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PubMed1997

Adrenocortical and adrenomedullary homologs in eight species of adult and developing teleosts: morphology, histology, and immunohistochemistry.

Morphology, histology, and immunohistochemistry of the adrenocortical and adrenomedullary homologs (adrenal glands) of the following developing and adult teleosts were examined: Salmoniformes-Oncorhynchus mykiss (rainbow trout), Salmo trutta fario (brown trout), Coregonus lavaretus (white fish); Cyprinodontiformes-Gambusia affinis (mosquito fish). Perciformes-Dicentrarchus labrax (sea bass), Sparus aurata (sea bream), Diplodus sargus (white bream), Oblada melanura (saddled bream). The anatomical relationships of the gland with the renal system and venous vessels were also noted. In adults of all species steroidogenic and catecholaminergic chromaffin cells were found in the head kidney, which is pronephric in origin and subsequently transformed into a hematopoietic lymphatic organ. In Perciformes, chromaffin cells are distributed around the anterior and posterior cardinal veins and ducts of Cuvier; in Salmoniformes, around the posterior cardinal veins and in the hematopoietic tissue; and in G. affinis, around the ducts of Cuvier and posterior cardinal veins, while a few are visible also around the sinus venosus. In Perciformes and Salmoniformes, numerous chromaffin cells are also present in the posterior kidney, derived from the opisthonephros, in contact with the caudal vein. Steroidogenic cells are always confined to the head kidney. During development chromaffin and steroidogenic cells appear early after hatching in the pronephric kidney, at the level of the ducts of Cuvier and of the cephalic part of the posterior cardinal veins. Later, chromaffin cells in Perciformes reach the anterior cardinal veins, and subsequently, in both Perciformes and Salmoniformes, they reach the developing posterior kidney. Their localization along the posterior kidney is still in progress about 4 months after hatching and is completed about a year after hatching. These findings support the concept that the structure of the adrenal gland in teleosts is intermediate between that of the other actinopterygians and that of tetrapods. The development differs from that of tetrapods in that it occurs mainly in the pronephros and only later do chromaffin cells reach the opisthonephric kidney.

3-Hydroxysteroid Dehydrogenases↗
PubMed2001

3-Methylcholanthrene increases phorbol 12-myristate 13-acetate-induced respiratory burst activity and intracellular calcium levels in common carp (Cyprinus carpio L) macrophages.

Phagocytic cells play a key role in the fish immune system. They secrete reactive oxygen species (ROS) involved in their bactericidal activity. These cells are highly sensitive to pollution by polycyclic aromatic hydrocarbons and other organic pollutants. We have investigated the intracellular mechanisms by which 3-methylcholanthrene (3-MC) increased bactericidal activity of carp phagocytes. Macrophages isolated from head kidney (pronephros) and incubated 1 h with 3-MC enhanced their production of ROS when they were stimulated 1.25 h with phorbol 12-myristate 13-acetate (PMA), a direct activator of protein kinase C (PKC). 3-MC also produced a rapid and a sustained increase in [Ca(2+)](i) (2 h minimum). However, the cytochrome P450 1A and Ah receptor inhibitor, alpha-naphtoflavone (alpha-NF), inhibited the potentiation of PMA-induced ROS production, suggesting 3-MC metabolic activation. Moreover, alpha-NF increased [Ca(2+)](i) without macrophage ROS production, suggesting that some mechanism other than calcium release is playing a role in the stimulation of the macrophages by 3-MC. The rise in [Ca(2+)](i) induced by 3-MC was potentiated by the inhibitor of the endoplasmic reticulum calcium ATPases, thapsigargin. And treating the cells with 3-MC decreased the calcium mobilization caused by thapsigargin. These results suggest that 3-MC acts on the endoplasmic reticulum, perhaps directly on calcium ATPases, to increase intracellular calcium levels in carp phagocytes.

Animals↗
PubMed1990

The distribution of mesenchyme proteoglycan (PG-M) during wing bud outgrowth.

This study utilizes immunofluorescence to describe the distribution of several extracellular matrix molecules in the chick embryo during the process of limb outgrowth and the formation of precartilage condensations. A large chondroitin sulfate proteoglycan (PG-M) is detected at the wing level at Hamburger and Hamilton stage 14 in and under the dorsal ectoderm, and is associated with the basement membranes around the neural tube, notochord and pronephros, but not with other basement membranes. The galactose-specific lectin, peanut agglutinin (PNA), has a similar distribution except that it also binds to the dorsal side of the neural tube. PG-M is not detected in the limb mesenchyme until after stage 17, when it is present in the distal region, as is PNA-binding material. With further development of the wing bud, PG-M is present in the subectodermal mesenchyme, the mesenchyme at the distal tip and in the prechondrogenic core. After stage 22 PNA-binding material becomes localized in the prechondrogenic core, the basement membranes under the apical ectodermal ridge, and the ventral sulcus. The distribution of these components (PG-M and PNA binding material) overlaps, but differs from that of type I collagen and fibronectin and basement membrane components, such as laminin, basement membrane heparan sulfate proteoglycan, and type IV collagen. Tenascin, on the other hand, is not detected in the limb bud until stage 25, after the appearance of cartilage matrix components such as type II collagen and cartilage proteoglycan (PG-H). These results are considered in relation to the formation of precartilage aggregates, and indicate that PNA binds to components in precartilage aggregates other than PG-M or tenascin.

Aggrecans↗
PubMed1993

Zebrafish Mhc class II alpha chain-encoding genes: polymorphism, expression, and function.

Its small size and short generation time renders the zebrafish (Brachydanio rerio) an ideal vertebrate for immunological research involving large populations. A prerequisite for this is the identification of the molecules critical for an immune response in this species. In earlier studies, we cloned the zebrafish genes coding for the beta chains of the class I and class II major histocompatibility complex (Mhc) molecules. Here, we describe the cloning of the zebrafish alpha chain-encoding class II gene, which represents the first identification of a class II A gene in teleost fishes. The gene, which is less than 3 kilobases (kb) distant from one of the beta chain-encoding genes, is approximately 1.2 kb long and consists of four exons interrupted by very short (< 200 base pairs) introns. Its organization is similar to that of the mammalian class II A genes, but its sequence differs greatly from the sequence of the latter (36% sequence similarity). Among the most conserved parts is the promoter region, which contains X, Y, and TATA boxes with high sequence similarity to the corresponding mammalian boxes. The observed striking conservation of the promoter region suggests that the regulatory system of the class II genes was established more than 400 million years ago and has, principally, remained the same ever since. Like the DMA, but unlike all other mammalian class II A genes, the zebrafish gene codes for two cysteine residues which might potentially be involved in the formation of a disulfide bond in the alpha 1 domain. The primary transcript of the gene is 1196 nucleotides long and contains 708 nucleotides of coding sequence. The gene is expressed in tissues with a high content of lymphoid/myeloid cells (spleen, pronephros, hepatopancreas, and intestine). The analyzed genomic and cDNA sequences are probably derived from different loci (their overall sequence similarity in the coding region is 73% and their 3' untranslated regions are highly divergent from each other). The genes are apparently functional. Comparison of genes from different zebrafish populations reveals high exon 2 variability concentrated in positions coding for the putative peptide-binding region. Phylogenetic analysis suggests that the zebrafish class II A genes stem from a different ancestor than the mammalian class II A genes and the recently cloned shark class II A gene.

Amino Acid Sequence↗
PubMed1993

In vitro immunomodulation by pentachlorophenol in phagocytes from an estuarine teleost, Fundulus heteroclitus, as measured by chemiluminescence activity.

Chemiluminescence (CL) was used to assay effects of pentachlorophenol (PCP) on the production of reactive oxygen intermediates (ROIs) generated by leukocytes from an estuarine teleost, Fundulus heteroclitus. Two parameters were measured during the phagocytically-induced respiratory burst: peak CL (maximal value) and total CL (temporal summation of induced values). Phagocytes obtained from the pronephros were incubated in the presence of sublethal doses of analytical (A-) or technical (T-) grades of PCP. Technical PCP has been reported to be more highly immunosuppressive than A-PCP in mammals; this effect was attributed to dioxins and other contaminants produced during manufacture. In this fish, both grades of the compound produced significant dose-dependent inhibition of peak and total CL. However, there were few significant differences between the effects of the two PCP grades on the magnitude of the CL responses at identical, sublethal concentrations, although T-PCP did produce a more marked reduction in the relative level of leukocytic CL as a function of dose than did A-PCP. Because of the probable roles of ROIs in antimicrobial blood cell-mediated mechanisms, reduced CL activity was interpreted as an indication of xenobiotic-induced immunosuppression.

Adjuvants, Immunologic↗
PubMed1984

Plasma cells in adult Atlantic hagfish, Myxine glutinosa.

Hagfishes, the most primitive vertebrates, are of special interest for the evolution of immune responses. Eptatretus stoutii, the Pacific hagfish, is able to mount cellular and humoral immune responses but all attempts to demonstrate in them the presence of plasma cells have failed. In the present study we demonstrate for the first time plasma cells identifiable by ultrastructural criteria in the pronephros, a primitive lymphohaemopoietic organ, of Myxine glutinosa, the Atlantic hagfish.

Animals↗
PubMed1989

A major histocompatibility locus in fish: serological identification and segregation of transplantation antigens in the common carp (Cyprinus carpio L.).

In this study, experiments are described that were designed to investigate whether fish have an immune regulatory systems similar to the major histocompatibility complex (MHC) in higher vertebrate species. From combinations of gynogenetic carp showing either slow or fast rejection of skin transplants, the latter were chosen for alloantiserum production by hyperimmunization with peripheral blood leucocytes. The resulting alloantisera were analyzed for hemagglutinating reactivity with gynogenetic siblings and proved to be operationally monospecific in absorption experiments. The serologically determined carp erythrocyte specificities were shown to correspond to two codominantly expressed allelic products of a single locus and were designated K1 and K2, respectively. Flow cytometer analysis revealed that the same products are also present on leucocytes from peripheral blood, thymus, spleen, and pronephros. K1- and K2-homozygous second-generation gynogenetic siblings were used to study the histocompatibility nature of the K locus products. Skin transplants between K-allogeneic gynogenetic siblings were rejected significantly faster than within K-syngeneic combinations. Taken together, these data suggest that the K locus incorporates MHC class I-like characteristics.

Animals↗
PubMed2002

Characterization of immunoglobulin light chain isotypes in the common carp.

Two or three isotypes of immunoglobulin (Ig) light (L) chain have been demonstrated in several teleost species thus far. A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used with the aim of isolating cDNA clones encoding IgL chain from the common carp ( Cyprinus carpio. L.) different from the previously isolated isotype, designated L1A. cDNA clones representing two novel isotypes, designated L1B and L3, were obtained in this study. For CL segments, L1B sequences were similar to type L1/G in various teleost species, as well as L1A, while L3 sequences were closely related with type L3/F in catfish and zebrafish. For VL segments, however, L1A sequences demonstrated higher similarity to L3 rather than L1B. These results were also supported by the phylogenetic study. The similarity found between L1A and L3 VL sequences was based on the very high degree of nucleotide identity in FR1, and/or FR2 to FR3, strongly suggesting the involvement of interlocus recombination between them. Southern blot analyses suggested that the locus of L1B has a cluster-like organization, but that those of L3 and L1A are currently unknown due to cross-hybridization between those VL probes. Northern blot analyses showed that CL mRNA of each isotype was expressed in lymphoid tissues examined, particularly strongly in pronephros, and was predominantly composed of approximately 1-kb and 0.6-kb transcripts, possibly corresponding to fully spliced (LVJC) and truncated (JC/J-C) forms, respectively.

Amino Acid Sequence↗
PubMed2002

Induction of the rainbow trout MHC class I pathway during acute IHNV infection.

Interferons are essential for establishing cytotoxic T-lymphocyte immunity against viral pathogens through different mechanisms including the modulation of antigen presentation to T-cell subsets. At the present time, interferons have yet to be isolated from teleost fish. We have developed a salmonid model to examine whether MHC gene regulation is modulated during acute viral infection in trout, an event attributable to interferons in mammals. During peak infection with infectious hematopoietic necrosis virus, induction of STAT-1, PSMB9A and ABCB2 mRNA was evident in all tissues within infected fish, as compared with controls. In addition, MHC class Ia and beta(2) microglobulin (beta(2)m) transcript levels were enhanced within the experimental group but surprisingly, splenic and pronephric class IIB mRNA expression was virtually absent. A time-course study looking at 24, 72 and 192 h post-infection was then performed to determine the overall kinetics of this response. STAT-1 and PSMB9A message levels increased early during the immune response and remain at relatively high levels until the final time point. MHC class Ia expression is not consistently upregulated until midway in the response. MHC class IIB transcripts are downregulated by 72 h in the spleen and pronephros and then partially restored by 192 h. Finally, analysis of the putative promoter regions for PSMB9A and ABCB2 identified interferon (IFN) regulatory factory (IRF-1) and INF-gamma (GAS) activation sites that may be involved in the regulation of these genes during viral infection.

Animals↗
PubMed2003

Molecular cloning and characterization of calreticulin from rainbow trout ( Oncorhynchus mykiss).

Calreticulin (CRT) is a highly conserved, high-capacity, calcium-binding protein shared among vertebrates, invertebrates and higher plants. Its biological importance, highlighted by its highly conserved nature, is supported by its crucial physiological and immunological functions. Within the endoplasmic reticulum, CRT serves as a calcium modulator and a lectin-like chaperone for glycoproteins, especially class I major histocompatibility receptors. To date, CRT cDNA clones have been isolated from a wide range of phyla, yet little is known about this gene in fish species, the largest and most diverse group of jawed vertebrates. This report describes the cloning of a cDNA from a rainbow trout pronephros library that encodes a deduced 419-amino acid protein, which includes a predicted 20-amino acid signal peptide and has a 69% amino acid identity to both murine and human CRT. Like its mammalian counterparts, this cDNA contains conserved cysteine residues believed to form a disulphide bond, a proline-rich region which includes a potential N-glycosylation site, and a highly acidic C-terminal domain terminating with the endoplasmic reticulum retrieval sequence, KDEL. Reverse transcription tissue-distribution assays indicate it is ubiquitously expressed in all tissues tested with highest expression in liver, while Southern blotting indicates it is a single copy gene.

Amino Acid Sequence↗
PubMed1997

Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish.

The closely linked recombination activating genes, rag1 and rag2, encode components of the recombinase involved in V(D)J recombination of the immunoglobulin and T-cell receptor genes. These genes are expressed together exclusively in immature lymphocytes and are useful markers for following the development of lymphoid tissues. We cloned the rag locus of the zebrafish Danio rerio and sequenced the open reading frames of the rag1 and rag2 genes. Although the gene organization is similar to that in other species, the rag1 gene is unusual in possessing two introns within the coding region. In another teleost, the rainbow trout, the rag1 gene is interrupted by a single intron. Introns are not present in the rag1 gene of any other species examined to date. Expression of both rag1 and rag2 begins late in embryonic development, on day 4, by northern RNA blot analysis. Expression of rag1 was detected in the adult zebrafish thymus, pronephros, mesonephros, and ovary. This pattern of expression is consistent with previous histological studies of adult teleosts, which implicate the kidney as the major site of hematopoiesis and the thymus as the major lymphocyte-containing organ.

Amino Acid Sequence↗
PubMed2002

Platelet-derived growth factor A (pdgf-a) expression during zebrafish embryonic development.

Zebrafish pdgf-a gene was cloned and its expression pattern studied during zebrafish embryogenesis. Zebrafish pdgf-a mRNA was present at high levels in fertilized eggs as well as in all embryonic cells up to the end of gastrulation. Spatially restricted expression started after the onset of segmentation and was mainly localized in the developing pharyngeal arches. Transient expression was also detected in Kupffer's vesicle, a teleost-specific structure, and in lateral trunk and tail regions surrounding the neural keel, as well as areas of the developing pronephros.

Amino Acid Sequence↗
PubMed2002

XHRT-1, a hairy and Enhancer of split related gene with expression in floor plate and hypochord during early Xenopus embryogenesis.

We have isolated a Xenopus homologue of the mammalian hairy and Enhancer of split related gene HRT1. XHRT1 expression in late gastrula and early neurula embryos is restricted to two stripes of cells in the medial neural plate and in dorsal endodermal cells. At later stages, XHRT1 is expressed in the floor plate, in hypochord cells and in the somitogenic and anterior presomitic mesoderm. By tailbud stage, XHRT1 is also highly expressed in the dorsal hindbrain, telencephalon and eye vesicles, olfactory placodes, pronephros, branchial arches and tail fin. We also show that XHRT1 expression in medial neural cells is induced by Notch signaling and that there are differences in the way XHRT1 and other H/E(spl) genes are regulated.

Amino Acid Sequence↗
PubMed2003

Cloning and expression patterns of dystroglycan during the early development of Xenopus laevis.

Dystroglycan is a cell surface receptor involved in the pathogenesis of muscular dystrophy, and plays a critical role in the assembly and homeostasis of basement membranes. Since data about the amphibian homologue are limited, we have cloned the full-length dystroglycan cDNAs from the frog Xenopus laevis. Using in situ hybridization, we show that mRNA expression is dynamic, particularly in the notochord at the end of gastrulation and during neurulation, suggesting that the protein might play unexplored roles in the specification and/or formation of this tissue. Subsequently, the transcripts are detected in the otic vesicle, the developing brain, and in mesenchymal cells of the visceral arches, as well as in pharyngeal endoderm, the pronephros, pronephric ducts, proctodaeum and the somites.

Amino Acid Sequence↗
PubMed2004

Isolation and developmental expression of Xenopus FoxJ1 and FoxK1.

xFoxJ1 and xFoxK1, two novel members of the fork head/winged helix family of transcription factors in Xenopus, have been identified by sequence homology to their mammalian orthologues. We here report the complete amino acid sequences of xFoxJ1, its pseudo-allelic version xFoxJ1', and of xFoxK1, the latter also comprising a fork-head-associated (FHA) domain. We have further analysed the temporal and spatial expression of both genes. xFoxJ1 transcripts were detected during gastrulation in the dorsal blastopore lip and in the animal half. A predominant feature of xFoxJ1 expression until the tailbud stage is its spotty pattern within the epidermis. xFoxK1 is maternally expressed and localized within the animal half during early cleavage stages. Zygotic transcription starts during neurulation and is initially restricted to neuroectoderm. As development proceeds, xFoxK1 is expressed in ectoderm- and mesoderm-derived tissues, like the branchial arches, brain, eye, otic vesicle, pronephros, somites and abdominal muscle precursors.

Amino Acid Sequence↗
PubMed2005

Phylogenomic analysis and expression patterns of large Maf genes in Xenopus tropicalis provide new insights into the functional evolution of the gene family in osteichthyans.

We have performed an exhaustive characterization of the large Maf family of basic leucine zipper transcription factors in vertebrates using the genome data available, and studied the embryonic expression patterns of the four paralogous genes thus identified in Xenopus tropicalis. Our phylogenetic analysis shows that, in osteichthyans, the large Maf family contains four orthology classes, MafA, MafB, c-Maf and Nrl, which have emerged in vertebrates prior to the split between actinopterygians and sarcopterygians. It leads to the unambiguous assignment of the Xenopus laevis XLmaf gene, previously considered a MafA orthologue, to the Nrl class, the identification of the amphibian MafA and c-Maf orthologues and the identification of the zebrafish Nrl gene. The four X. tropicalis paralogues display partially redundant but nevertheless distinct expression patterns in the somites, developing hindbrain, pronephros, ventral blood island and lens. Comparisons with the data available in the mouse, chick and zebrafish show that these large Maf expression territories are highly conserved among osteichthyans but also highlight a number of differences in the timing of large Maf gene expression, the precise extent of some labelled territories and the combinations of paralogues transcribed in some organs. In particular, the availability of robust phylogenies leads to a reinterpretation of previous expression pattern comparisons, suggesting an important part for function shuffling within the gene family in the developing lens. These data highlight the importance of exhaustive characterizations of gene families for comparative analyses of the genetic mechanisms, which control developmental processes in vertebrates.

Animals↗
PubMed2006

The FoxP subclass in Xenopus laevis development.

We have investigated the sequences and the expression patterns of different members of the Xenopus laevis FoxP gene subfamily during embryogenesis. Low stringency hybridisation of a tadpole cDNA library with an xlFoxP2 fragment led to the isolation of several splice variants of xlFoxP1, xlFoxP2 and xlFoxP4. These variants do not only differ by utilisation of different leader exons, but also by alternative usage of coding exons thereby leading to functional alterations. For xlFoxP1b, we show that insertion of an additional exon disrupts binding to the co-repressor C-terminal binding protein1. Temporal and spatial expression patterns of xlFoxP2 and xlFoxP4 were analysed by RT-PCR and by whole mount in situ hybridisation. xlFoxP2 transcripts are detected from mid-gastrula to late tadpole stages and are found to be localised to pronephros, branchial arches and distinct structures of the hind-, mid- and forebrain, including the ciliary marginal zone of the retina. xlFoxP4 RNA is already present in early cleavage stage embryos and accumulates from midblastula until the end of embryogenesis. Localised expression is found within the anterior neural fold, in the mid- and hindbrain, in the branchial arches as well as in the pancreas.

Amino Acid Sequence↗
PubMed1999

The zebrafish eya1 gene and its expression pattern during embryogenesis.

The eyes absent-like genes encode a group of putative transcriptional coactivators with a sole representative in Drosophila and several members in mammals. Haploinsufficiency of the human EYA1 gene results in branchio-oto-renal syndrome characterized by developmental anomalies of the branchial arches, the three compartments of the ear and the kidney. As a first step towards a functional analysis of this gene in lower vertebrates, we isolated its zebrafish homologue, eya1, and studied its expression pattern during embryogenesis. The eya1 cDNA predicts a protein with 84.7% identity with the human homologue. Transcripts are first detected at the tailbud stage in presumptive cranial placodal precursor cells. Thereafter, eya1 expression continues in anterior pituitary, olfactory, otic, and lateral line placodes. Aside from these placodal sites of expression, eya1 transcripts were observed in the somites, developing pectoral fins, and branchial arches. No expression was found in pronephros or Wolffian duct of the zebrafish renal system. Within the developing ear, eya1 expression becomes confined to the ventral part of the otic vesicle from where the acoustic ganglion precursor cells arise and the sensory patches differentiate. In the lateral line, eya1 is expressed in the placodes, ganglia, migrating primordia, and receptive organs at all developmental stages, including both the differentiating hair and supporting cells. Taken together, these results indicate a remarkable similarity in both the structure and expression pattern of eya1 between higher and lower vertebrates, suggesting that the function of this gene has been conserved throughout vertebrate evolution.

Amino Acid Sequence↗