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PubMed2003

Tcf-1 expression during Xenopus development.

We report the cloning and expression of Xenopus Tcf-1. The amino acid sequence of Tcf-1 of Xenopus laevis and Xenopus tropicalis is closely related to that of chicken, mouse and man. Thus, the family of Tcf/Lef proteins in the amphibian Xenopus comprises four members as in higher vertebrates. RT-PCR analysis revealed that Tcf-1 RNA encoding a beta-catenin binding isoform is maternally present as well as throughout early development. Different transcripts are expressed by alternative splicing. In cleavage and blastula stage embryos, Tcf-1 RNA is present at high levels in the animal hemisphere. During gastrulation Tcf-1 is differentially expressed with high levels in the animal cap and most of the marginal zone except for a narrow domain around the blastopore. At neurula stages expression is predominant in the neural plate. At tailbud stages expression is localized in specific areas of the brain, in the eyes, the otic vesicle, branchial arches and head mesenchyme, somites, tailbud, pronephros and pronephric duct.

Amino Acid Sequence↗
PubMed2003

Inflammatory interactions in fish exposed to pollutants and parasites: a role for apoptosis and C reactive protein.

Although previous studies have highlighted the inflammatory responses of fish infected with parasites and exposed to pollutants, very little is known about how these two stressors interact within the fish. In this review, which also contains original data, the effect of these two parameters on the fish inflammatory response is assessed and, in particular, the role of apoptosis and the acute phase protein, C reactive protein, is evaluated. In Cyprinus carpio exposed to 0.5 mg NH4+ l(-1) or 0.1 mg Cd2+ l(-1) and experimentally infected with the blood fluke, Sanguinicola inermis, the pollutant type and the order in which the fish experiences the parasite and toxicant, significantly affects the ultrastructural appearance and cellular content of the pronephros and thymus. This is reflected in the intensity of infection where the pollutant appears to have less effect on an established infection. Both stressors, pollutant and infection, may mediate their effects via the endocrine system. Studies have revealed that cortisol at 100 ng ml(-1) is able to induce apoptosis in pronephric cells of carp and that an increase in apoptosis is associated with an increase in phagocytosis in this immune organ. In addition, C reactive protein, which is used as a biomarker of the inflammatory response in humans and other mammals, is evaluated as a possible indicator of physiological states in fish exposed to pathogens and pollutants.

Ammonia↗
PubMed2000

Growth of rainbow trout hemopoietic cells in methylcellulose and methods of monitoring their proliferative response in this matrix.

A technique for the clonal culture of rainbow trout leukocytes in a methylcellulose matrix can be used to identify growth factors and other substances affecting cell proliferation and development in fish. Methylcellulose supports colony formation by rainbow trout leukocytes isolated from the major hemopoietic organ, the pronephros. The addition of rainbow trout serum dramatically increased the number of colonies formed, scored by counting colonies. As an alternative measure of cell proliferation, 3H-thymidine incorporation by cells can be easily measured in methylcellulose cultures. This method requires only small amounts of test substances, is rapid, and is superior for assessing the growth-stimulating ability of some substances, such as bacterial lipopolysaccharide, which stimulated growth but not the formation of discreet colonies by rainbow trout cells.

Animals↗
PubMed1993

Secreted noggin protein mimics the Spemann organizer in dorsalizing Xenopus mesoderm.

A dorsalizing signal acts during gastrulation to change the specification of lateral mesodermal tissues from ventral (blood, mesenchyme) to more dorsal fates (muscle, heart, pronephros). This signal, from Spemann's organizer, cannot be mimicked by the mesoderm inducers activin and fibroblast growth factor. The gene noggin is expressed in the organizer, and could be the dorsalizing signal. Here we show that soluble noggin protein added to ventral marginal zones during gastrulation induces muscle, but that activin does not. Dorsal pattern can be partially rescued in ventralized embryos by injection of a plasmid that expresses noggin during gastrulation. The results suggest that the noggin product may be the dorsalizing signal from the organizer.

Activins↗
PubMed2006

Syndecan-4 regulates non-canonical Wnt signalling and is essential for convergent and extension movements in Xenopus embryos.

Early shaping of Xenopus laevis embryos occurs through convergent and extension movements, a process that is driven by intercalation of polarized dorsal mesodermal cells and regulated by non-canonical Wnt signalling. Here, we have identified Xenopus syndecan-4 (xSyn4), a cell-surface transmembrane heparan sulphate proteoglycan. At the gastrula stage, xSyn4 is expressed in the involuting dorsal mesoderm and the anterior neuroectoderm. Later, it is found in the pronephros, branchial arches, brain and tailbud. Both gain- and loss-of-function of xSyn4 impaired convergent extension movements in Xenopus embryos and in activin-treated ectodermal explants. xSyn4 interacts functionally and biochemically with the Wnt receptor Frizzled7 (xFz7) and its signal transducer Dishevelled (xDsh). Furthermore, xSyn4 is necessary and sufficient for translocation of xDsh to the plasma membrane - a landmark in the activation of non-canonical Wnt signalling. Our results suggest that the ability of xSyn4 to translocate xDsh is regulated by fibronectin, a component of the extracellular matrix required for proper convergent extension movements. We propose a model where xSyn4 and fibronectin cooperate with xFz7 and Wnt in the specific activation of the non-canonical Wnt pathway.

Adaptor Proteins, Signal Transducing↗
PubMed1997

The zebrafish homologue of the ret receptor and its pattern of expression during embryogenesis.

The c-ret proto-oncogene, a member of the receptor tyrosine kinase gene superfamily, plays a critical role in the development of the excretory system and the enteric and autonomic nervous systems of mammalian embryos. To study the potential function of the c-ret locus in lower vertebrates, we have isolated its zebrafish homologue, ret1 and established its expression pattern during embryogenesis. Ret1 mRNA first appears during early somitogenesis in the presumptive brain, spinal cord and excretory system. Within the CNS, expression of ret1 is detected in primary motor and sensory (Rohon-Beard) neurons. Ret1 transcripts are also expressed in subsets of neural crest cells and cranial ganglia as well as in the enteric nervous system. In the excretory system, expression is detected in the developing nephric duct and the pronephros. Our findings reveal a remarkable similarity in the expression pattern of c-ret between higher and lower vertebrates, suggesting that the function of this locus has been conserved throughout vertebrate evolution. Furthermore, the conservation of ret1 expression in cell types which remain unaffected by the mammalian c-ret mutations, such as motor and sensory neurons, suggests a function of this receptor in these cell lineages.

Amino Acid Sequence↗
PubMed1997

A novel Xenopus homologue of bone morphogenetic protein-7 (BMP-7).

We identified a Xenopus gene closely related to mammalian bone morphogenetic protein (BMP)-7 (also termed osteogenic protein-1 or OP-1). It resembles the mammalian gene in primary structure and expression pattern much more closely than does a previously described Xenopus homologue, originally termed XBMP-7 [Nishimatsu, Suzuki, Shoda, Murakami and Ueno (1992) Biochem. Biophys. Res. Commun. 186, 1487-1495]. The novel gene has therefore been designated XBMP-7 and the gene described earlier has been renamed XBMP-7R (M. Moos and N. Ueno, unpublished work). It has a broad distribution, primarily in the anterior and posterior ventral regions during gastrulation, subsequently becoming prominent at different stages in a wide variety of structures (eyes, neural structures, heart, pronephros, posterior ventral region and other structures), paralleling the distribution of XBMP-4 closely. However, its expression begins later than that of XBMP-4 during gastrulation. Lithium treatment of embryos concentrates the XBMP-7 expression in the expanded eye and heart structures. Ventral overexpression of XBMP-7 produces large protrusions that ultimately develop colouration characteristic of haemoglobin, which is confirmed by markedly expanded expression of alpha-globin. Dorsal overexpression suppresses dorsal anterior structures. Molecular analysis of animal caps overexpressing XBMP-7 reveals induction of markers associated with ventral and haematopoietic tissue, which is consistent with whole-embryo overexpression results. Globin induction by XBMP-7 can be blocked by a truncated BMP receptor previously shown to interrupt BMP-4 signalling, indicating XBMP-7 also interacts with this receptor. Our data support the concept that XBMP-7 may play a variety of roles during embryogenesis, and suggest a possible role in haematogenesis.

Amino Acid Sequence↗
PubMed1994

Comparative analysis of the tissue distribution of three fibroblast growth factor receptor mRNAs during amphibian morphogenesis.

We have used in situ hybridization to survey the expression pattern of three fibroblast growth factor receptor (FGFR) mRNAs (PFR-1, PFR-3 and PFR-4, which we previously identified as the amphibian Pleurodeles waltl homologs of human FGFR-1, FGFR-3 and FGFR-4, respectively) during morphogenesis. Previous work suggests that these FGFR mRNAs exhibit a distinct pattern of expression at early developmental stages. In the present study we have tested the functional activity of these receptors and shown that both FGF-1 (acidic FGF) and FGF-2 (basic FGF), but not FGF-7 (keratinocyte growth factor), can lead to their activation, suggesting that the three cDNAs encode functional receptors. Results from in situ hybridization indicate that various FGFRs are involved in various developmental events. Their involvement in these processes is both overlapping and distinct. During the differentiation of the central nervous system (CNS), PFR-1 and PFR-4 mRNAs show high levels of redundant expression, while the sites of expression of PFR-3 mRNA correlate with regions, such as the diencephalon and the rhombencephalon, undergoing important anatomic changes. The three FGFR mRNAs are distinctly expressed in the cranial ganglia, the pigmented epithelia of retina and the otic vesicles. Most significantly, we found that they are strongly expressed at cranial and branchial mesenchymal condensation sites. PFR-3 mRNA is expressed earlier in this process than PFR-1 and PFR-4 mRNAs. Furthermore PFR-3 mRNA is detected in the mesenchyme of the limb bud, while PFR-1 and PFR-4 mRNAs are found in the primordia of the skeletal elements. In addition, PFR-1 mRNA is expressed in axial mesenchyme and PFR-4 mRNA is detected in the melanophores, xanthophores and in the pronephros. These results suggest that various FGFRs may be involved in distinct developmental events including cell proliferation and differentiation. We also discuss the functional redundancy of the FGFR system during amphibian morphogenesis.

Animals↗
PubMed1997

The Na+,K+-ATPase alpha subunit requires gastrulation in the Xenopus embryo.

Na+,K+-ATPase participates in reabsorption of ions and water and produces an electrochemical gradient between the intra- and extracellular spaces across the cell membrane. It also plays an important role in many developmental phenomena such as a blastocoele formation and neural formation. To elucidate the expression pattern of Na+,K+-ATPase in the Xenopus embryo, the spatial expression patterns of the Na+,K+-ATPase alpha subunit were studied in a normal embryo by whole-mount in situ hybridization. These transcripts were localized around the dorsal blastopore at the gastrula stage, in the neural tube at the neurula stage, and then in the pronephros and cloaca at the tail-bud stage. To study the function of Na+,K+-ATPase in embryogenesis after mid-blastula transition, the expression of the Na+,K+-ATPase alpha subunit was inhibited by the injection of specific antisense RNA. Embryos injected with Na+,K+-ATPase antisense RNA showed inhibition of gastrulation. When antisense RNA was injected into the dorsal blastomeres, head differentiation was markedly inhibited. These results suggest that this transcript plays an important role during gastrulation and head differentiation.

Activins↗
PubMedDate not supplied

Influence of HMB (beta-hydroxy-beta-methylbutyrate) on antibody secreting cells (ASC) after in vitro and in vivo immunization with the anti-Yersinia ruckeri vaccine of rainbow trout (Oncorhynchus mykiss).

In practice, protection of fish against disease by immunization is of limited effectiveness. Therefore, research is concentrated on how to improve the potency and efficacy of vaccines and how to optimally activate the cell-mediated immunity and the specific antibody response. In the present study, the influence of HMB (beta-Hydroxy-beta-methylbutyrate) on the antibody secreting cells (ASC) after both in vitro and in vivo immunization of rainbow trout (Oncorhynchus mykiss) with the anti-yersiniosis vaccine was studied. For in vitro immunization, the spleens from 160 fish were sampled and placed each in 35 mm sterile wells with medium containing HMB at concentrations of 0, 0.1, 1, 5, 10, 25, 50 or 100 microg/mL of medium. The spleens from 80 fish were injected with the vaccine and incubated at 14 degrees C for 10 days. For the in vivo study, fish were fed pellets containing HMB at doses of 0, 10, 25 and 50 mg/kg bw per day. After 2 weeks of HMB supplementation, the fish were immunized by intraperitoneal injection of the vaccine. At 7, 14, 18, 21, 28 and 35 days after immunization, pronephros were taken from 10 fish in each group for testing. When analyzed by the ELISPOT assay, HMB increased the number of splenic ASC after in vitro immunization at concentrations between 10 and 100 microg/mL (P < 0.05). Dietary HMB also increased the number of total and specific ASC when the fish were vaccinated in vivo. In conclusion, the results of the present study showed that HMB increases the levels of specific ASC after both in vitro and in vivo immunization of rainbow trout with the anti-Yersinia ruckeri vaccine.

Animals↗
PubMed2005

Discovery of a unique Ig heavy-chain isotype (IgT) in rainbow trout: Implications for a distinctive B cell developmental pathway in teleost fish.

During the analysis of Ig superfamily members within the available rainbow trout (Oncorhynchus mykiss) EST gene index, we identified a unique Ig heavy-chain (IgH) isotype. cDNAs encoding this isotype are composed of a typical IgH leader sequence and a VDJ rearranged segment followed by four Ig superfamily C-1 domains represented as either membrane-bound or secretory versions. Because teleost fish were previously thought to encode and express only two IgH isotypes (IgM and IgD) for their humoral immune repertoire, we isolated all three cDNA isotypes from a single homozygous trout (OSU-142) to confirm that all three are indeed independent isotypes. Bioinformatic and phylogenetic analysis indicates that this previously undescribed divergent isotype is restricted to bony fish, thus we have named this isotype "IgT" (tau) for teleost fish. Genomic sequence analysis of an OSU-142 bacterial artificial chromosome (BAC) clone positive for all three IgH isotypes revealed that IgT utilizes the standard rainbow trout V(H) families, but surprisingly, the IgT isotype possesses its own exclusive set of D(H) and J(H) elements for the generation of diversity. The IgT D and J segments and tau constant (C) region genes are located upstream of the D and J elements for IgM, representing a genomic IgH architecture that has not been observed in any other vertebrate class. All three isotypes are primarily expressed in the spleen and pronephros (bone marrow equivalent), and ontogenically, expression of IgT is present 4 d before hatching in developing embryos.

Amino Acid Sequence↗
PubMed2001

Expression patterns of HNK-1 carbohydrate and serotonin in sea urchin, amphioxus, and lamprey, with reference to the possible evolutionary origin of the neural crest.

We examined deuterostome invertebrates, the sea urchin and amphioxus, and an extant primitive vertebrate, the lamprey, for the presence of structures expressing the HNK-1 carbohydrate and serotonin. In sea urchin embryos and larvae, HNK-1 positive cells were localized in the ciliary bands and in their precursor ectoderm. Serotonergic cells were exclusively observed in the apical organs. In juvenile amphioxus, primary sensory neurons in the dorsal nerve cords were HNK-1 immunoreactive. The juvenile amphioxus nerve cords contained anti-serotonin immunoreactive nerve fibers that seem to be the Rohde axons extending from amphioxus interneurons, the Rohde cells. In lamprey embryos, migrating neural crest cells and primary sensory neurons, including Rohon-Beard cells, expressed the HNK-1 carbohydrate. Lamprey larvae (ammocoetes) contained cell aggregates expressing both the HNK-1 carbohydrate and serotonin in the pronephros and in the regions adjacent to the gut epithelium. Some of these cell aggregates were present in the anti-serotonin positive visceral motor nerve net. Motor neurons and Müller fibers were serotonergic in ammocoetes. Comparison of the expression patterns of HNK-1 carbohydrate among sea urchins, amphioxus and lampreys seem to suggest the possible evolutionary origin of the neural crest, that is, ciliary bands in dipleurula-type ancestors evolved into primary sensory neurons in chordate ancestors, as inferred from Garstang's auricularia hypothesis, and the neural crest originated from the primary sensory neurons.

Journal Article↗
PubMed2003

Cadmium-induced cellular and immunological responses in Cyprinus carpio infected with the blood parasite, Sanguinicola inermis.

Little is known about immune responses in teleosts as linked to the aetiology of pollutants and parasitic diseases and in particular their combined effects on the host. Cadmium(Cd)-mediated immunological responses in the thymus and pronephros of juvenile carp (Cyprinus carpio), experimentally infected with the blood parasite, Sanguinicola inermis (Trematoda: Sanguinicolidae) for 30 days followed by an exposure to 0.1 mg Cd2+ l(-1) for 48 or 168 h were investigated. Differential organ-specific changes occurred in both organs examined. In carp exposed to Cd, intracelluar spaces, vacuolation in the eosinophils, dissociation of cell membranes together with the formation of concentric whorls occurred. The thymus of infected carp exposed to Cd had a granular cytosol which contained vesicles with electron-dense inclusions, swollen mitochondria with distended cristae and condensed nuclei in the erythrocytes. Cell counts on the two organs revealed a differential response to cadmium exposure in S. inermis infected carp compared to control infected fish. A significant increase in the neutrophil, eosinophil and thrombocyte components occurred in the thymus in contrast to a significant decrease in pronephric neutrophils. In addition, there was a differential blastogenesis response in infected and Cd-exposed infected carp fry exposed to cercarial antigens and the mitogens, concanavalin A and pokeweed mitogen.

Animals↗
PubMed2004

Developmental relation between palatine tonsils and kidney.

The evolutionary changes of the immune process in the whole line of representatives of the organisms were followed. The immune organs responsible for processing the antigens evolved from the derivatives of coelomic lining over the gut folds to the immune organs developed in the gill region of the digestive tract. Among them the tonsils have to be considered as a very modern evolutionary immune organ. They develop also as the latest in ontogeny even though their primordium contributes very early to the IgA production. The results of the host-antigen reactions have to be eliminated by the excretory organs, which are in invertebrates represented by body poruses and various types of simple segmentally arranged nephridia which are in contact with the gills. These nephridia have evolved in lower vertebrates in pronephros, further in mesonephros and finally in kidneys with filtrating nephrons. In case of defective elimination, the more voluminous immunocomplexes can form the subendothelial or mesangial deposits.

Animals↗
PubMed2003

Distinct molecular and morphogenetic properties of mutations in the human HNF1beta gene that lead to defective kidney development.

The homeobox transcription factor hepatocyte nuclear factor 1beta (HNF1beta) is a tissue-specific regulator that plays an essential role in early vertebrate development. In humans, heterozygous mutations in the HNF1beta gene are associated with young-onset diabetes as well as a variety of disorders of renal development with cysts as the most consistent feature. This report compares and classifies nine different HNF1beta mutations that lead in humans to distinct renal diseases, including solitary functioning kidney, renal dysplasia, glomerulocystic kidney disease, and oligomeganephronia. Analysis of these mutants in vitro identifies mutants that either retain or lack DNA binding. Investigation of the transactivation potential in transfected cell lines reveals a strict correlation between DNA binding and transactivation. Introduction of these mutants into developing Xenopus embryos shows that these mutants interfere with pronephros development, the first kidney form in amphibian. Whereas three mutants lead in Xenopus to a reduction or agenesis of the pronephric tubules and the anterior part of the duct, six mutants generate an enlargement of the pronephric structures. The differential morphogenetic potential in the developing embryo does not strictly correlate with the properties observed in vitro or in transfected cell lines. This suggests that the functional test in the developing embryo defines features of the HNF1beta protein that cannot be assessed in cell cultures. The distinct properties observed in the various HNF1beta mutants may guide the classification of the phenotypes observed in patients with a mutated HNF1beta gene.

Animals↗
PubMed1992

The chicken limb deformity gene encodes nuclear proteins expressed in specific cell types during morphogenesis.

The chicken limb deformity (ld) mutation affects morphogenesis of both limbs and kidneys and is one of few murine mutations for which the affected gene has been isolated. Analysis of the chicken homolog reveals evolutionary conservation of large parts of the encoded ld gene products. This is the first study of these proteins, their intracellular localization, and their temporal and spatial distribution during embryogenesis. A major 180-kD protein is expressed in chicken embryos and certain adult tissues. The proteins are localized in the nuclei of different embryonic cell types in a characteristic punctate pattern. In the developing chicken limb bud, they are expressed in the newly differentiated apical ectodermal ridge and the mesenchymal compartment, where an unequal distribution along the anteroposterior and, subsequently, the dorsoventral axes, is observed. During kidney morphogenesis, expression is initially restricted to the epithelial compartment of the pronephros and mesonephros. These results correlate well with the previous analysis of the murine ld phenotype and imply determinative roles for ld gene products during the morphogenesis of limbs and kidneys. Unexpected expression in the notochord, floor plate, and ventral horns suggests an involvement of the ld gene products in establishment of the dorsoventral polarity of the neural tube.

Amino Acid Sequence↗
PubMed1998

The renin-angiotensin system and the development of the kidney and adrenal in sheep.

1. The earliest form of the kidney, the pronephros, does not really occur in the ovine embryo; instead, a giant glomerulus forms at the anterior end of the mesonephros. 2. In the sheep, the mesonephros is present from 11-38% of total gestation (150 days) and produces a dilute urine, as well as expressing the genes for erythropoietin, renin, angiotensinogen, angiotensin-converting enzyme and the angiotensin II (AngII) receptors AT1 and AT2. 3. The ovine metanephros begins to develop at 18% of gestation and nephrogenesis is complete several weeks before birth. All components of the renin-angiotensin system (RAS) are expressed from at least 27% of gestation. 4. Both AT1 and AT2 receptors are expressed by the adrenocortical cells early in gestation but, at mid-gestation, exogenous AngII does not stimulate aldosterone secretion in vivo. 5. Preliminary results suggest that AngII has important roles in renal development in the ovine foetus but the role(s), if any, in adrenal development, remains to be investigated.

Adrenal Glands↗
PubMed2005

Fish mesonephric model of polycystic kidney disease in medaka (Oryzias latipes) pc mutant.

BACKGROUND: Polycystic kidney disease (PKD) is a common hereditary disease. A number of murine and zebrafish mutants have been generated and used for the study of PKD as metanephric and pronephric models, respectively. Here, we report a medaka (Oryzias latipes) mutant that develops numerous cysts in the kidney in adulthood fish in an autosomal-recessive manner as a mesonephric model of PKD. METHODS: The phenotypes of the medaka pc mutant were described in terms of morphologic, histologic, and ultrastructural features. The pc see-through stock was produced by crossing a pc mutant and a fish from the see-through stock and used for observing the kidney through the transparent body wall of a live fish. RESULTS: The mutant developed bilateral massive enlargement of the kidney in adulthood. They sexually matured normally within 2 months of age and died within 6 months of age. The affected kidney was occupied by numerous, fluid-filled cysts, which were lined by attenuated squamous epithelial cells. Developmentally, cystic formation began in the pronephros in 10-day-old fry and in the mesonephros in 20-day-old fry at the microscopic level. The pc see-through stock was useful in observing disease progression in live fish. CONCLUSION: The kidney disorder that develops in the medaka pc mutant is a mesonephric counterpart of PKD, particularly an autosomal-dominant PKD, based on its morphologic, histologic, and ultrastructural features, and slow progression.

Animals↗