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PubMed2005

The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis.

The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing embryo.

Amino Acid Sequence↗

PubMed2005

Regulation of the lmo2 promoter during hematopoietic and vascular development in zebrafish.

The Lmo2 transcription factor, a T-cell oncoprotein, is required for both hematopoiesis and angiogenesis. To investigate the fate of lmo2-expressing cells and the transcriptional regulation of lmo2 in vivo, we generated stable transgenic zebrafish that express green fluorescent protein (EGFP) or DsRed under the control of an lmo2 promoter. A 2.5-kb fragment contains the cis-regulatory elements required to recapitulate endogenous lmo2 expression in embryonic hematopoietic and vascular tissues. We further characterized embryonic Lmo2+ cells through transplantation into vlad tepes (vlt), an erythropoietic mutant. These Lmo2+ primitive wave donor cells differentiated into circulating hematopoietic cells and extended the life span of vlt recipients, but did not demonstrate long-term repopulation of the erythroid lineage. Promoter analysis identified a 174-bp proximal promoter that was sufficient to recapitulate lmo2 expression. This element contains critical ETS-binding sites conserved between zebrafish and pufferfish. Furthermore, we show that ets1 is coexpressed with lmo2, and overexpression experiments indicate that ets1 can activate the lmo2 promoter through this element. Our studies elucidate the transcriptional regulation of this key transcription factor, and provide a transgenic system for the functional analysis of blood and blood vessels in zebrafish.

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PubMed2005

Gli function is essential for motor neuron induction in zebrafish.

The Gli family of zinc-finger transcription factors mediates Hedgehog (Hh) signaling in all vertebrates. However, their roles in ventral neural tube patterning, in particular motor neuron induction, appear to have diverged across species. For instance, cranial motor neurons are essentially lost in zebrafish detour (gli1(-)) mutants, whereas motor neuron development is unaffected in mouse single gli and some double gli knockouts. Interestingly, the expression of some Hh-regulated genes (ptc1, net1a, gli1) is mostly unaffected in the detour mutant hindbrain, suggesting that other Gli transcriptional activators may be involved. To better define the roles of the zebrafish gli genes in motor neuron induction and in Hh-regulated gene expression, we examined these processes in you-too (yot) mutants, which encode dominant repressor forms of Gli2 (Gli2(DR)), and following morpholino-mediated knockdown of gli1, gli2, and gli3 function. Motor neuron induction at all axial levels was reduced in yot (gli2(DR)) mutant embryos. In addition, Hh target gene expression at all axial levels except in rhombomere 4 was also reduced, suggesting an interference with the function of other Glis. Indeed, morpholino-mediated knockdown of Gli2(DR) protein in yot mutants led to a suppression of the defective motor neuron phenotype. However, gli2 knockdown in wild-type embryos generated no discernable motor neuron phenotype, while gli3 knockdown reduced motor neuron induction in the hindbrain and spinal cord. Significantly, gli2 or gli3 knockdown in detour (gli1(-)) mutants revealed roles for Gli2 and Gli3 activator functions in ptc1 expression and spinal motor neuron induction. Similarly, gli1 or gli3 knockdown in yot (gli2(DR)) mutants resulted in severe or complete loss of motor neurons, and of ptc1 and net1a expression, in the hindbrain and spinal cord. In addition, gli1 expression was greatly reduced in yot mutants following gli3, but not gli1, knockdown, suggesting that Gli3 activator function is specifically required for gli1 expression. These observations demonstrate that Gli activator function (encoded by gli1, gli2, and gli3) is essential for motor neuron induction and Hh-regulated gene expression in zebrafish.

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PubMed2005

Exocrine pancreas development in zebrafish.

Although many of the genes that regulate development of the endocrine pancreas have been identified, comparatively little is known about how the exocrine pancreas forms. Previous studies have shown that exocrine pancreas development may be modeled in zebrafish. However, the timing and mechanism of acinar and ductal differentiation and morphogenesis have not been described. Here, we characterize zebrafish exocrine pancreas development in wild type and mutant larvae using histological, immunohistochemical and ultrastructural analyses. These data allow us to identify two stages of zebrafish exocrine development. During the first stage, the exocrine anlage forms from rostral endodermal cells. During the second stage, proto-differentiated progenitor cells undergo terminal differentiation followed by acinar gland and duct morphogenesis. Immunohistochemical analyses support a model in which the intrapancreatic ductal system develops from progenitors that join to form a contiguous network rather than by branching morphogenesis of the pancreatic epithelium, as described for mammals. Contemporaneous appearance of acinar glands and ducts in developing larvae and their disruption in pancreatic mutants suggest that common molecular pathways may regulate gland and duct morphogenesis and differentiation of their constituent cells. By contrast, analyses of mind bomb mutants and jagged morpholino-injected larvae suggest that Notch signaling principally regulates ductal differentiation of bipotential exocrine progenitors.

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PubMed2005

sox4b is a key player of pancreatic alpha cell differentiation in zebrafish.

Pancreas development relies on a network of transcription factors belonging mainly to the Homeodomain and basic Helix-Loop-Helix families. We show in this study that, in zebrafish, sox4, a member of the SRY-like HMG-box (SOX) family, is required for proper endocrine cell differentiation. We found that two genes orthologous to mammalian Sox4 are present in zebrafish and that only one of them, sox4b, is strongly expressed in the pancreatic anlage. Transcripts of sox4b were detected in mid-trunk endoderm from the 5-somite stage, well before the onset of expression of the early pancreatic gene pdx-1. Furthermore, by fluorescent double in situ hybridization, we found that expression of sox4b is mostly restricted to precursors of the endocrine compartment. This expression is not maintained in differentiated cells although transient expression can be detected in alpha cells and some beta cells. That sox4b-expressing cells belong to the endocrine lineage is further illustrated by their absence from the pancreata of slow-muscle-omitted mutant embryos, which specifically lack all early endocrine markers while retaining expression of exocrine markers. The involvement of sox4b in cell differentiation is suggested firstly by its up-regulation in mind bomb mutant embryos displaying accelerated pancreatic cell differentiation. In addition, sox4b knock-down leads to a drastic reduction in glucagon expression, while other pancreatic markers including insulin, somatostatin, and trypsin are not significantly affected. This disruption of alpha cell differentiation is due to down-regulation of the homeobox arx gene specifically in the pancreas. Taken together, these data demonstrate that, in zebrafish, sox4b is expressed transiently during endocrine cell differentiation and plays a crucial role in the generation of alpha endocrine cells.

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PubMed2005

Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes.

Podocytes are specialized cells of the kidney that form the blood filtration barrier in the kidney glomerulus. The barrier function of podocytes depends upon the development of specialized cell-cell adhesion complexes called slit-diaphragms that form between podocyte foot processes surrounding glomerular blood vessels. Failure of the slit-diaphragm to form results in leakage of high molecular weight proteins into the blood filtrate and urine, a condition called proteinuria. In this work, we test whether the zebrafish pronephros can be used as an assay system for the development of glomerular function with the goal of identifying novel components of the slit-diaphragm. We first characterized the function of the zebrafish homolog of Nephrin, the disease gene associated with the congenital nephritic syndrome of the Finnish type, and Podocin, the gene mutated in autosomal recessive steroid-resistant nephrotic syndrome. Zebrafish nephrin and podocin were specifically expressed in pronephric podocytes and required for the development of pronephric podocyte cell structure. Ultrastructurally, disruption of nephrin or podocin expression resulted in a loss of slit-diaphragms at 72 and 96 h post-fertilization and failure to form normal podocyte foot processes. We also find that expression of the band 4.1/FERM domain gene mosaic eyes in podocytes is required for proper formation of slit-diaphragm cell-cell junctions. A functional assay of glomerular filtration barrier revealed that absence of normal nephrin, podocin or mosaic eyes expression results in loss of glomerular filtration discrimination and aberrant passage of high molecular weight substances into the glomerular filtrate.

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PubMed2006

The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis.

The hox genes play a central role in organogenesis and are implicated in the formation of hematopoietic stem cells (HSCs). The cdx genes encode homeodomain transcription factors that act as master regulators of the hox genes. In zebrafish, mutations in cdx4 cause a severe, but not complete, deficit in embryonic blood cells. Here, we report the expression and function of cdx1a, a zebrafish Cdx1 paralogue. Using morpholino-mediated knockdown of cdx1a in a cdx4 mutant background, we show that a deficiency in both cdx genes causes a severe perturbation of hox gene expression and a complete failure to specify blood. The hematopoietic defect in cdx-deficient embryos does not result from a general block in posterior mesoderm differentiation as endothelial cells and kidney progenitors are still formed in the doubly deficient embryos. In addition, cdx-deficient embryos display a significant reduction in runx1a(+) putative HSCs in the zebrafish equivalent to the aorta-gonad-mesonephros (AGM) region. Overexpressing hoxa9a in cdx-deficient embryos rescues embryonic erythropoiesis in the posterior mesoderm as well as the formation of HSCs in the AGM region. Taken together, these results suggest that the cdx-hox pathway plays an essential role in the formation of both embryonic erythroid cells and definitive HSCs during vertebrate embryogenesis.

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PubMed2006

Zebrafish Staufen1 and Staufen2 are required for the survival and migration of primordial germ cells.

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to the cells of the germ line, the gametes. In many animals, PGCs are set apart from somatic cells early during embryogenesis. Work in Drosophila, C. elegans, Xenopus, and zebrafish has shown that maternally provided localized cytoplasmic determinants specify the germ line in these organisms (Raz, E., 2003. Primordial germ-cell development: the zebrafish perspective. Nat. Rev., Genet. 4, 690--700; Santos, A.C., Lehmann, R., 2004. Germ cell specification and migration in Drosophila and beyond. Curr. Biol. 14, R578-R589). The Drosophila RNA-binding protein, Staufen is required for germ cell formation, and mutations in stau result in a maternal effect grandchild-less phenotype (Schupbach,T., Weischaus, E., 1989. Female sterile mutations on the second chromosome of Drosophila melanogaster:1. Maternal effect mutations. Genetics 121, 101-17). Here we describe the functions of two zebrafish Staufen-related proteins, Stau1 and Stau2. When Stau1 or Stau2 functions are compromised in embryos by injecting antisense morpholino modified oligonucleotides or dominant-negative Stau peptides, germ layer patterning is not affected. However, expression of the PGC marker vasa is not maintained. Furthermore, expression of a green fluorescent protein (GFP):nanos 3'UTR fusion protein in germ cells shows that PGC migration is aberrant, and the mis-migrating PGCs do not survive in Stau-compromised embryos. Stau2 is also required for survival of neurons in the central nervous system (CNS). These phenotypes are rescued by co-injection of Drosophila stau mRNA. Thus, staufen has an evolutionarily conserved function in germ cells. In addition, we have identified a function for Stau proteins in PGC migration.

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PubMed2006

Scube2 mediates Hedgehog signalling in the zebrafish embryo.

The Hedgehog family of secreted morphogens specifies the fate of a large number of different cell types within invertebrate and vertebrate embryos, including the muscle cell precursors of the embryonic myotome of zebrafish. Formation of Hedgehog-sensitive muscle fates is disrupted within homozygous zebrafish mutants of the "you"-type class, the majority of which disrupt components of the Hedgehog (HH) signal transduction pathway. We have undertaken a phenotypic and molecular characterisation of one of these mutants, you, which we show results from mutations within the zebrafish orthologue of the mammalian gene scube2. This gene encodes a member of the Scube family of proteins, which is characterised by several protein motifs including EGF and CUB domains. Epistatic and molecular analyses position Scube2 function upstream of Smoothened (Smoh), the signalling component of the HH receptor complex, suggesting that Scube2 may act during HH signal transduction prior to, or during, receipt of the HH signal at the plasma membrane. In support of this model we show that scube2 has homology to cubilin, which encodes an endocytic receptor involved in protein trafficking suggesting a possible mode of function for Scube2 during HH signal transduction.

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PubMed2006

Zebrafish Trap230/Med12 is required as a coactivator for Sox9-dependent neural crest, cartilage and ear development.

The vertebrate Sox9 transcription factor directs the development of neural crest, otic placodes, cartilage and bone. In zebrafish, there are two Sox9 orthologs, Sox9a and Sox9b, which together perform the functions of the single-copy tetrapod Sox9. In a large-scale genetic screen, we have identified a novel zebrafish mutant that strongly resembles the Sox9a/Sox9b double mutant phenotype. We show that this mutation disrupts the zebrafish Trap230/Med12 ortholog, a member of the Mediator complex. Mediator is a coactivator complex transducing the interaction of DNA-binding transcription factors with RNA polymerase II, and our results reveal a critical function of the Trap230 subunit as a coactivator for Sox9.

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PubMed2006

Conserved co-regulation and promoter sharing of hoxb3a and hoxb4a in zebrafish.

The expression of zebrafish hoxb3a and hoxb4a has been found to be mediated through five transcripts, hoxb3a transcripts I-III and hoxb4a transcripts I-II, driven by four promoters. A "master" promoter, located about 2 kb downstream of hoxb5a, controls transcription of a pre-mRNA comprising exon sequences of both genes. This unique gene structure is proposed to provide a novel mechanism to ensure overlapping, tissue-specific expression of both genes in the posterior hindbrain and spinal cord. Transgenic approaches were used to analyze the functions of zebrafish hoxb3a/hoxb4a promoters and enhancer sequences containing regions of homology that were previously identified by comparative genomics. Two neural enhancers were shown to establish specific anterior expression borders within the hindbrain and mediate expression in defined neuronal populations derived from hindbrain rhombomeres (r) 5 to 8, suggesting a late role of the genes in neuronal cell lineage specification. Species comparison showed that the zebrafish hoxb3a r5 and r6 enhancer corresponded to a sequence within the mouse HoxA cluster controlling activity of Hoxa3 in r5 and r6, whereas a homologous region within the HoxB cluster activated Hoxb3 expression but limited to r5. We conclude that the similarity of hoxb3a/Hoxa3 regulatory mechanisms reflect the shared descent of both genes from a single ancestral paralog group 3 gene.

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PubMed2006

Global analysis of hematopoietic and vascular endothelial gene expression by tissue specific microarray profiling in zebrafish.

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.

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PubMed2006

Discontinuous organization and specification of the lateral floor plate in zebrafish.

The floor plate is a signaling center in the ventral neural tube of vertebrates with important functions during neural patterning and axon guidance. It is composed of a centrally located medial floor plate (MFP) and a bilaterally positioned lateral floor plate (LFP). While the role of the MFP as source of signaling molecules like, e.g., Sonic Hedgehog (Shh) is well understood, the exact organization and function of the LFP are currently unclear. Based on expression analyses, the one cell wide LFP in zebrafish has been postulated to be a homogenous structure. We instead show that the zebrafish trunk LFP is discontinuously arranged. Single LFP cells alternate with p3 neuronal precursor cells, which develop V3 interneurons along the anteroposterior (AP) axis. Our mutant analyses indicate that both, formation of LFP and p3 cells require Delta-Notch signaling. Importantly, however, the two cell types are differentially regulated by Hedgehog (HH) and Nkx2.2 activities. This implicates a novel mechanism of neural tube patterning, in which distinct cell populations within one domain of the ventral neural tube are differently specified along the AP axis. We conclude that different levels of HH and Nkx2.2 activities are responsible for the alternating appearance of LFP and p3 neuronal progenitor cells in the zebrafish ventral neural tube.

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PubMed2006

Fibin, a novel secreted lateral plate mesoderm signal, is essential for pectoral fin bud initiation in zebrafish.

We identified a novel secreted protein, fibin, in zebrafish, mice and humans. We inhibited its function in zebrafish embryos by injecting antisense fibin morpholino oligonucleotides. A knockdown of fibin function in zebrafish resulted in no pectoral fin bud initiation and abolished the expression of tbx5, which is involved in the specification of pectoral fin identification. The lack of pectoral fins in fibin-knockdown embryos was partially rescued by injection of fibin RNA. fibin was expressed in the lateral plate mesoderm of the presumptive pectoral fin bud regions. Its expression region was adjacent to that of tbx5. fibin expression temporally preceded tbx5 expression in presumptive pectoral fin bud regions, and not abolished in tbx5-knockdown presumptive fin bud regions. In contrast, fibin expression was abolished in retinoic acid signaling-inhibited or wnt2b-knockdown presumptive fin bud regions. These results indicate that fibin is a secreted signal essential for pectoral fin bud initiation in that it potentially acts downstream of retinoic acid and wnt signaling and is essential for tbx5 expression. The present findings have revealed a novel secreted lateral plate mesoderm signal essential for fin initiation in the lateral plate mesoderm.

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PubMed2006

Loss of selenoprotein N function causes disruption of muscle architecture in the zebrafish embryo.

Mutations in the gene coding for selenoprotein N (SelN), a selenium containing protein of unknown function, cause different forms of congenital muscular dystrophy in humans. These muscular diseases are characterized by early onset of hypotonia which predominantly affect in axial muscles. We used zebrafish as a model system to understand the function of SelN in muscle formation during embryogenesis. Zebrafish SelN is highly homologous to its human counterpart and amino acids corresponding to the mutated positions in human muscle diseases are conserved in the zebrafish protein. The sepn1 gene is highly expressed in the somites and notochord during early development. Inhibition of the sepn1 gene by injection of antisense morpholinos does not alter the fate of the muscular tissue, but causes muscle architecture disorganization and greatly reduced motility. Ultrastructural analysis of the myotomes reveals defects in muscle sarcomeric organization and in myofibers attachment, as well as altered myoseptum integrity. These studies demonstrate the important role of SelN for muscle organization during early development. Moreover, alteration of myofibrils architecture and tendon-like structure in embryo deficient for SelN function provide new insights into the pathological mechanism of SelN-related myopathy.

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PubMed2006

The effects of the members of growth hormone family knockdown in zebrafish development.

Growth hormone (GH), prolactin (PRL), and somatolactin (SL) are members of GH/PRL superfamily. These hormones are involved in the regulation of an array of physiological processes, including growth, lactation, and osmoregulation. While recent evidence has shown the GH, PRL, and SL gene transcripts and protein products are expressed during early zebrafish development, their functions at this time of embryogenesis remain unknown. In the current study, antisense morpholino oligonucleotide (MO) inhibition of gh, prl, and sl gene translation was used to examine the effects of gene knockdown on hormone function in zebrafish development. We observed that PRL, SLalpha and SLbeta MO treatment all affected development. PRL MO-treated embryos showed defects in gas bladder inflation, reduced head and eye size, shorter body length and fewer melanophores than untreated controls, whereas SLalpha and SLbeta MO-treated embryos were only defective in gas bladder inflation, GH MO-inhibition of GH specific translation did not lead to any discernable morphological changes within 10 days post fertilization (dpf). The effects of PRL knockdown were further verified using a second PRL morpholino antisense and by a rescue experiment with in vitro transcribed prl mRNA containing 5 nucleotide mismatch within the PRL-MO binding region. These results provide the first evidence that members of the GH/PRL superfamily play a role in proper development of various structures including the head, eyes, melanophores and the gas bladder in zebrafish.

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PubMed2006

Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.

The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental time points and in specific regions of the organism. The focal application of current allows macromolecules to be efficiently introduced into a targeted region at any time in the life cycle. Here we describe a rapid protocol by which DNA, RNA and morpholinos can all be precisely electroporated into zebrafish in a temporally and spatially controlled manner. This versatile technique allows gene function to be determined by both gain and loss of function analyses in specific regions at specific times. This is the first report that describes the electroporation of three different molecules into embryonic and larval zebrafish cells.

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PubMed2005

The repertoire of trace amine G-protein-coupled receptors: large expansion in zebrafish.

Trace amines, such as tyramine, beta-phenylethylamine, tryptamine, and octopamine, are present in trace levels in nervous systems and bind a specific family of G-protein-coupled receptors (GPCR), but the function or origin of this system is not well understood. We searched the genomes of several eukaryotic species for receptors similar to the mammalian trace amine (TA) receptor subfamily. We identified 18 new receptors in rodents that are orthologous to the previously known TA-receptors. Remarkably, we found 57 receptors (and 40 pseudogenes) of this type in the zebrafish (Danio rerio), while fugu (Takifugu rubripes) had only eight receptors (and seven pseudogenes). We mapped 47 of the zebrafish TA-receptors on chromosomes using radiation hybrid panels and meiotic mapping. The results, together with the degree of conservation and phylogenetic relationships displayed among the zebrafish receptors suggest that the family arose through several different mechanisms involving tetraploidization, block duplications, and local duplication events. Interestingly, these vertebrate TA-receptors do not show a close evolutionary relationship to the invertebrate TA-binding receptors in fruitfly (Drosophila melanogaster), indicating that the ability to bind TA have evolved at least twice in animal evolution. We collected in total over 100 vertebrate TA-receptor sequences, and our phylogenetic analysis shows that several TA-receptors have evolved rapidly with remarkable species variation and that the common ancestor of vertebrate TA-receptors arose before the split of the ray-finned and lobe-finned fishes. The evolutionary history of the TA-receptors is more complex than for most other GPCR families and here we suggest a mechanism by which they may have arisen.

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