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At least 991 records · Page 55Linked to original sources
PubMed2003

Essential role for puma in development of postembryonic neural crest-derived cell lineages in zebrafish.

Multipotent neural crest stem cells have been identified in late gestation amniote embryos. Yet, significant questions remain about the mechanisms by which these cells are generated, maintained, and recruited during postembryonic development. The zebrafish, Danio rerio, offers an opportunity to identify genes essential for these processes, by screening for mutants with defects in traits likely to depend on these cells during metamorphosis and adult life. One such trait is the pigment pattern formed by neural crest-derived pigment cells, or chromatophores, which include black melanophores, yellow xanthophores, and iridescent iridophores. Previous analyses have demonstrated that the adult zebrafish pigment pattern depends on the de novo differentiation of latent precursor cells during both early and late phases of pigment pattern metamorphosis. To better understand the development of these cells, in this study, we analyze the zebrafish puma mutant, which ablates most of the adult melanophores that differentiate during metamorphosis, but leaves intact early larval melanophores that differentiate during embryogenesis. We use epistasis analyses to show that puma promotes the development of both early-appearing metamorphic melanophores that depend on the kit receptor tyrosine kinase, as well as late-appearing metamorphic melanophores that depend on both the G-protein-coupled endothelin receptor b1 (ednrb1) and the kit-related fms receptor tyrosine kinase. We further demonstrate that, during pigment pattern metamorphosis, puma mutants have deficiencies in the numbers of cells expressing transcripts for kit, ednrb1, and fms, as well as the HMG domain transcription factor sox10. Because the puma mutant phenotype is temperature-sensitive, we use temperature-shift experiments to identify a critical period for puma activity during pigment pattern metamorphosis. Finally, we use cell transplantations to show that puma acts cell-autonomously to promote the expansion of pigment cell lineages during metamorphosis. These results suggest a model for the lineage diversification of neural crest stem cells during zebrafish postembryonic development.

Animals↗
PubMed2005

Aryl hydrocarbon receptor activation inhibits regenerative growth.

There is considerable literature supporting the conclusion that inappropriate activation of the aryl hydrocarbon receptor (AHR) alters cellular signaling. We have established previously that fin regeneration is specifically inhibited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in adult zebrafish and have used this in vivo endpoint to evaluate interactions between AHR and growth-controlling pathways. Because there are experimental limitations in studying regeneration in adult animals, we have developed a larval model to evaluate the effect of AHR activation on tissue regeneration. Two-day-old zebrafish regenerate their amputated caudal fins within 3 days. Here, we demonstrate that TCDD specifically blocks regenerative growth in larvae. The AHR pathway in zebrafish is considerably more complex than in mammals, with at least three zebrafish AHR genes (zfAHR1a, zfAHR1b, and zfAHR2) and two ARNT genes (zfARNT1 and zfARNT2). Although it was presumed that the block in regeneration was mediated by AHR activation, it had not been experimentally demonstrated. Using antisense morpholinos and mutant fish lines, we report that zfAHR2 and zfARNT1 are the in vivo dimerization partners that are required for inhibition of regeneration by TCDD. Several pathways including fibroblast growth factor (FGF) signaling are essential for fin regeneration. Even though impaired FGF signaling and TCDD exposure both inhibit fin regeneration, their morphometric response is distinct, suggesting that the mechanisms of impairment are different. With the plethora of molecular and genetic techniques that can be applied to larval-stage embryos, this in vivo regeneration system can be further exploited to understand cross-talk between AHR and other signaling pathways.

Animals↗
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↗
PubMed2006

Inhibition of Notch signaling induces myotube hypertrophy by recruiting a subpopulation of reserve cells.

During muscle differentiation, a population of quiescent undifferentiated myoblasts (reserve cells) emerges among mature muscle cells. However, the molecular mechanisms underlying such cell segregation and the characterization of this subpopulation of myoblasts remain to be determined. Notch is known to control the behavior and fate of murine muscle stem cells. In this study, we examined the role of Notch in myoblast segregation. We showed that inhibition of Notch activity by either overexpressing Numb or by using a pharmacological gamma-secretase inhibitor (DAPT) enhanced differentiation of murine and human myoblasts. This effect was not restricted to in vitro culture systems since DAPT-treated zebrafish embryos also showed increased differentiation. Using C2.7 myoblasts as a model, we showed that inhibition of Notch induced myotube hypertrophy by recruiting reserve cells that do not normally fuse. We further showed that endogenous Notch-signaling components were differentially expressed and activated in reserve cells with respect to Notch 1 and CD34 expression. We identified CD34 negative reserve cells as the subpopulation of myoblasts recruited to fuse into myotubes during differentiation in response to Notch inhibition. Therefore, we showed here that the activation of Notch 1 is important to maintain a subpopulation of CD34 negative reserve cells in an undifferentiated state.

Animals↗
PubMed2001

Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.

The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.

Animals↗
PubMed2026

Hypoxia-activated scleraxis a mediates epicardial progenitor differentiation into a unique cardiac perivascular cell type.

The epicardium provides progenitor cells and paracrine signals essential for heart development and regeneration, yet the mechanisms regulating epicardial cell fate remain poorly understood. Here, we identify the transcription factor Scleraxis a (scxa) as a key regulator of epicardial progenitor differentiation in zebrafish. Single-cell transcriptomics, genetic lineage tracing, and cardiac injury models reveal transient scxa expression in activated epicardial progenitor cells (aEPCs) during developmental coronary angiogenesis and heart regeneration. scxa+ epicardial cells predominantly differentiate into a previously uncharacterized col18a1a+ perivascular population, termed epicardial-derived perivascular mesenchymal cells (Epi-PMCs), which is distinct from pericytes, vascular smooth muscle cells, and mammalian adventitial fibroblasts. Epi-PMCs closely associate with coronary vessels and may contribute to vascular stabilization and remodeling, potentially through collagen XVIII. Loss of scxa increases coronary vessel density. Hypoxia and Hif signaling induce scxa expression, identifying a hypoxia-responsive mechanism that promotes epicardial differentiation toward a vascular-supportive fate during heart development and regeneration.

Animals↗
PubMed2006

Genomic alterations upon integration of zebrafish L1 elements revealed by the TANT method.

Transposable elements, or transposons, constitute substantial portions of eukaryotic genomes and have contributed to the diversity and functions of the genomes. Bioinformatic analysis of target junctions of genomic transposon copies can provide insights into their mobility mechanisms and consequent genomic alterations, but definitive identification of the target junctions remains difficult despite the steady accumulation of genomic sequence information. To overcome this difficulty, we recently developed a method termed "the target analysis of nested transposons" (TANT), which anatomizes junction features of numerous genomic copies of transposons that reside within other transposons. Whereas the mammalian long interspersed nuclear element (LINE)-1 (L1), a retrotransposon, has been proposed to make a considerable impact on host genomes, the mobility and impact of non-mammalian L1s are poorly understood. In the present study, we analyzed genomic copies of zebrafish L1 elements by using the TANT method. Some copies exhibited the features of integration that are similar to those of mammalian L1s. The zebrafish L1 retrotransposition reaction, however, frequently truncated the target-site DNA by up to 0.6 kb and produced a new sequence at LINE-target junctions. Moreover, our data suggest that L1 retrotransposition can be used to repair double-strand DNA breaks (DSBs). These results imply that L1s have had considerable impact on the evolution of the zebrafish genome.

Animals↗
PubMed2005

High-affinity peptide transporter PEPT2 (SLC15A2) of the zebrafish Danio rerio: functional properties, genomic organization, and expression analysis.

Solute carrier 15 (SLC15) membrane proteins PEPT1 (SLC15A1) and PEPT2 (SLC15A2) have been described in great detail in mammals. In contrast, information in lower vertebrates is limited. We characterized the functional properties of a novel zebrafish peptide transporter orthologous to mammalian and avian PEPT2, described its gene (pept2) structure, and determined mRNA tissue distribution. An expressed sequence tag (EST) cDNA (Integrated Molecular Analysis of Gene Expression; IMAGE) corresponding to zebrafish pept2 was completed by inserting a stretch of 75 missing nucleotides in the coding sequence to obtain a 3,238-bp functional clone. The complete open reading frame (ORF) was 2,160 bp and encoded a 719-amino acid protein. Electrophysiological analysis after cRNA injection in Xenopus laevis oocytes suggested that zebrafish PEPT2 is a high-affinity/low-capacity transporter (K(0.5) for glycyl-L-glutamine approximately 18 microM at -120 mV and pH 7.5). Zebrafish pept2 gene was 19,435 kb, thus being the shortest vertebrate pept2 fully characterized so far. Also, zebrafish pept2 exhibited 23 exons and 22 introns, whereas human and rodent pept2 genes contain 22 exons and 21 introns only. Zebrafish pept2 mRNA was mainly detected in brain, kidney, gut, and, interestingly, otic vesicle, the embryonic structure that develops into the auditory/vestibular organ, homolog to the higher vertebrate inner ear, of the adult fish. Characterization of zebrafish pept2 will contribute to the investigation of peptide transporters using a well-established genetic model and will allow the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful marker to screen mutations that affect choroid plexus and inner ear development.

Amino Acid Sequence↗
PubMed2002

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation.

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.

Animals↗
PubMedDate not supplied

Sensitivity of zebrafish to environmental toxins implicated in Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. Whereas the etiology and pathogenesis of PD is still poorly understood, studies in animal models are providing important insights. One valuable type of animal model for PD is established by treating animals with PD-inducing neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, and paraquat. These neurotoxins are thought to inhibit mitochondrial complex I activity leading to oxidative stress, impaired energy metabolism, proteasomal dysfunction, and, eventually, dopamine neuronal loss. However, the genes and pathways that underlie the neurotoxicity of these agents are not known. In this study, we explored the effect of MPTP, rotenone, and paraquat in both adult and larval zebrafish, which are highly amenable to genetic analysis that can lead to the identification of the underlying genes and pathways. Here, we report that adult zebrafish display behavioral alterations, including decreased locomotor activity in response to MPTP, whereas larval zebrafish exhibited developmental, behavioral, and DA sensitivity to these agents. Taken together, these findings suggest that zebrafish could be a valuable model for genetically dissecting the molecular mechanisms underlying the neurotoxicity of PD-inducing agents.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1992

Diisopropylfluorophosphate inhibits acetylcholinesterase activity and disrupts somitogenesis in the zebrafish.

Acetylcholinesterase (AChE) activity, localized histochemically, appeared in the nuclei of presumptive somitic mesodermal cells prior to the onset of somitogenesis. AChE activity appeared in a rostro-caudal sequence, in cells located the equivalent of five somite lengths caudal to the last formed somite. To investigate whether AChE activity was required for somitogenesis, several inhibitors of AChE activity were tested for their ability to block somitogenesis. Diisopropylfluorophosphate (DFP), a broad spectrum inhibitor of serine proteases and related enzymes, was the only AChE inhibitor tested that disrupted somitogenesis. Gastrulae at 50% epiboly exposed continuously to DFP at concentrations between 40 microM and 90 microM completed epiboly, but exhibited a dose-dependent decrease in the number of somites formed, and a parallel decrease in the caudal extent of somite innervation, by 24 hours post-fertilization (h). Fifteen somite (15h) embryos exposed to DFP at the ED50 of 70 microM for 3 hours, followed by recovery to 24h, developed abnormal somites. Approximately five normal somites formed after drug treatment before the first abnormal somite formed. The abnormal somites corresponded in location to that area of the presumptive somitic mesoderm that would have initiated AChE activity while the DFP was present. While exposed to 70 microM DFP, presumptive somites formed and motoneurons extended processes that had initiated AChE activity at the time of treatment with DFP, although at a slower than normal rate. However, embryos exposed to 1 mM DFP for 30 minutes at both the 5 and 15 somite stages, followed by recovery to 24h, developed the normal number of somites but were reduced in the caudal extent of somite innervation, and occasionally developed abnormal primary motoneurons. As with the abnormal somites, the abnormal motoneurons would have initiated AChE activity while the DFP was present. Presumptive somitic mesoderm unable to initiate AChE activity due to inhibition by DFP developed abnormally. While the effects of DFP are not limited to inhibiting AChE, the data support the "clock and wavefront" model proposed for somite formation, and support the hypothesis that AChE activity has a role in somitogenesis in zebrafish.

Animals↗
PubMed2004

Signals derived from the underlying mesoderm are dispensable for zebrafish neural crest induction.

Signals from the non-neural ectoderm, the neural ectoderm, and the underlying mesoderm have all been implicated in the induction of neural crest. Bone morphogenetic protein (BMP) signaling in particular has an important role in this process; however, it is unclear whether this activity of BMP is due to its effects on patterning the underlying mesoderm, to its ability to establish a competent neural plate boundary zone, or to the direct specification of neural crest at intermediate levels of activity within a BMP gradient. We show neural crest induction occurs in zebrafish in the absence of involuted mesoderm, indicating that this tissue and signals derived from it are dispensable for the formation of neural crest. Dorsal-involuted mesoderm is a major source of secreted BMP antagonists, and the activity of BMP signaling is thought to depend on the presence of the opposing activity of these antagonists. We find that the three BMP antagonists known to be expressed during gastrulation in zebrafish, noggin1, follistatin, and chordin, are dispensable for neural crest induction. These results suggest that mechanisms for restricting the spatio-temporal pattern of BMP expression may compensate for the loss of secreted BMP antagonist activity in establishing dorso-ventral patterning, neural induction, and the neural crest.

Animals↗
PubMed2006

EBP, a program for protein identification using multiple tandem mass spectrometry datasets.

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.

Algorithms↗
PubMed2006

Developing dendrites demonstrate unexpected specificity.

Our knowledge of how developing dendrites attain their mature state is still rudimentary. In this issue of Neuron, Mumm et al. rely on time-lapsed analysis of ingrowing dendrites of retinal ganglion cells in transgenic zebrafish to show that this process is much more specific than has been suspected.

Animals↗
PubMed2004

Comparative analysis of a 229-kb medaka genomic region, containing the zic1 and zic4 genes, with Fugu, human, and mouse.

Medaka is one of the prominent model animals, which also include other fishes such as Fugu and zebrafish. Its genome is relatively compact but has not been well characterized. Here we have sequenced a 229-kb region of medaka, containing the Double anal fin (Da) locus, and compared its structure to those in Fugu, human, and mouse. This region, representing a gene-poor region, contains no major rearrangements and can be readily compared among different species. Comparison of G+C contents and repeats suggested that medaka and Fugu are highly related as expected and that medaka is more similar to mammals than Fugu is. Sequence comparisons of developmental genes zic1 and zic4, identified within this region, revealed that zic1, but not zic4, is highly conserved among vertebrates. The 5' coding region of zic4 is, however, extremely homologous among fishes with little synonymous substitutions, implying its distinct function in fish.

Amino Acid Sequence↗
PubMed2006

Transgenic analysis of Dlx regulation in fish tooth development reveals evolutionary retention of enhancer function despite organ loss.

It has been considered a "law" that a lost structure cannot reappear in evolution. The common explanation, that genes required for the development of the lost structure degrade by mutation, remains largely theoretical, however. Additionally, the extent to which this mechanism applies to systems of repeated parts, where individual modules are likely to exhibit few unique aspects of genetic control, is unclear. We investigated reversibility of evolution in one such system, the vertebrate dentition, using as a model loss of oral teeth in cypriniform fishes, which include the zebrafish. This evolutionary event, which occurred > 50 million years ago, has not been reversed despite subsequent diversification of feeding modes and retention of pharyngeal teeth. We asked whether the cis-regulatory region of a gene whose expression loss parallels cypriniform tooth loss, Dlx2b, retains the capacity for expression in oral teeth. We first created a zebrafish reporter transgenic line that recapitulates endogenous dlx2b expression. We then showed that this zebrafish construct drives reporter expression in oral teeth of the related characiform Astyanax mexicanus. This result, along with our finding that Dlx genes are required for normal tooth development, suggests that changes in trans-acting regulators of these genes were responsible for loss of cypriniform oral teeth. Preservation of oral enhancer function unused for > 50 million years could be the result of pleiotropic function in the pharyngeal dentition. If enhancers of other genes in the tooth developmental pathway are similarly preserved, teeth lost from specific regions may be relatively easy to reacquire in evolution.

Animals↗