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PubMed1999

Getting a-head of the organizer: anterior-posterior patterning of the forebrain.

The molecular mechanisms that drive the development of embryonic tissues are being uncovered rapidly. One such fascinating example is the development of the forebrain, the most anterior part of the nervous system. In this review, we will discuss the mechanisms that induce the formation of the forebrain in multiple vertebrate systems, placing emphasis on a recent article published by Grinblat et al. ((1)) Using zebrafish as a model system, these authors combine elegant embryological manipulations with the use of early markers of the presumptive forebrain, to show that initial induction and patterning of this tissue occurs near the onset of gastrulation. In addition, their results confirm observations made in other systems that planar signals, those traveling in the plane of the ectoderm, are involved in forebrain induction and patterning.

Animals↗

PubMed2001

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Acetylcholinesterase↗

PubMed1996

Class III POU genes of zebrafish are predominantly expressed in the central nervous system.

POU genes encode a family of transcription factors involved in a wide variety of cell fate decisions and in the regulation of differentiation pathways. We have searched for POU genes in the zebrafish, a popular model organism for the study of early development of vertebrates. Besides five putative pseudogenes we have identified five POU genes that are expressed during embryogenesis. Probes obtained by PCR were used to isolate full-length cDNAs. Four of the isolated genes encode proteins with class III POU domains. Analysis of genomic clones suggests that the fish genes in general do not contain introns, similar to class III genes of mammals. However, the C-termini of two of the encoded proteins vary due to facultative splicing of a short intervening sequence. These two genes show very strong similarities in their sequence. They have probably arisen by gene duplication, possibly as part of a larger scale duplication of part of the zebrafish genome. Analysis of the expression of the class III genes shows that they are predominantly expressed in the central nervous system and that they may play important roles in patterning the embryonic brain.

Amino Acid Sequence↗

PubMed2025

Studying the Role of HOX Genes in Thrombocyte Development.

In our laboratory, we study thrombopoiesis and hemostasis using zebrafish as a model organism to unravel the mechanisms of differentiation and development of thrombocytes. We have shown in our earlier work that thrombocytes are functional equivalents of platelets and have transcriptional machinery similar to megakaryocytes. We recently found evidence that hox genes play a role in their development. We used piggyback gene knockdown and thrombocyte quantification assays to understand the influence of these ancient developmental regulators on thrombopoiesis. In this chapter, we describe methods used to discover these hox genes.

Animals↗

PubMed2006

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

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

Amino Acid Sequence↗

PubMed2002

Characterization of a type IIb sodium-phosphate cotransporter from zebrafish (Danio rerio) kidney.

Zebrafish (Danio rerio) express two isoforms of the type IIb Na-dependent P(i) cotransporter (NaPi). Type NaPi-IIb1 has previously been cloned and characterized. Here, we report the cloning of the NaPi-IIb2 transcript from zebrafish kidney, its localization, and its functional characterization. RT-PCR with renal RNA and degenerate NaPi-IIb-specific primers resulted in a specific fragment. 3'-Rapid amplification of cDNA ends yielded a product that contained typical NaPi-IIb characteristics such as a cysteine-rich COOH terminus and a PDZ (PSD95- Dlg-zona occludens-1) binding motif. Several approaches were unsuccessful at cloning the 5' end of the transcript; products lacked an in-frame start codon. The missing information was obtained from an EST (GenBank accession number ). The combined clone displayed a high degree of homology with published type IIb cotransporter sequences. Specific antibodies were raised against a COOH-terminal epitope of both NaPi-IIb1 and NaPi-IIb2 isoforms. Immunohistochemical mapping revealed apical expression of both isoforms in zebrafish renal and intestinal epithelia, as well as in bile ducts. The novel clone was expressed in oocytes, and function was assayed by the two-electrode voltage-clamp technique. The function of the new NaPi-IIb2 clone was found to be significantly different from NaPi-IIb1 despite strong structural similarities. NaPi-IIb2 was found to be strongly voltage sensitive, with higher affinities for both sodium and phosphate than NaPi-IIb1. Also, NaPi-IIb2 was significantly less sensitive to external pH than NaPi-IIb1. The strong structural similarity but divergent function makes these zebrafish transporters ideal models for the molecular mapping of functionally important regions in the type II NaPi-cotransporter family.

Amino Acid Sequence↗

PubMed2006

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio.

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K(m) value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Amino Acid Sequence↗

PubMed2006

Developmental and evolutionary origins of the vertebrate dentition: molecular controls for spatio-temporal organisation of tooth sites in osteichthyans.

The rainbow trout (Oncorhynchus mykiss) as a developmental model surpasses both zebrafish and mouse for a more widespread distribution of teeth in the oro-pharynx as the basis for general vertebrate odontogenesis, one in which replacement is an essential requirement. Studies on the rainbow trout have led to the identification of the initial sequential appearance of teeth, through differential gene expression as a changing spatio-temporal pattern, to set in place the primary teeth of the first generation, and also to regulate the continuous production of replacement tooth families. Here we reveal gene expression data that address both the field and clone theories for patterning a polyphyodont osteichthyan dentition. These data inform how the initial pattern may be established through up-regulation at tooth loci from a broad odontogenic band. It appears that control and regulation of replacement pattern resides in the already primed dental epithelium at the sides of the predecessor tooth. A case is presented for the developmental changes that might have occurred during vertebrate evolution, for the origin of a separate successional dental lamina, by comparison with an osteichthyan tetrapod dentition (Ambystoma mexicanum). The evolutionary origins of such a permanent dental lamina are proposed to have occurred from the transient one demonstrated here in the trout. This has implications for phylogenies based on the homology of teeth as only those developed from a dental lamina. Utilising the data generated from the rainbow trout model, we propose this as a standard for comparative development and evolutionary theories of the vertebrate dentition.

Ambystoma↗

PubMed2002

Epidermal growth factor and TGFalpha promote zebrafish oocyte maturation in vitro: potential role of the ovarian activin regulatory system.

Epidermal growth factor (EGF) and TGFalpha are well known for their activities in the ovary. Both factors initiate signal transduction by binding to the common EGF receptor that has been demonstrated in the ovary across vertebrates from fish to humans. Using zebrafish as the model, we demonstrated in the present study that recombinant human EGF and TGFalpha significantly enhanced final maturation of the fully grown, follicle-enclosed oocytes (0.58-0.65 mm) in vitro in a clear time- and dose-dependent manner. The effect of EGF/TGFalpha was additive to that of hCG at low concentrations, but the additivity diminished when the concentration increased. Both actinomycin D and cycloheximide completely blocked the effect of EGF/TGFalpha, indicating that the promotion of oocyte maturation by EGF/TGFalpha requires de novo mRNA transcription and protein synthesis. Interestingly, the effect of EGF/TGFalpha could be blocked by cotreatment with follistatin, a potent binding protein for activin, an ovarian growth factor belonging to the TGFbeta superfamily. Semiquantitative RT-PCR assays showed that both EGF and TGFalpha significantly stimulated the expression of activin betaA and activin type II receptor in the cultured zebrafish ovarian follicle cells in a clear time- and dose-dependent manner. This together with our previous report that activin had a potent stimulatory effect on zebrafish oocyte maturation strongly suggests that the intrinsic ovarian activin system is probably a downstream mediator of EGF/TGFalpha actions in the zebrafish ovary.

Activin Receptors, Type II↗

PubMed2006

sall4 acts downstream of tbx5 and is required for pectoral fin outgrowth.

Okihiro syndrome (OS) is defined by forelimb defects associated with the eye disorder Duane anomaly and results from mutations in the gene SALL4. Forelimb defects in individuals with OS range from subtle thumb abnormalities to truncated limbs. Mutations in the T-box transcription factor TBX5 cause Holt-Oram syndrome (HOS), which results in forelimb and heart defects. Although mutations in TBX5 result in HOS, it has been predicted that these mutations account for only approximately 30% of all individuals with HOS. Individuals with OS and HOS limb defects are very similar, in fact, individuals with mutations in SALL4 have in some cases previously been diagnosed with HOS. Using zebrafish as a model, we have investigated the function of sall4 and the relationship between sall4 and tbx5, during forelimb development. We demonstrate that sall4 and a related gene sall1 act downstream of tbx5 and are required for pectoral fin development. Our studies of Sall gene family redundancy and tbx5 offer explanations for the similarity of individuals with OS and HOS limb defects.

Animals↗

PubMed2004

Hedgehog and Fgf signaling pathways regulate the development of tphR-expressing serotonergic raphe neurons in zebrafish embryos.

Serotonin (5HT) plays major roles in the physiological regulation of many behavioral processes, including sleep, feeding, and mood, but the genetic mechanisms by which serotonergic neurons arise during development are poorly understood. In the present study, we have investigated the development of serotonergic neurons in the zebrafish. Neurons exhibiting 5HT-immunoreactivity (5HT-IR) are detected from 45 h postfertilization (hpf) in the ventral hindbrain raphe, the hypothalamus, pineal organ, and pretectal area. Tryptophan hydroxylases encode rate-limiting enzymes that function in the synthesis of 5HT. As part of this study, we cloned and analyzed a novel zebrafish tph gene named tphR. Unlike two other zebrafish tph genes (tphD1 and tphD2), tphR is expressed in serotonergic raphe neurons, similar to tph genes in mammalian species. tphR is also expressed in the pineal organ where it is likely to be involved in the pathway leading to synthesis of melatonin. To better understand the signaling pathways involved in the induction of the serotonergic phenotype, we analyzed tphR expression and 5HT-IR in embryos in which either Hh or Fgf signals are abrogated. Hindbrain 5HT neurons are severely reduced in mutants lacking activity of either Ace/Fgf8 or the transcription factor Noi/Pax2.1, which regulates expression of ace/fgf8, and probably other genes encoding signaling proteins. Similarly, serotonergic raphe neurons are absent in embryos lacking Hh activity confirming a conserved role for Hh signals in the induction of these cells. Conversely, over-activation of the Hh pathway increases the number of serotonergic neurons. As in mammals, our results are consistent with the transcription factors Nk2.2 and Gata3 acting downstream of Hh activity in the development of serotonergic raphe neurons. Our results show that the pathways involved in induction of hindbrain serotonergic neurons are likely to be conserved in all vertebrates and help establish the zebrafish as a model system to study this important neuronal class.

Animals↗

PubMed2004

Expression analysis of the Toll-like receptor and TIR domain adaptor families of zebrafish.

The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.

Animals↗

PubMed2005

Critical roles of CD146 in zebrafish vascular development.

In this report, we use zebrafish as a model system to understand the importance of CD146 in vascular development. Endothelial-specific expression of CD146 was verified by whole-mount in situ hybridization. Suppression of CD146 protein expression by antisense morpholino oligonucleotides (MO) resulted in poorly developed intersomitic vessels (ISVs). In CD146 morphants, we observed a lack of blood flow through the ISV region, despite that fluorescence microangiography showed that the ISVs were present. This finding suggests that the lumens of the developing ISVs may be too narrow for proper circulation. Additionally, remodeling of the caudal vein plexus into functional vascular tubes appeared to be affected. Suppression of CD146 protein expression resulted in a circulation shunt that caused the circulation to by-pass part of the caudal artery/vein system. The same vascular defects were recapitulated by using a second morpholino oligonucleotide. This morphant expressed a truncated CD146 protein with amino acids V32 to T57 at the N terminus deleted in an in-frame manner. This region, therefore, is likely to contain elements critical for CD146 function. This study provides the first in vivo functional assessment of CD146 in embryonic development by showing that knockdown of CD146 protein expression severely hinders vascular development in zebrafish.

Amino Acid Sequence↗

PubMed1997

Jak1 kinase is required for cell migrations and anterior specification in zebrafish embryos.

Establishment of the vertebrate body plan requires a variety of signaling molecules. In a search for tyrosine kinases expressed in early zebrafish embryos, a model system for the study of vertebrate development, we discovered Jak1 kinase to be maternally encoded and the mRNA evenly distributed among the cells of blastula-stage embryos. Injection of RNA-encoding dominant-negative Jak1 kinases reduces a specific cell migration, epiboly, and results in the reduction of goosecoid expression and of anterior structures. This work establishes that, in addition to its role in signal transduction of cytokines in adult tissues, Jak1 kinase has a role in early vertebrate development.

Animals↗

PubMed1996

Identification of the retinoic acid-inducible all-trans-retinoic acid 4-hydroxylase.

Retinoic acid (RA) metabolites of vitamin A are key regulators of gene expression involved in embryonic development and maintenance of epithelial tissues. The cellular effects of RA are dependent upon the complement of nuclear receptors expressed (RARs and RXRs), which transduce retinoid signals into transcriptional regulation, the presence of cellular retinoid-binding proteins (CRABP and CRBP), which may be involved in RA metabolism, and the activity of RA metabolizing enzymes. We have been using the zebrafish as a model to study these processes. To identify genes regulated by RA during exogenous RA exposure, we utilized mRNA differential display. We describe the isolation and characterization of a cDNA, P450RAI, encoding a novel member of the cytochrome P450 family. mRNA transcripts for P450RAI are expressed normally during gastrulation, and in a defined pattern in epithelial cells of the regenerating caudal fin in response to exogenous RA. In COS-1 cells transfected with the P450RAI cDNA, all-trans-RA is rapidly metabolized to more polar metabolites. We have identified 4-oxo-RA and 4-OH-RA as major metabolic products of this enzyme. P450RAI represents the first enzymatic component of RA metabolism to be isolated and characterized at the molecular level and provides key insight into regulation of retinoid homeostasis.

Amino Acid Sequence↗

PubMed2006

GRK1 and GRK7: unique cellular distribution and widely different activities of opsin phosphorylation in the zebrafish rods and cones.

Retinal cone cells exhibit distinctive photoresponse with a more restrained sensitivity to light and a more rapid shutoff kinetics than those of rods. To understand the molecular basis for these characteristics of cone responses, we focused on the opsin deactivation process initiated by G protein-coupled receptor kinase (GRK) 1 and GRK7 in the zebrafish, an animal model suitable for studies on retinal physiology and biochemistry. Screening of the ocular cDNAs identified two homologs for each of GRK1 (1A and 1B) and GRK7 (7-1 and 7-2), and they were classified into three GRK subfamilies, 1 A, 1B and 7 by phylogenetic analysis. In situ hybridization and immunohistochemical studies localized both GRK1B and GRK7-1 in the cone outer segments and GRK1A in the rod outer segments. The opsin/GRKs molar ratio was estimated to be 569 in the rod and 153 in the cone. The recombinant GRKs phosphorylated light-activated rhodopsin, and the Vmax value of the major cone subtype, GRK7-1, was 32-fold higher than that of the rod kinase, GRK1A. The reinforced activity of the cone kinase should provide a strengthened shutoff mechanism of the light-signaling in the cone and contribute to the characteristics of the cone responses by reducing signal amplification efficiency.

Animals↗

PubMed2006

Inhibition of zebrafish fin regeneration using in vivo electroporation of morpholinos against fgfr1 and msxb.

Increased interest in using zebrafish as a model organism has led to a resurgence of fin regeneration studies. This has allowed for the identification of a large number of gene families, including signaling molecules and transcription factors, which are expressed during regeneration. However, in cases where no specific inhibitor is available for the gene product of interest, determination of a functional role for these genes has been difficult. Here we demonstrate that in vivo electroporation of morpholino oligonucleotides is a feasible approach for protein knock-down during fin regeneration. Morpholino oligonucleotides against fgfr1 and msxb were utilized and knock-down of both proteins resulted in reduced fin outgrowth. Importantly, Fgfr1 knock-down phenocopied outgrowth inhibition obtained with an Fgfr1 inhibitor. Furthermore, this method provided direct evidence for a functional role for msxb in caudal fin regeneration. Finally, knock-down of Fgfr1, but not Msxb, affected the blastemal expression of msxc, suggesting this technique can be used to determine epistasis in genetic pathways affecting regeneration. Thus, this convenient reverse genetic approach allows researchers to quickly (1) assess the function of genes known to be expressed during fin regeneration, (2) screen genes for functional relevance during fin regeneration, and (3) assign genes to the molecular pathways underlying fin regeneration.

Animals↗

PubMed2006

Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity.

The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of approximately 9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum DeltaRD1 mutants are attenuated in adult zebrafish and extend this finding to show that DeltaRD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with DeltaRD1 attenuation in zebrafish embryos is ongoing during adult infection.

Animals↗