the singular, promiscuous and probably unique Cg-TGFbetasfR2 would be the shared type II receptor interface for both BMP and activin ligands.

Activin Receptors, Type I↗

PubMed2004

The biochemical and genetic odyssey to the function of a nicastrin-like protein.

Gamma-secretase is a high-molecular-weight protein complex required for the proteolytic processing of various transmembrane proteins including the Alzheimer's disease-associated amyloid precursor protein and the signaling receptor Notch. One of the gamma-secretase complex components is the type I transmembrane protein nicastrin. Here we review the odyssey to a cyclopic fish, which at the end allowed the functional analysis of nicalin, a novel member of the nicastrin protein family. This 60-kDa protein is part of a previously unknown membrane protein complex unrelated to gamma-secretase and binds to Nomo (Nodal modulator, previously known as pM5), a novel 130-kDa transmembrane protein. Both proteins are highly conserved in metazoans and show almost identical tissue distribution in humans. Functional studies in zebrafish embryos and cultured human cells revealed that nicalin and Nomo collaborate to antagonize the Nodal/TGFbeta signaling pathway. Thus, nicastrin and nicalin are both associated with protein complexes involved in cell fate decisions during early embryonic development.

Amyloid Precursor Protein Secretases↗

PubMed1998

Anterior-posterior subdivision of the somite in embryonic zebrafish: implications for motor axon guidance.

The ventral branches of the segmental peripheral nerves in the zebrafish embryo are pioneered by the caudal primary (CaP) motor axons, which extend midsegmentally at the interface of the somite and the notochord. The signals that define the CaP pathway are not well understood. To gain insight into the nature of the guidance cues, we have examined the environment of the CaP motor axons by using electron microscopy and histochemistry. Specifically, we have mapped the distribution of the transcription factor engrailed, of a chondrotin sulfate epitope, and of the recognition molecules zebrafish semaphorin z1b and zebrafish tenascin C. Ultrastructural examination of dye-labeled CaP motor axons revealed a close association with the medial surface of the somite but not with the notochord. The CaP axons were always accompanied by cells that appeared to migrate at the interface of somite and notochord. These cells were confined to the posterior half of the somite. Some of the cells may be neural crest derived, many others are probably of sclerotomal origin. The putative migratory cells expressed a chondroitin sulfate epitope that is a marker of sclerotome in the chick. The pathway of the CaP axon and the distribution of the putative neural crest and sclerotome cells correlated with a subdivision of the myotome into an anterior and posterior components, which were evident at the histological level and by the expression of the markers engrailed, semaphorin z1b, chondroitin sulfate, and tenascin C. We suggest that both the pathway choice of the CaP axon and the route of migratory cells reflect this anterior-posterior bipartition of the myotome.

Animals↗

PubMed2002

Eye defects in receptor protein-tyrosine phosphatase alpha knock-down zebrafish.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is highly expressed in the developing retina of different species, but little is known about its function there. Here, we report that injection of antisense morpholinos in zebrafish embryos reduced RPTP alpha expression to almost nondetectable levels up to 3 days postfertilization (dpf). RPTP alpha was detectable again from 4 dpf onward. RPTP alpha knock-down resulted in smaller eyes. Examination of sections of the retina at different developmental stages demonstrated that already at 28 hours postfertilization (hpf) fewer cells were present in the retina of RPTP alpha-morpholino-injected embryos. At 3 dpf, the layered organization of the retina was absent. In addition, the morphology and labeling with an axon specific antibody, acetylated tubulin, demonstrated that most cells appeared to be undifferentiated. Strikingly, at 5 dpf the lamination of the retina was partially restored, concomitant with re-expression of RPTP alpha protein. Although cells in the retina were now differentiated, the layering of the retina remained disrupted and significant gaps were observed in the amacrine cell layer. Therefore, knock-down of RPTP alpha protein provides evidence that RPTP alpha is essential for normal retinal development.

Animals↗

PubMed2005

Functional genomic analysis of the Wnt-wingless signaling pathway.

The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.

Animals↗

PubMed2006

Repression of smoothened by patched-dependent (pro-)vitamin D3 secretion.

The developmentally important hedgehog (Hh) pathway is activated by binding of Hh to patched (Ptch1), releasing smoothened (Smo) and the downstream transcription factor glioma associated (Gli) from inhibition. The mechanism behind Ptch1-dependent Smo inhibition remains unresolved. We now show that by mixing Ptch1-transfected and Ptch1 small interfering RNA-transfected cells with Gli reporter cells, Ptch1 is capable of non-cell autonomous repression of Smo. The magnitude of this non-cell autonomous repression of Smo activity was comparable to the fusion of Ptch1-transfected cell lines and Gli reporter cell lines, suggesting that it is the predominant mode of action. CHOD-PAP analysis of medium conditioned by Ptch1-transfected cells showed an elevated 3beta-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3beta-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3beta-hydroxysteroid (pro-)vitamin D3 to the medium effectively inhibited Gli activity. Vitamin D3 bound to Smo with high affinity in a cyclopamine-sensitive manner. Treating zebrafish embryos with vitamin D3 mimicked the smo(-/-) phenotype, confirming the inhibitory action in vivo. Hh activates its signalling cascade by inhibiting Ptch1-dependent secretion of the 3beta-hydroxysteroid (pro-)vitamin D3. This action not only explains the seemingly contradictory cause of Smith-Lemli-Opitz syndrome (SLOS), but also establishes Hh as a unique morphogen, because binding of Hh on one cell is capable of activating Hh-dependent signalling cascades on other cells.

Animals↗

PubMed2006

Biomolecular imaging based on far-red fluorescent protein with a high two-photon excitation action cross section.

Received October 14, 2005; revised January 7, 2006; accepted January 9, 2006; posted January 12, 2006 (Doc. ID 65391) The two-photon excitation action cross section of Hc-Red fluorescent proteins (Hc-RFPs) is measured and found to be of the same order as that of enhanced green fluorescent proteins. With a 618 nm emission wavelength in the far-red region and with an excitation wavelength around 1200 nm, Hc-RPF-based two-photon fluorescence microscopy (2PFM) can offer deep penetration capability inside live samples and is ideal for in vivo gene expression study and biomolecular imaging in live objects. In vivo 2PFM of the developing heart deep inside a transgenic zebrafish embryo tagged by Hc-RFP is also successfully demonstrated.

Animals↗