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PubMed2007

Dynamic expression of the basic helix-loop-helix transcription factor neuroD in the rod and cone photoreceptor lineages in the retina of the embryonic and larval zebrafish.

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for determining neuronal cell fate and regulating withdrawal from the cell cycle. We showed previously that, in goldfish, neuroD is expressed in the rod photoreceptor lineage, and we inferred that neuroD is also expressed in a subset of amacrine cells and nascent cone photoreceptors. Here we extended that study by examining the temporal and spatial expression pattern of neuroD in the embryonic and larval zebrafish and by identifying the cell types that express this gene. NeuroD expression in the developing zebrafish retina is dynamic, spanning early retinogenesis and the maturation of cone photoreceptors. In early retinogenesis neuroD expression expands from a small patch in the ventronasal retina, through the remaining retinal neuroepithelium. As retinogenesis progresses, neuroD expression becomes restricted to amacrine cells, immature cones, and cells of rod and cone lineages. This expression achieves an adult pattern by 96 hours postfertilization (hpf), whereupon the temporal pattern of neuroD expression in central retina is spatially recapitulated at the germinative margin. The cellular pattern of expression suggests that neuroD regulates aspects of rod and cone genesis, but through separate cellular lineages. Furthermore, neuroD is coexpressed with the cone-rod-homeobox transcription factor (Crx) in putative cone progenitors and nascent cone photoreceptors, suggesting that, in the zebrafish retina, as in other vertebrate retinas, similar genetic cascades regulate photoreceptor genesis and maturation.

Animals↗

PubMed2003

The Wnt/beta-catenin pathway regulates cardiac valve formation.

Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/beta-catenin signalling pathway. Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation. To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor, blocks cushion formation. In wild-type hearts, nuclear beta-catenin, the hallmark of activated canonical Wnt signalling, accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear beta-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial-mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/beta-catenin signalling in determining endocardial cell fate.

Adenomatous Polyposis Coli Protein↗

PubMed2000

The Translocon-Associated Protein beta (TRAPbeta) in zebrafish embryogenesis. I. Enhanced expression of transcripts in notochord and hatching gland precursors.

The normal translocation of nascent polypeptides into the lumen of the endoplasmic reticulum (ER) is thought to be aided in part by a translocon-associated protein (TRAP) complex consisting of 4 protein subunits. The association of mature proteins with the ER and Golgi, or other intracellular locales, such as lysosomes, depends on the initial targeting of the nascent polypeptide to the ER membrane. A similar scenario must also exist for proteins destined for secretion. We have identified a member of the TRAP complex using a two hybrid screen to isolate proteins that bind to zebrafish (Danio) Ran binding protein 1. The polypeptide predicted from the largest open reading frame contains 183 amino acids with a 86 and 87% sequence identity to the TRAPbeta subunits in human and chicken, respectively. Sequence analysis identified a cleavable amino-terminal signal peptide in the zebrafish TRAPbeta subunit and a region of the protein spans the membrane of the endoplasmic reticulum. A reverse transcriptase-polymerase chain reaction assay showed that TRAPbeta mRNA is expressed in the developing zebrafish embryo. TRAPbeta mRNA is maternally supplied to the egg and is expressed constitutively throughout development and in the adult. This pattern of expression indicates that the message encoding part of the machinery targeting nascent polypeptides to the ER lumen is available at the onset of embryogenesis when the rate of translation increases exponentially over that occurring in the oocyte. In situ hybridization was used to test whether or not TRAPbeta transcripts might become localized and/or enriched in the developing embryo. Homogeneous staining is seen in the blastula and early gastrula stages. At mid-to-late gastrula stages, however, the message becomes enriched in the developing notochord and polster, or hatching gland rudiment. The TRAPbeta gene, mapped using the LN54 mouse-zebrafish radiation hybrid panel to linkage group 19, resides next to a gene (Z15451) which has sequence homology to notch2 and vascular endothelial growth factor. TRAPbeta, however, does not appear to belong to a group of genes which are syntenic with orthologues or paralogues on human chromosomes.

Amino Acid Sequence↗

PubMed2005

Xenopus as a model organism in developmental chemical genetic screens.

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.

Animals↗

PubMed2000

The zebrafish pronephros: a genetic system for studies of kidney development.

The zebrafish, as a model system for vertebrate development, offers distinct experimental advantages for studies of organogenesis. The simplicity of the zebrafish pronephros, the feasibility of isolating large numbers of mutants, and the growth in infrastructure for genomics makes the zebrafish an attractive system for the analysis of kidney development. Mutants affecting several aspects of nephrogenesis, including differentiation of the intermediate mesoderm, nephron patterning, epithelial polarity, and angiogenesis, have been isolated. Analysis of mutant phenotypes and the cloning of mutant genes has revealed: (1) a role for bone morphogenetic proteins in patterning the ventral mesoderm, (2) an essential role for the pax2.1 gene in pronephric development, (3) multiple loci required for establishing epithelial polarity in the pronephric duct, (4) a central role for podocytes in directing glomerulogenesis, and (5) 15 loci associated with cystic maldevelopment in the pronephros. The striking similarities of pronephric cell types to those found in higher vertebrates, as well as the conservation of kidney-specific gene expression patterns, suggest that insights gained from studies in zebrafish will be broadly applicable to cell differentiation in the kidney.

Animals↗

PubMed2005

AP2-dependent signals from the ectoderm regulate craniofacial development in the zebrafish embryo.

AP2 transcription factors regulate many aspects of embryonic development. Studies of AP2a (Tfap2a) function in mice and zebrafish have demonstrated a role in patterning mesenchymal cells of neural crest origin that form the craniofacial skeleton, while the mammalian Tfap2b is required in both the facial skeleton and kidney. Here, we show essential functions for zebrafish tfap2a and tfap2b in development of the facial ectoderm, and for signals from this epithelium that induce skeletogenesis in neural crest cells (NCCs). Zebrafish embryos deficient for both tfap2a and tfap2b show defects in epidermal cell survival and lack NCC-derived cartilages. We show that cartilage defects arise after NCC migration during skeletal differentiation, and that they can be rescued by transplantation of wild-type ectoderm. We propose a model in which AP2 proteins play two distinct roles in cranial NCCs: an early cell-autonomous function in cell specification and survival, and a later non-autonomous function regulating ectodermal signals that induce skeletogenesis.

Animals↗

PubMed2006

Embryonic expression of zebrafish AMPA receptor genes: zygotic gria2alpha expression initiates at the midblastula transition.

The AMPA-preferring receptors (AMPARs) mediate rapid excitatory synaptic transmission in the central nervous system of vertebrates. Expression profiles of 8 AMPAR genes were studied by RT-PCR analyses to elucidate the properties of AMPARs during early zebrafish development. Transcripts of all AMPAR genes are detected at the time of fertilization, suggesting maternal transcriptions of zebrafish AMPAR genes. The amounts of gria1 and gria2 transcripts are several-fold higher than that of gria3 and gria4 between 10 and 72 hpf (hour postfertilization). The edited gria2alpha transcript decreases during gastrulation period, suggesting that zygotic expression of gria2alpha begins around the time of midblastula transition. Relative to the amount of beta-actin, the amounts of AMPAR transcripts increase significantly after the completion of neurulation. The amounts of gria2 transcripts exceed the total amounts of the remaining AMPAR transcripts after 36 hpf, suggesting increases in the representation of low Ca2+ permeable AMPARs during neuronal maturation. Many but not all of the known mammalian protein-protein interaction motifs are preserved in the C-terminal domains (CTD) of zebrafish AMPARs. Before 16 hpf, the embryos express predominantly the alternative splice forms encoding longer CTD. Representations of the short CTD splice forms of gria2 and gria4alpha increase after 24 hpf, when neurulation is nearly completed.

Animals↗

PubMed2000

Characterization of a zebrafish (Danio rerio) desmin cDNA: an early molecular marker of myogenesis.

Desmin is a muscle-specific protein and a constitutive subunit of the intermediate filaments (IF) in skeletal, cardiac and smooth muscles. It is an early marker of skeletal muscle myogenesis. We have characterized a clone of desmin cDNA from an embryonic zebrafish (Danio rerio) cDNA library. The full-length cDNA comprised 1798 nucleotides, encoding a protein of 473 amino acids. The predicted amino acid sequence of the zebrafish desmin shares a high degree of similarity to other vertebrate desmins, but also contains a sequence at the carboxyl terminal of the tail domain that is unique to the zebrafish. It carries many features which are distinctive of IF subunit proteins. These include the T/SSYRRXF/Y motif in the head domain, and the intermediate filament signature consensus, [I/V]-X-[T/A/C/I]-Y-[R/K/H]-X-[L/M]-L-[D/E], located in the carboxyl terminus of the central helical rod. Unlike other 3' UTR sequences, the 3' UTR of the zebrafish cDNA sequence has two CAYUG elements flanking a single polyadenylation site. The temporal and spatial expression patterns of desmin mRNA during early zebrafish development were studied. The onset of desmin expression occurred at the 1-3 somite stage (11 hpf). It increased throughout somitogenesis, with maximum expression at the Prim-6 stage (25 hpf), and decreasing expression towards the protruding-mouth stage (72 hpf). Desmin mRNA was initially localised exclusively to the somites, but was subsequently also detected in other musculature in the developing heart and fins. The onset of expression and the spatial localization of desmin mRNA in the zebrafish coincides with that reported for MyoD and myogenin.

Amino Acid Sequence↗

PubMed2006

Target of rapamycin (TOR) signaling controls epithelial morphogenesis in the vertebrate intestine.

The target of rapamycin (TOR) signaling pathway regulates cell growth and proliferation, however the extent to which TOR signaling mediates particular organogenesis programs remains to be determined. Here we report an examination of TOR signaling during zebrafish development, using a combination of small molecule treatment and morpholino-mediated gene knockdown. First, we amplified and sequenced the full-length cDNA for the zebrafish TOR ortholog (ztor). By in situ hybridization, we found that ztor is expressed ubiquitously in the early embryo, but displays a dynamic pattern in the gut between 48 and 72 h post-fertilization (hpf). Treatment of zebrafish embryos with rapamycin induced only a mild general developmental delay up to 72 hpf, but digestive tract development became arrested at the primitive gut tube stage. Rapamycin inhibited intestinal epithelial growth, morphogenesis and differentiation. Using morpholino-mediated gene knockdown of TOR pathway components, we show that this effect is mediated specifically by the rapamycin-sensitive TOR complex 1 (TORC1). Thus, in addition to regulating cell growth and proliferation, TOR signaling controls the developmental program guiding epithelial morphogenesis in the vertebrate intestine.

Animals↗

PubMed1995

Stages of embryonic development of the zebrafish.

We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis--the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts.

Animals↗

PubMed1998

Expression of Rhizobium chitin oligosaccharide fucosyltransferase in zebrafish embryos disrupts normal development.

In this report we present data about the effect of the Rhizobium NodZ enzyme on zebrafish development. We injected zebrafish embryos with a plasmid expressing NodZ protein, and we confirmed that the enzyme is active and has chitin oligosaccharide fucosyltransferase (NodZ) activity in vitro. In addition, the embryos injected with the NodZ-expressing plasmid, but not with a control plasmid, showed malformations or bends in the tail, and in some cases shunted tail structures and fused somites. These results clearly indicate that the likely substrates for this enzyme, chitin oligosaccharides and free N-glycans, have essential functions during early vertebrate embryogenesis.

Animals↗

PubMed1997

Vessel patterning in the embryo of the zebrafish: guidance by notochord.

We have cloned the zebrafish homolog of the receptor tyrosine kinase flk-1 to provide us with a tool to study normal vascular pattern formation in the developing zebrafish embryo and to compare it to mutants in which vascular pattern is perturbed. We find that during normal development the first angioblasts arise laterally in the mesoderm and then migrate medially to form the primordia of the large axial vessels, the dorsal aorta (axial artery) and the axial vein. Lumen formation occurs shortly before onset of circulation at 24 hr postfertilization. We examined the specification of vascular progenitors in the mutant cloche, which fails to form both vessels and blood. cloche lacks all flk-expressing cells and therefore appears to lack angioblasts. The axial vessels of the trunk form in close proximity to notochord and endoderm, which may provide cues for their formation. The dorsal aorta is normally just ventral to the notochord; the axial vein is just below the dorsal aorta and above the endoderm. floating head (flh) and no tail (ntl) mutants both have defects in the formation of notochord. Both are cell-autonomous lesions, flh abolishing notochord and ntl preventing its differentiation. In both mutants the dorsal aorta fails to form, while formation of the axial vein is less affected. Mosaic analysis of mutant embryos shows that transplanted wild-type cells can become notochord in mutant flh embryos. In these mosaic embryos flh cells expressing flk assemble at the midline, beneath the wild-type notochord, and form an aortic primordium. This suggests that signals from the notochord may guide angioblasts in the fashioning of the dorsal aorta. The notochord seems to be less important for the formation of the vein.

Amino Acid Sequence↗

PubMed2003

The zebrafish band 4.1 member Mir is involved in cell movements associated with gastrulation.

Cellular processes rely on dynamic events occurring between the cortical cytoskeleton and plasma membrane. Members of the Band 4.1 superfamily, which are best known for their ability to tether the cytoskeleton to the plasma membrane, play prominent structural and regulatory roles that influence cell-cell and cell-substrate interactions, endo- and exocytosis, cell polarity, migration, proliferation, and differentiation. We have identified a new member of the zebrafish Band 4.1 superfamily, which is the homolog of human myosin regulatory light chain interacting protein (MIR), and have examined its role in embryonic development. Zebrafish Mir contains the conserved amino-terminal plasma membrane-binding FERM (Band 4.1/ezrin/radixin/moesin) domain as well as other putative protein-protein interacting domains, including a RING finger. Overall, zebrafish Mir is 71% identical to human MIR located at chromosome 6p23-p22.3, and maps on linkage group 19 to a region of synteny with human chromosome 6. In situ hybridization and RT-PCR revealed that mir is expressed maternally and ubiquitously throughout development. Blocking Mir translation using a mir-specific, morpholino-based, knock-down strategy or expressing Mir constructs lacking the RING finger domain disrupts gastrulation and leads to subsequent trunk and tail defects. In severe cases, morphants exogastrulate. The synergistic effect seen when two mir-specific morpholinos are used in conjunction reflects the specific knock-down of mir. In addition, morphant phenotypes induced by mir-specific morpholinos are rescued by overexpression of the full-length Mir. In situ hybridization analysis with mesodermal- and neural-specific markers shows that morphants exhibit a delay in cell movements associated with gastrulation, epiboly, convergence, and extension. A yeast two-hybrid analysis was performed to identify binding partners that may participate with Mir during gastrulation, and Annexin V, a calcium channel protein, was isolated. At early developmental stages, annexin V transcripts colocalize with mir, but after gastrulation, annexin V mRNA becomes localized to the distal tail region and an area in the olfactory placode. At the protein level, Mir colocalizes with Annexin V when expressed in COS cells. Together, these results indicate that Mir is essential for embryonic development and that its role in early embryonic development likely involves calcium-dependent mechanisms essential during the extensive cell movements associated with gastrulation.

Amino Acid Sequence↗

PubMed1997

Heat shock protein gene expression during embryonic development of the zebrafish.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.

Animals↗

PubMed2004

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

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

Amino Acid Sequence↗

PubMed2000

Isolation, characterization, expression and functional analysis of the zebrafish ortholog of MEN1.

Mutations in the MEN1 gene lead to an autosomal dominant disorder, multiple endocrine neoplasia type 1 (MEN1), which is characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine, and pituitary tissues. The protein encoded by MEN1, 610-amino acid menin, resides primarily in the nucleus and binds to the transcription factor JunD, resulting in the repression of JunD-induced transcription. We report here a detailed characterization of the zebrafish men1 gene and its full-length (2551 nt) transcript, encoding a 617-amino acid protein with 67% identity and 80% similarity to human menin. Of the 81 missense mutations and in-frame deletions reported in MEN1 patients, 72 occur in residues that are identical in zebrafish, suggesting the importance of the conserved regions. The zebrafish men1 gene maps 61 cM from the top of linkage group 7 (LG7), a region that appears to show conserved synteny to the MEN1 loci at human 11q13. A 2.7-kb men1 message is detected at all stages of zebrafish development analyzed, from one-cell embryos to adult fish. Whole-mount in situ hybridization showed ubiquitous distribution of men1 message in zebrafish embryos at cleavage, blastula, gastrula, and early segmentation stages, with relatively abundant expression in blood cell progenitors (24 h post fertilization) and mesenchymal tissues (48 h post fertilization) at later stages. Zebrafish menin binds both human and mouse JunD, and represses JunD-induced transcription, indicating that the JunD-binding ability of menin is evolutionarily conserved.

Amino Acid Sequence↗

PubMed1995

COUP-TFI is a potential regulator of retinoic acid-modulated development in Xenopus embryos.

Isomers of retinoic acid are considered likely regulators of developmental pattern formation in vertebrate embryos. The orphan receptor COUP-TFI, which can alter cellular responses to retinoic acid in cultured cells, is expressed in distinct regions of the developing zebrafish and mouse anterior central nervous system. We asked if COUP-TFI can modulate retinoic acid signaling and anterior neural development in a vertebrate embryo by examining: (1) whether COUP-TFI could alter transcriptional responses to retinoic acid in Xenopus embryonic explants, and (2) whether misexpression of COUP-TFI could regulate anterior neural gene expression during early Xenopus development. The results from these studies show that COUP-TFI is a potent regulator of retinoic acid-induced gene expression in Xenopus embryonic cells, and that misexpression of COUP-TFI causes deficiencies in anterior neural structures and head development in Xenopus embryos with a concomitant change in anterior neural gene expression. These results support the proposition that COUP-TFI has a role in the elaboration and patterning of anterior neural gene expression in vertebrates, possibly via effects on the retinoic acid signaling pathways.

Activins↗

PubMed2004

Tenascin-R as a repellent guidance molecule for newly growing and regenerating optic axons in adult zebrafish.

In adult fish, in contrast to mammals, new optic axons are continuously added to the optic projection, and optic axons regrow after injury. Thus, pathfinding of optic axons during development, adult growth, and adult regeneration may rely on the same guidance cues. We have shown that tenascin-R, a component of the extracellular matrix, borders the optic pathway in developing zebrafish and acts as a repellent guidance molecule for optic axons. Here we analyze tenascin-R expression patterns along the unlesioned and lesioned optic pathway of adult zebrafish and test the influence of tenascin-R on growing optic axons of adult fish in vitro. Within intraretinal fascicles of optic axons and in the optic nerve, newly added optic axons grow in a tenascin-R immunonegative pathway, which is bordered by tenascin-R immunoreactivity. In the brain, tenascin-R expression domains in the ventral diencephalon, in non-retinorecipient pretectal nuclei and in some tectal layers closely border the optic pathway in unlesioned animals and during axon regrowth. We mimicked these boundary situations with a sharp substrate border of tenascin-R in vitro. Optic axons emanating from adult retinal explants were repelled by tenascin-R substrate borders. This is consistent with a function of tenascin-R as a repellent guidance molecule in boundaries for adult optic axons. Thus, tenascin-R may guide newly added and regenerating optic axons by a contact-repellent mechanism in the optic pathway of adult fish.

Animals↗