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PubMed2005

Developmental expression of sema3G, a novel zebrafish semaphorin.

The semaphorins are a large, evolutionarily conserved family of signaling molecules with broad functions during development. The class 3 semaphorins are a subclass of secreted semaphorins found in vertebrates. There have been six class 3 semaphorins identified to date (sema3A to sema3F) and some have been shown to function in axon guidance and cardiovascular development. However, the functions of many class 3 semaphorins and their potential interactions in vivo are still not well understood. As a step toward understanding the actions of all class 3 semaphorins in vivo, we have cloned and analyzed the developmental expression pattern of a novel zebrafish class 3 semaphorin, sema3H [corrected] sema3H [corrected] is expressed in a dynamic pattern throughout the first 3 days of development. It is expressed in the adaxial cells of the somite during somitogenesis. In the brain, sema3H [corrected] is expressed in cell clusters in the midbrain and diencephalon, and is expressed in the telencephalon in close proximity to the olfactory epithelium. sema3H [corrected] also is expressed in the pharyngeal arches, the pectoral fin bud, and the developing pronephros. These results provide a basis for studying how expression of multiple semaphorins could be essential for aspects of early development.

Animals↗
PubMed2005

Amino acid cotransporter SLC3A2 is selectively expressed in the early proximal segment of Xenopus pronephric kidney nephrons.

The transport of amino acids across membranes is critical to all cells. As amino acids freely pass through the glomerular filtration barrier of the kidney, they must be efficiently resorbed to avoid depletion of circulating amino acid reserves. Not only do defects in amino acid resorption lead to costly wastage, they also cause congenital aminoacidurias. A clone encoding Xenopus SLC3A2 was identified and shown to be expressed at high levels in the early segment of the pronephric proximal tubules in developing tadpoles. The type II membrane glycoprotein encoded by this gene can associate with a wide variety of protein partners and participates in a broad spectrum of biological processes. In this report, the first whole-mount analysis of SLC3A2 during early embryonic development is presented. The expression pattern of SLC3A2 in the early proximal segment of the Xenopus pronephros is analogous to that of a previously described SLC7A8/XAA2 amino acid transporter. In mammals, SLC3A2 and SLC7A8/XAA2 associate to form a functional neutral amino acid transporter complex and coexpression of these two genes in a small domain within the pronephric tubules indicates that this is also the situation in the developing Xenopus kidney.

Amino Acid Sequence↗
PubMed2006

Developmental expression of FoxJ1.2, FoxJ2, and FoxQ1 in Xenopus tropicalis.

Members of the Fox gene family exhibit remarkably restricted patterns of expression where they have interesting, required functions during development. We have analyzed the developmental expression patterns of three members of the Fox gene family, FoxJ1.2, FoxJ2, and FoxQ1, which have not been previously described in Xenopus. FoxJ1.2 is expressed in the otic vesicle during late neurula stages and is then also expressed in the presumptive nephrostomes of the pronephros during tailbud stages. FoxJ2 is expressed in the notochord and ventral portion of the neural tube. FoxQ1 is expressed specifically in the pharyngeal pouches as early as neurula stages and remains on in pharyngeal tissue throughout the tailbud stages. At later stages, FoxQ1 is also expressed in the anterior gut. FoxJ1.2, FoxJ2, and FoxQ1 may prove to be useful tissue-specific markers of these embryonic structures.

Animals↗
PubMed2006

Duplicate sfrp1 genes in zebrafish: sfrp1a is dynamically expressed in the developing central nervous system, gut and lateral line.

The secreted frizzled-related proteins (Sfrp) are a family of soluble proteins with diverse biological functions having the capacity to bind Wnt ligands, to modulate Wnt signalling, and to signal directly via the Wnt receptor, Frizzled. In an enhancer trap screen for embryonic expression in zebrafish we identified an sfrp1 gene. Previous studies suggest an important role for sfrp1 in eye development, however, no data have been reported using the zebrafish model. In this paper, we describe duplicate sfrp1 genes in zebrafish and present a detailed analysis of the expression profile of both genes. Whole mount in situ hybridisation analyses of sfrp1a during embryonic and larval development revealed a dynamic expression profile, including: the central nervous system, where sfrp1a was regionally expressed throughout the brain and developing eye; the posterior gut, from the time of endodermal cell condensation; the lateral line, where sfrp1a was expressed in the migrating primordia and interneuromast cells that give rise to the sensory organs. Other sites included the blastoderm, segmenting mesoderm, olfactory placode, developing ear, pronephros and fin-bud. We have also analysed sfrp1b expression during embryonic development. Surprisingly this gene exhibited a divergent expression profile being limited to the yolk syncytium under the elongating tail-bud, which later covered the distal yolk extension, and transiently in the tail-bud mesenchyme. Overall, our studies provide a basis for future analyses of these developmentally important factors using the zebrafish model.

Amino Acid Sequence↗
PubMed2006

GDNF expression during Xenopus development.

Glial cell line-derived neurotrophic factor (GDNF) has multiple roles in kidney morphogenesis, spermatogenesis, and neurogenesis during development. In this study, we report the cloning and expression pattern of Xenopus laevis GDNF. The X. laevis GDNF cDNA sequence has a complete open reading frame of 684 bases, predicting 227 amino acid residues at the protein level. Comparison of the X. laevis GDNF amino acid sequence with those of chick, human, mouse, rat and zebrafish indicates that X. laevis GDNF has 60%-52% and 75%-62% identity over the whole amino acid sequence and for the putative mature forms, respectively. All known functional motifs of GDNF were conserved in the X. laevis sequence. Temporal expression analysis by RT-PCR indicated that GDNF transcripts were first detectable at stage 12 at a low level, and gradually increased up to stage 22. From stage 24, the expression sharply increased and continued at a similar level as development progressed. Spatial expression analysis by whole-mount in situ hybridization showed that the GDNF mRNA was predominantly detected in somites, pronephros, pharyngeal arches, epibranchial placodes, digestive tract and some of the lateral line structure. These results suggest that this X. laevis gene is the orthologue for GDNF.

Amino Acid Sequence↗
PubMed2005

Molecular cloning of the BCL-6 gene, a transcriptional repressor for B-cell differentiation, in torafugu (Takifugu rubripes).

B-cell lymphoma-6 (BCL-6) is a transcriptional repressor that prevents the terminal differentiation of mature B-cells to plasma cells, and is essential for germinal center formation in the primary lymphoid organs of mammals. In this study, we identified the BCL-6 gene in torafugu (Takifugu rubripes) using the torafugu genome database, and analyzed the expression of BCL-6 mRNA in various tissues of torafugu, using RT-PCR. The BCL-6 gene consisted of eight exons and seven introns spanning a genome of ca. 3.3 kb. BCL-6 mRNA contained a 2112 bp open reading frame encoding 703 amino acids, with a predicted protein size of 78.8 kDa. The predicted torafugu BCL-6 primary structure contains two conserved specific motifs, the BTB/POZ domain at the N-terminus and the sixC2H2-type zinc finger motifs at the C-terminal region. The homology of torafugu BCL-6 to those of zebrafish (Danio rerio), Xenopus laevis, mouse (Mus musculus) and human (Homo sapiens) is 76, 59, 60 and 60%, respectively. RT-PCR analysis revealed that BCL-6 mRNA is highly expressed in pronephros, thymus, intestine, ovary, brain, nasal cavity and muscle. These results imply that torafugu BCL-6 is involved in regulation of B-cell differentiation in torafugu.

Amino Acid Motifs↗
PubMed2003

Cloning and characterization of Xenopus Id4 reveals differing roles for Id genes.

We have identified Xenopus Id4, a member of the Id (inhibitor of differentiation/DNA binding) class of helix-loop-helix proteins. Id factors dimerize with general bHLH factors, preventing their interaction with tissue-specific bHLH factors, to inhibit premature differentiation. The presence of several Id proteins could reflect simple redundancy in function, or more interestingly, might suggest different activities for these proteins. During embryonic development, Xenopus Id4 is expressed in a number of neural tissues, including Rohon-Beard neurons, olfactory placode, eye primordia, and the trigeminal ganglia. It is also expressed in other organs, such as the pronephros and liver primordium. As embryogenesis progresses, it is expressed in the migrating melanocytes and lateral line structures. We compare the expression of Id4 mRNA with that of Id2 and Id3 and find that the Id genes are expressed in complementary patterns during neurogenesis, myogenesis, kidney development, in the tailbud, and in the migrating neural crest. To examine the regulation of Id gene expression during Xenopus neural development, we show that expression of Id3 and Id4 can be induced by overexpression of BMP4 in the whole embryo and in ectodermal explants. Expression of Id2, Id3, and Id4 in these explants is unaffected by the expression of FGF-8 or a dominant-negative Ras (N17ras), suggesting that Id genes are not regulated by the FGF signaling pathway in naive ectoderm. We also show that Notch signaling can activate Id2 and Id3 expression in the whole embryo. In contrast, Id4 expression in the Rohon-Beard cells is inhibited by activated Notch and increased by a dominant-negative Delta. This may reflect an increase in Rohon-Beard cells in response to inhibition of Notch signaling rather than transcriptional regulation of Id4. Finally, to compare the activities of Id2, Id3, and Id4, we use animal cap explants and in vivo overexpression to show that Id proteins can differentially inhibit the activities of neurogenin and neuroD, both neurogenic bHLH molecules and MyoD, a myogenic bHLH protein. Id4 is able to inhibit the activity all these bHLH molecules, Id2 inhibits MyoD and neuroD, while Id3 blocks only neuroD activity in our assays.

Amino Acid Sequence↗
PubMed2004

Expression of Pax2 in the intermediate mesoderm is regulated by YY1.

The transcription factor Pax2 is essential for the development of the urogenital system. Pax2 expression can be detected by mouse embryonic day 8.5 (E8.5) in the region of intermediate mesoderm fated to become the nephric duct, pronephros, and mesonephros. Elements that direct Pax2 expression to nephrogenic precursor cells must be responding to positional information that controls nephrogenic fate. A 4.1-kb Pax2 promoter/enhancer fragment directs expression of a lacZ reporter to the nephrogenic region and the midbrain-hindbrain junction in transgenic mice. As kidney development proceeds, transgene expression is limited to the nephric duct and its derivatives, but not the metanephric mesenchyme. The early expression driven by the 4.1 promoter does not require the Pax2 protein, demonstrating that it receives positional information in the absence of a developing pro- or mesonephros. We have identified two DNAseI hypersensitive regions within this promoter, one at the start site of transcription initiation and a second approximately 2-kb upstream. Deletion of the more distal site significantly attenuates lacZ expression in the nephrogenic region but not in the midbrain-hindbrain region. DNA footprinting of this fragment revealed a highly conserved sequence between mouse and human Pax2 promoter sequences. Using fractionated nuclear extracts, we identified the transcription factor Yin Yang 1 (YY1) as the protein that binds to this conserved sequence. Deletion of the YY1 element significantly attenuated expression of a full-length 4.1 promoter. Moreover, inclusion of this YY1 binding element significantly enhanced expression of a minimal Pax2 promoter/enhancer transgene in E12 embryos. These data point to a novel role for YY1 in the establishment of high level tissue-specific expression within the intermediate mesoderm.

Animals↗
PubMed2004

Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules.

The embryonic kidneys of larval aquatic vertebrates such as fish and frogs serve as excellent model systems for exploring the early development of nephric organs. These experimental systems can easily be manipulated by microsurgery, microinjection, genetics, or combinations of these approaches. However, little is known about how physiologically similar these simple kidneys are to the more complex mammalian adult kidneys. In addition, almost nothing is known about proximo-distal patterning of nephrons in any organism. In order begin to explore the physiological specialization of the pronephric tubules along the proximo-distal axis, a combination of uptake assays using fluorescently tagged proteins, LDL particles and dextrans, and an informatics-targeted in situ screen for transport proteins have been performed on embryos of the frog, Xenopus laevis. Genes identified to be expressed within unique subdomains of the pronephric tubules include an ABC transporter, two amino acid cotransporters, two sodium bicarbonate cotransporters, a novel sodium glucose cotransporter, a sodium potassium chloride cotransporter (NKCC2), a sodium chloride organic solute cotransporter (ROSIT), and a zinc transporter. A novel combination of colorimetric and fluorescent whole-mount in situ hybridization (FCIS) was used to precisely map the expression domain of each gene within the pronephros. These data indicate specialized physiological function and define multiple novel segments of the pronephric tubules, which contain at least six distinct transport domains. Uptake studies identified functional transport domains and also demonstrated that early glomeral leakage can allow visualization of protein movement into the pronephric tubules and thus establish a system for investigating experimentally induced proteinuria and glomerulonephritis.

Animals↗
PubMed2005

bloodthirsty, an RBCC/TRIM gene required for erythropoiesis in zebrafish.

The Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) constitute the only vertebrate taxon that fails to produce red blood cells. These fishes can be paired with closely related, but erythrocyte-producing, notothenioids to discover erythropoietic genes via representational difference analysis. Using a B30.2-domain-encoding DNA probe so derived from the hematopoietic kidney (pronephros) of a red-blooded Antarctic rockcod, Notothenia coriiceps, we discovered a related, novel gene, bloodthirsty (bty), that encoded a 547-residue protein that contains sequential RING finger, B Box, coiled-coil, and B30.2 domains. bty mRNA was expressed by the pronephric kidney of N. coriiceps at a steady-state level 10-fold greater than that found in the kidney of the icefish Chaenocephalus aceratus. To test the function of bty, we cloned the orthologous zebrafish gene from a kidney cDNA library. Whole-mount in situ hybridization of zebrafish embryos showed that bty mRNA was present throughout development and, after the mid-blastula transition, was expressed in the head and in or near the site of primitive erythropoiesis in the tail just prior to red cell production. One- to four-cell embryos injected with two distinct antisense morpholino oligonucleotides (MOs) targeted to the 5'-end of the bty mRNA failed to develop red cells, whereas embryos injected with 4- and 5-bp mismatch control MOs produced wild-type quantities of erythrocytes. The morphant phenotype was rescued by co-injection of synthetic bty mRNA containing an artificial 5'-untranslated region (UTR) with the antisense MO that bound the 5'-UTR of the wild-type bty transcript. Furthermore, the expression of genes that mark terminal erythroid differentiation was greatly reduced in the antisense-MO-treated embryos. We conclude that bty is likely to play a role in differentiation of the committed red cell progenitor.

Amino Acid Sequence↗
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.

Amino Acid Sequence↗
PubMed2005

Fishing for prostanoids: deciphering the developmental functions of cyclooxygenase-derived prostaglandins.

Prostaglandin G/H synthases (PGHS), commonly referred to as cyclooxygenases (COX-1 and COX-2), catalyze a key step in the synthesis of biologically active prostaglandins (PGs), the conversion of arachidonic acid (AA) into prostaglandin H(2) (PGH(2)). PGs have important functions in a variety of physiologic and pathologic settings, including inflammation, cardiovascular homeostasis, reproduction, and carcinogenesis. However, an evaluation of prostaglandin function in early development has been difficult due to the maternal contribution of prostaglandins from the uterus. The emergence of zebrafish as a model system has begun to provide some insights into the roles of this signaling cascade during vertebrate development. In zebrafish, COX-1 derived prostaglandins are required for two distinct stages of development, namely during gastrulation and segmentation. During gastrulation, PGE(2) signaling promotes cell motility, without altering the cell shape or directional migration of gastrulating cells. During segmentation, COX-1 signaling is also required for posterior mesoderm development, including the formation of vascular tube structures, angiogenesis of intersomitic vessels, and pronephros morphogenesis. We propose that deciphering the role for prostaglandin signaling in zebrafish development could yield insight and ultimately address the mechanistic details underlying various disease processes that result from perturbation of this pathway.

Animals↗
PubMed2006

FGF is essential for both condensation and mesenchymal-epithelial transition stages of pronephric kidney tubule development.

The pronephros is a transient embryonic kidney that is essential for the survival of aquatic larvae. It is also absolutely critical for adult kidney development, as the pronephric derivative the wolffian duct forms the ductal system of the adult kidney and also triggers the condensation of metanephric mesenchyme into the adult nephrons. While exploring Xenopus pronephric patterning, we observed that epidermally delivered hedgehog completely suppresses pronephric kidney tubule development but does not effect development of the pronephric glomus, the equivalent of the mammalian glomerulus or corpuscle. This effect is not mediated by apoptosis. Microarray analysis of microdissected primordia identified FGF8 as one of the potential mediators of hedgehog action. Further investigation demonstrated that SU5402-sensitive FGF signaling plays a critical role in the very earliest stages of pronephric tubule development. Modulation of FGF8 activity using a morpholino has a later effect that blocks condensation of pronephric mesenchyme into the pronephric tubule. Together, these data show that FGF signaling plays a critical role at two stages of embryonic kidney development, one in the condensation of the pronephric primordium from the intermediate mesoderm and a second in the later epithelialization of this mesenchyme into the pronephric nephron. The data also show that in Xenopus, development of the glomus/glomerulus can be uncoupled from nephron formation via ectopic hedgehog expression and provides an experimental avenue for investigating glomerulogenesis in the complete absence of tubules.

Animals↗
PubMed2006

ADMP2 is essential for primitive blood and heart development in Xenopus.

We describe here the cloning of a new member of the TGF-beta family with similarity to the anti-dorsalizing morphogenetic proteins (ADMPs). This new gene, ADMP2, is expressed in a broad band of mesendoderm cells that appear to include the progenitors of the endoderm and the ventral mesoderm. Antisense morpholino oligonucleotide knockdown of ADMP2 results in near-complete disruption of primitive blood and heart development, while the development of other mesoderm derivatives, including pronephros, muscle and lateral plate is not disrupted. Moreover, the development of the primitive blood in ADMP2 knockdown embryos cannot be rescued by BMP. These results suggests that ADMP2 plays an early role in specifying presumptive ventral mesoderm in the leading edge mesoderm, and that ADMP2 activity may be necessary to respond to BMP signaling in the context of ventral mesoderm induction.

Amino Acid Sequence↗
PubMed2006

Odd-skipped genes encode repressors that control kidney development.

Odd-skipped family of proteins (Odd in Drosophila and Osr in vertebrates) are evolutionarily conserved zinc finger transcription factors. Two Osr genes are present in mammalian genomes, and it was recently reported that Osr1, but not Osr2, is required for murine kidney development. Here, we show that in Xenopus and zebrafish both Osr1 and Osr2 are necessary and sufficient for the development of the pronephros. Osr genes are expressed in early prospective pronephric territories, and morphants for either of the two genes show severely impaired kidney development. Conversely, overexpression of Osr genes promotes formation of ectopic kidney tissue. Molecularly, Osr proteins function as transcriptional repressors during kidney formation. We also show that Drosophila Odd induces kidney tissue in Xenopus. This might be accomplished through recruitment of Groucho-like co-repressors. Odd genes may also be required for proper development of the Malpighian tubules, the Drosophila renal organs. Our results highlight the evolutionary conserved involvement of Odd-skipped transcription factors in the development of kidneys.

Animals↗
PubMed2005

ACTH response to LPS in the first stages of development of the fish Dicentrarchus labrax L.

ACTH and ACTH receptor-like molecules were found at the examined stages of development (2, 4, 8, 12, 18, and 24 days post-hatching) in yolk sac, pronephros tubules, interrenal tissue, thymus, liver, spleen, cardinal veins, and skin of the teleost fish Dicentrarchus labrax. ACTH and the related receptor-like molecules show a similar distribution. LPS treatment at two different stages (8 and 24 days post-hatching) provoked both a release and an induction of ACTH-like molecules, suggesting an important role of this peptide to control the modifications in body homeostasis during the first period of the sea bass' life, i.e., 30 days post-hatching, before the lymphoid cells have reached complete maturation.

Adaptation, Physiological↗
PubMed1995

Pronephric carcinoma: chromosomes of cells rescued from apoptosis by an oncogenic herpesvirus detected with a polymerase chain reaction.

The amphibian pronephros is fated to die during early development. Pronephric cells undergo apoptosis and their function is replaced by the mesonephros, which becomes the functional kidney of the adult frog. Tadpoles of the northern leopard frog, Rana pipiens, were inoculated with a Lucké tumour herpesvirus (LTV) preparation. Most of the animals developed typical Lucké renal carcinomas at metamorphosis. Fewer developed carcinomas of the pronephric cell type. A pronephric carcinoma, rescued from apoptosis by the herpesvirus, was harvested from a post-metamorphic frog. The tumour was judged to be pronephric by its anatomical location (in the anterior part of the body) and because both mesonephric kidneys were intact and tumour-free upon removal of the tumour mass. A tumour fragment was fixed for histological examination, which confirmed that the tissue was a renal carcinoma. A further fragment was subjected to short-term culture in order to produce metaphase cells for cytogenetical analysis. Based upon silverstained nucleolar organizing region numbers, 14 of 15 metaphase cells were estimated to have the diploid number (2N = 26) of chromosomes and a karyotype was constructed which did not appear to differ from that of normal cells. A single cell was estimated to be tetraploid (4N = 52). This is the first report of chromosomes of a pronephric Lucké carcinoma. LTV replicates only in tumour tissue maintained in the cold. Because the frog in this study had been maintained in the laboratory at 22 degrees C for about 10 months, no viruses would have been detectable with electron microscopy. However, the presence of Lucké herpesvirus DNA was detected in tumour homogenates by polymerase chain reaction amplification of a 1.2 kbp Hind III restriction fragment of the LTV DNA. The presence of LTV DNA provided assurance that the rescued pronephric tumour was indeed a Lucké carcinoma.

Animals↗
PubMed2001

Cloning and developmental expression of Xenopus Stat1.

Proteins of the STAT family (Signal Transducers and Activators of Transcription) are latent cytoplasmic factors which, upon phosphorylation, are translocated to the nucleus where they participate in gene activation. In this report, we describe the cloning and developmental expression of a Xenopus homolog of Stat1. XStat1 is highly conserved, exhibiting greater than 90% identity in the critical DNA binding, SH2, SH3 and trans-activation domains with both human and murine Stat1. Using RT-PCR, we show that XStat1 is present as a maternal message during early development of Xenopus. The maternal message is translated during cleavage and its product is phosphorylated on tyrosine, a prerequisite for functional activation. During cleavage and gastrula stages, XStat1 is widely expressed throughout the developing embryo. During neurulation and early tailbud stages, XStat1 is expressed in both dorsal axial and ventral tissues. By late tailbud, dorsal XStat1 expression domains are associated with the developing pharyngeal arches and pronephros. These regions of the embryo correspond to the future location of the thymus, sites of dorsal hematopoietic activity, and one location where melanocytes differentiate.

Amino Acid Sequence↗