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PubMed1999

Expression of Xenopus T-box transcription factor, tbx2 in Xenopus embryo.

We report here the cloning and expression of the Xenopus orthologue of the T-box transcription factor gene Tbx2 (optomotor-blind in Drosophila). Tbx2 is first detected in the ventral mesodermal cells just above the yolk plug at late gastrula. At the neurula stage it is strongly expressed in the cement gland, dorsal root ganglia, and otic vesicle region. At the tailbud stage strong Tbx2 expression is observed in the dorsal part of the optic cup and trigeminal ganglia, and it is also expressed in the branchial arches, heart anlage, nasal pit, proctodeum, and the region around the pronephros.

Amino Acid Sequence↗
PubMed1997

Emx2 developmental expression in the primordia of the reproductive and excretory systems.

The development of the urogenital system has always attracted many investigators owing to the peculiar aspects of the embryology of the reproductive and excretory organs and to the high number of congenital anomalies related to these structures. It is remarkable because of the common origin of the kidneys, gonads, and genital tracts from the intermediate mesoderm and because differentiation of these organs involves extensive mesenchyme to epithelium transition. Our knowledge about the molecular mechanisms controlling the differentiation of these diverse structures from the same precursor has taken advantage of gene expression data and gene-targeting experiments using genes with a specific expression pattern in the urogenital system. A more detailed function in kidney development has been postulated for transcription factors such as WT-1, Pax-2 or other molecules such as glial cell line-derived neurotrophic factor (GDNF), Wnt-4, c-ret. In the present work we have described the expression pattern of the homeobox-containing gene Emx2 during the development of the urogenital system in mouse embryos. We have found that Emx2 is expressed in the early primordia of the organs that will form the excretory and reproductive systems. In particular we have found that Emx2 is expressed in the epithelial components of pronephros and mesonephros, in Wolffian and Müllerian ducts, in the ureteric buds with their branches and in the early epithelial structures derived from metanephrogenic mesenchyme. Emx2 is also intensely expressed in the "bipotential" or "indifferent" gonads and ovaries. These data and the recent finding that Emx2 homozygous mutant mice die soon after birth because of the absence of kidneys indicate an essential role of Emx2 in the morphogenesis of the urogenital system.

Animals↗
PubMed2000

Overexpression of a Zebrafish ARNT2-like Factor Represses CYP1A Transcription in ZLE Cells.

Aryl hydrocarbon receptor nuclear translocator (ARNT) factors belong to a novel basic-helix-loop-helix-PAS (bHLH-PAS) transcription factor family that controls a variety of physiological and developmental processes. In a previous study, we obtained a partial complementary DNA fragment of an ARNT2-like factor from zebrafish embryo, liver, and other tissues by reverse transcription-polymerase chain reaction. In an effort to characterize the function of this factor, we screened an embryonic complementary DNA library and obtained a complete cDNA of this ARNT2-like factor, zARNT2A. The deduced protein sequence of zARNT2A encompasses the basic-helix-loop-helix and PAS-A/B motifs and shares highest sequence similarity with the amino-terminal half of mouse ARNT2 factor. However, it lacks a carboxy-terminal transactivation motif following the PAS-A/B motifs. Transient expression of zARNT2A in cultured cells resulted in repression of TCDD-dependent CYP1A transcription. Whole-mount in situ hybridization revealed that zARNT2A is expressed in brain and pronephros at prime-5 stages. In adult fish, zARNT2A messenger RNA is transcribed in a wide range of tissues, which indicates that zARNT2A and its corresponding signal transduction mechanisms have important roles in fish development and other physiological aspects.

Journal Article↗
PubMed1988

Dorsal and ventral cells of cleavage-stage Xenopus embryos show the same ability to induce notochord and somite formation.

Cells in the dorsal marginal zone of the amphibian embryo acquire the potential for mesoderm formation during the first few hours following fertilization. An examination of those early cell interactions may therefore provide insight on the mechanisms important for organization of axial structures. The formation of mesoderm (notochord, somites, and pronephros) was studied by combining blastomeres from the animal pole region of Xenopus embryos (32- to 512-cell stages) with blastomeres from different regions of the vegetal hemisphere. The frequency of notochord and somite development was similar in combinations made with dorsal or ventral blastomeres, or with both. Our results show that during early cleavage stages the ventral half of the vegetal hemisphere has the potential to organize axial structures, a property previously believed to be limited to the dorsal region.

Animals↗
PubMed1988

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers.

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Spectrophotometric analyses of hydroxysteroid dehydrogenase activity in presumed steroid-producing tissues of the brook lamprey (Lampetra planeri bloch) in different developmental stages.

Hydroxysteroid dehydrogenase (HSD) activity was determined spectrophotometrically on homogenates of the pronephros, the opisthonephros, and the gonads of metamorphosing, immature, and mature adult male and female brook lampreys (Lampetra planeri Bloch), employing pregnenolone, dehydroepiandrosterone, androsterone, epiandrosterone, and 3 beta,17 beta-dihydroxy-5 alpha-androstane as substrates and NAD or NADP as coenzymes in October, December, February, and April. With NADP a higher HSD-dependent HSD activity was detected in presumed adrenocortical tissue (PAT) homogenates with pregnenolone and 3 beta,17 beta-dihydroxy-5 alpha-androstane and in gonad homogenates only with 3 beta,17 beta-dihydroxy-5 alpha-androstane. With 3 beta,17 beta-dihydroxy-5 alpha-androstane as substrate the highest enzyme activities were measured in PAT and gonad homogenates from animals of all investigated life stages. A varying intensity of HSD activity in the examined tissue homogenates probably dependent on stages of the individual development was observed.

3-Hydroxysteroid Dehydrogenases↗
PubMed1983

T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: I. T-lymphocyte proportions, distribution and ontogeny, as measured by E-rosetting, nylon wool adherence, postmetamorphic thymectomy, and non-specific esterase staining.

We used sheep erythrocyte (SRBC) E-rosetting, nylon wool fractionation thymectomy and nonspecific esterase staining to assess T-lymphocyte characteristics in Rana pipiens during various developmental stages. T-lymphocytes appear in the spleens of premetamorphic tadpoles. We found significant levels of lymphocytes classifiable as T-cells in the peripheral blood of adults, but fewer numbers of T-cells in the thymus, jugular bodies, spleen, pronephros, mesonephros, liver and bone marrow. In addition, thymectomized Xenopus laevis showed a sharp decrease in T-cells as evidenced by E-rosetting, ANAE stain and nylon wool fractionation. The presence of T-lymphocytes in anuran amphibians and the existence of a receptor for SRBC on a population of these cells, suggest conservation of T-cells during evolution.

Age Factors↗
PubMed1985

Hydrocortisone suppresses the chemiluminescent response of striped bass phagocytes.

Phagocytic cells obtained from the pronephros of striped bass (Morone saxatilis) were exposed in vitro to various levels of hydrocortisone acetate. This treatment reduced the normal ability of the cells to generate a chemiluminescent response when exposed to bacteria or phorbol myristate acetate. Although the levels of hydrocortisone were higher than is physiological for fish, suppression was dose dependent and was not attributable to a reduction in cell viability. Whereas phagocytic chemiluminescence has been linked to the respiratory burst and bactericidal activity, the possibility exists that stress-induced elevations in serum corticosteroids lead to increased susceptibility to infection.

Animals↗
PubMed1987

A chemokinetic factor in the carp Cyprinus carpio.

The results describe experiments which demonstrate that the teleost Cyprinus carpio is capable of elaborating a substance which has analogous in vitro effects to that of mammalian chemokinetic lymphokines. In vitro stimulation of leucocytes from carp pronephros, both with antigens and mitogens, was used to provide culture supernatants which were applied to an in vitro chemokinetic assay. The chemokinetic factor-like activity could be detected in supernatants after one day of culture and at a dilution of 25%. The presence of lymphokines in fish supports the theory that lymphokine production is a fundamental property of vertebrate leucocytes and is of central importance in vivo cell-mediated immunity.

Animals↗
PubMedDate not supplied

Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor.

Carp, Cyprinus carpio L, macrophages and neutrophilic granulocytes obtained from pronephros were cultured. Supernatant was harvested after 48 h and tested for interleukin-1 (IL-1) bioactivity. A concentration-dependent stimulation of proliferation was found of carp Ig- lymphocytes as well as of the murine IL-1 dependent D10 (N4) M cell line. A 4 h treatment of cells with phorbol myristate acetate prior to culture gave a two- to fourfold enhancement of the bioactivity in the supernatant. Antibodies raised in sheep against human recombinant IL-1 alpha or IL-1 beta added to the supernatant annulled the IL-1 bioactivity. Western blot analysis of supernatants with sheep or rabbit polyclonal antisera against human IL-1s revealed 22 kDa and 15 kDa protein species. The predominant newly synthesized protein that was immunoprecipitated with these antisera was a 15 kDa molecular species. We conclude that carp macrophages and neutrophilic granulocytes produce an IL-1-like molecule with T-cell proliferating potency that shares structural similarities with mammalian IL-1. This is the first evidence for the IL-1 signal protein in carp immunocompetent cells.

Animals↗
PubMedDate not supplied

Two different subpopulations of Ig-bearing cells in lymphoid organs of rainbow trout.

In a previous report we described the existence of two structural and antigenic variants of light chains in serum trout Igs, defined by mAbs 2H9 and 2A1. In this report, we analyse the cell distribution, the "in vitro" kinetics of production, and the ontogenic appearance of these two variants. Both variants were already present at day 8 before hatching and their kinetics of appearance correlated with that of total Ig, showing a peak around hatching. These mAbs stained two distinct lymphoid cell populations in the pronephros, mesonephros, spleen, and peripheral blood. Both mAbs reacted with approximately 1% of thymocytes. The ratio between these two cell populations (2A1+/2H9+) was about 2/1 in the lymphoid organs analysed. No differences were found between the profiles of the "in vitro" production of these variants in pronephric cell cultures stimulated with LPS. In these cultures, the 2A1 and 2H9 Igs together accounted for 10-70% of the total trout Ig produced, suggesting the existence of at least one additional L chain variant.

Animals↗
PubMed1991

Effects of organophosphorus insecticides: effects of trichlorfon and dichlorvos on the immune response of carp (Cyprinus carpio). III. In vitro effects on lymphocyte proliferation and phagocytosis and in vivo effects on humoral response.

Cells isolated from the pronephros of carp were incubated in vitro with the organophosphorus insecticide trichlorfon or dichlorvos, each of which is used in aquaculture to eliminate fish ectoparasites. Dose-dependent suppressive effects were observed in assays for lymphocyte proliferation and myeloid cell respiratory burst activities. Dichlorvos given by bath in vivo did not affect antibody production against Yersinia ruckeri even if the spleen and kidney were obviously contaminated according to acetylcholinesterase activity analysis. Some blood parameters of nonspecific immunity (ceruloplasmin, lysozyme, hemagglutinins) were slightly affected.

Animals↗
PubMed1986

Pronephric leucocytes of Cyprinus carpio: isolation, separation and characterization.

Since the teleost pronephros is an important source of diverse immunocytes, suspensions of pronephric cells from young adult carp have been characterized. In freshly prepared suspensions, adherent, spreading cells (macrophages?) constituted less than 3% of the total population. Granulocytes and lymphocytes were co-dominant (less than 80%) leucocyte types. Continuous Percoll density gradient centrifugation yielded discrete subpopulations with these rho values and cytological characteristics: Fraction I & II rho = 1.055-1.070 thrombocytes, monocytes, macrophages, and lymphocytes. Fraction III rho = 1.080-1.090 granulocytes, type 1. Fraction IV rho = 1.105-1.110 erythrocytes and granulocytes, type 2. Fraction V rho = 1.118-1.125 granulocytes, type 3. Fraction VI rho = 1.140-1.150 granulocytes, type 4. Granulocyte motility increased markedly over the first 24 hr in vitro, and was enhanced by components washed from intact yeast. The subtypes of granulocytes were distinguishable by not only the rho values, but also on the basis of cell size, ultra-structure of the granules, and their histochemical and phagocytic characteristics. After simultaneous in vivo injection of Bacillus megaterium (Gram + ve), Aeromonas hydrophila (Gram - ve) and Saccharomyces cerevisiae (yeast), individual pronephric leucocytes were found capable of phagocytosing all three types of particle. Granulocytes which had phagocytosed B. megaterium were slower than macrophages in their ability to kill the bacteria. Encounter with B. megaterium or S. cerevisiae in vitro elicited a clumping reaction which involved mostly the larger leucocytes [granulocytes]. Both adherent cells and non-adherent cells were phagocytic in vitro.

Animals↗
PubMed1986

Effects of metals on the chemiluminescent response of rainbow trout (Salmo gairdneri) phagocytes.

Quantification of an induced chemiluminescent (CL) response in phagocytes is currently being evaluated as an indicator system for determining those environmental pollutants that may predispose fish to disease. A CL assay was developed using phagocytes from the pronephros of rainbow trout (Salmo gairdneri). The CL response of phagocytes to phorbol myristate acetate, a chemical inducer of CL, was shown to be dose-dependent. The response to five species of bacteria was also evaluated. Staphylococcus aureus and Aeromonas hydrophila produced the most intense CL responses and the longest duration of response (100 min.) Yersinia ruckeri induced an immediate strong CL response of short duration (20 min.) whereas Vibrio anguillarum and Aerococcus viridans failed to stimulate CL under the test conditions employed. The effect of sub-toxic levels of Cu, Al, and Cd on the CL response of phagocytes to S. aureus was examined using phagocytes exposed to the metals immediately before assay or after 1 hr or 24 hr exposure times. Copper caused a significant decrease in CL to the baseline level under all treatment conditions upon stimulation with S. aureus. Similar results were obtained with Al except that the decrease in CL, although significant, was not to the baseline level. In contrast, Cd caused a significant increase in CL when added 1 hr prior to or immediately before the assay; but, following a 24 hr exposure, the results were variable, in that either no change or a decrease was observed. The addition of Cu to phagocytes already exhibiting a strong CL response to S. aureus caused an immediate decrease in CL to that seen with the negative controls.

Aeromonas↗
PubMed1986

Effect of an immunopotentiator on Aeromonas salmonicida infection in rainbow trout (Salmo gairdneri).

The immunoactive peptide FK-565 (heptanoyl-y-D-glutamyl-(L)-mesodiaminopimelyl-(D)-alanine) was found to induce protection against intraperitoneal Aeromonas salmonicida infection in rainbow trout (Salmo gairdneri Richardson). The survival rate was as high as 60% when FK-565 was given intraperitoneally as a single dose (1mg/kg) one day before bacterial challenge. A non-specific stimulation of phagocytic cells by FK-565 at an early stage of the bacterial infection may contribute to the resistance observed. The phagocytic activity of peritoneal phagocytic cells as well as phagocytic cells of the pronephros were stimulated by FK-565 in vivo and in vitro, respectively, as compared to untreated control fish. Furthermore, decreased activity of phagocytic cells previously immunosuppressed with cyclophosphamide was rapidly restored by application of FK-565.

Aeromonas↗
PubMed1986

Does Aeromonas salmonicida affect the immune system of carp, Cyprinus carpio L.?

Aeromonas salmonicida is a significant bacterial pathogen of cyprinid and salmonid fishes causing the systemic disease furunculosis. Several observations led us to believe that A. salmonicida was able to evade or suppress the immune system of the fish: injection of whole bacteria or surface antigens was unsuccessful at protecting fish against lethal challenges; memory did not develop in survivors of sublethal infections; diseased fish often carried other opportunistic bacterial pathogens in addition to A. salmonicida, and serum protein and particularly immunoglobulin significantly decreased during A. salmonicida infections. We tested the ability of fish sublethally infected with virulent and avirulent A. salmonicida to mount a humoral immune response to sheep erythrocytes and found fewer plaque forming cells in the pronephros and lower serum anti-SRBC antibodies in infected fish as compared to controls. We also monitored the cellular immune response of diseased fish by skin allograft rejection and found an enhancement of the response that increased as the disease progressed. However, the extend of inflammation was reduced in infected fish as compared to non-infected animals. At this moment these preliminary observations are difficult to explain. Our future research will focus more specifically on cell populations that may be affected by A. salmonicida.

Aeromonas↗
PubMed1996

Multiple regulation of carp (Cyprinus carpio L.) macrophages and neutrophilic granulocytes by serum factors: influence of infection with atypical Aeromonas salmonicida.

Normal carp serum contains inhibitory and stimulatory factors for macrophage and neutrophilic granulocyte respiratory burst activity. As stimulatory factors were only effective in combination with phorbol myristate actetate (PMA) activation, it is concluded that they are probably linked to protein kinase C activation. Both the stimulatory and inhibitory factors are heat stable. Macrophage- and neutrophilic granulocyte-enriched cell fractions from the pronephros of carp had high respiratory burst- and high bactericidal in vitro responses to virulent atypical Aeromonas salmonicida bacteria. Serum factors were inhibitory for the A. salmonicida induced respiratory burst activity. No change in inhibitory or stimulatory serum factors could be observed during a 12-day challenge experiment with A. salmonicida, or during a rechallenge of survivors from a previous sub-lethal infection. The sensitivity of macrophages and neutrophilic granulocytes to stimulation of respiratory burst activity by PMA was not significantly altered. Culture supernatants from PHA pre-treated lymphocytes stimulated the respiratory burst activity of macrophages and neutrophilic granulocytes suggesting that serum factors may partially be lymphocyte derived.

Aeromonas↗
PubMed1992

Comparative analysis of Pax-2 protein distributions during neurulation in mice and zebrafish.

Members of different vertebrate species share a number of developmental mechanisms and control genes, suggesting that they have similar genetic programs of development. We compared the expression patterns of the Pax-2 protein in Mus musculus and Brachydanio rerio to gain a better understanding of the evolution of developmental control genes. We found that the tissue specificity and the time course of Pax-2 expression relative to specific developmental processes are remarkably similar during the early development of the two organisms. The brain, the optic stalk, the auditory vesicle, the pronephros, and single cells in the spinal cord and the hindbrain express Pax-2 in both species. The Pax-2 expression domain in the prospective brain of E8 mouse embryos has not been described previously. Expression appears first during early neurulation at the junction between the midbrain and hindbrain. However, there are some differences in Pax-2 expression between the two species. Most notable, expression at the midbrain/hindbrain boundary is no longer detectable after E11 in the mouse. Using monoclonal antibodies, we could exclude that primary neurons express Pax-2 in the zebrafish spinal cord. Our results confirm that Pax genes are highly conserved both in sequences and in expression patterns, indicating that they may have a function during early development that has been conserved during vertebrate evolution.

Animals↗