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Expression of Xfz3, a Xenopus frizzled family member, is restricted to the early nervous system.

Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.

Amino Acid Sequence↗

The specification of the pronephric tubules and duct in Xenopus laevis.

We have examined the timing of specification of the pronephric tubules and duct in Xenopus laevis by explanting the presumptive pronephric rudiments into blastula ectodermal wraps. We have established the time point of specification using the monoclonal antibody markers 3G8 and 4A6 which recognize antigens in pronephric tubule and duct, respectively. We show that, by experimental analysis in explants, kidney tubules are specified by stage 12.5 in the pronephric anlagen whereas pronephric duct is specified later between stages 13 and 14. Furthermore we show that signals involved in tubulogenesis of the pronephric tubules are normally received between stage 12.5 and 13. These experiments unambiguously pinpoint the timing of pronephros specification analyzed by explant experimentation to a developmental stage prior to that demonstrated for urodele amphibia, and provide an essential biological backdrop to a search for the molecular nature of pronephric inducers.

Animals↗

Expression pattern of the winged helix factor XFD-11 during Xenopus embryogenesis.

We have identified a cDNA encoding a novel Xenopus winged helix transcription factor termed as XFD-11 (Xenopus fork head domain). The DNA binding domain is most closely related to those of human or murine FREAC-3 (FKHL7/MF-1/FKH-1) proteins. The XFD-11 gene is activated at late blastula/early gastrula and transcription proceeds throughout embryogenesis. Early expression is found in ventral and lateral but not in dorsal mesoderm. At neurula stages, transcripts are found in posterior mesoderm except for the dorsal midline, and the gene is also transcribed at the lateral border of the neural plate and within anterior neuroectoderm. At later stages of development, transcripts are detected within the pronephros, the heart, within neural crest cells surrounding the eye, in the mandibular, hyoid and branchial arches, and within the tail.

Amino Acid Sequence↗

Expression of a cysteine-rich protein (CRP) encoding gene during early development of the trout.

Members of the cysteine-rich protein (CRP) define a subclass of LIM-only proteins implicated mainly in muscle differentiation. Until now, very little is known concerning the expression of CRP encoding genes during vertebrate development. We describe here the isolation of a trout (Oncorhynchus mykiss) gene encoding a cysteine-rich protein (TCRP) and the pattern of its mRNA accumulation during embryogenesis, focusing on somitogenesis. TCRP encodes a putative protein with two LIM domains linked to a short glycine-rich region that displays 86%, 76%, 67% identity with chicken CRP2, CRP1 and MLP/CRP3 proteins, respectively. Whole-mount in situ hybridisation showed that TCRP transcript is first detected just before somitogenesis in the paraxial mesoderm, while it is absent in the axial structures. During somitogenesis, the expression of TCRP was observed caudally in the elongating presomitic mesoderm and in the last formed somites. The labelling for TCRP was found to fade as the somites mature. At the end of the somitogenesis, TCRP transcripts accumulation was restricted to pronephros and bronchial arches.

Amino Acid Sequence↗

The Xenopus Ets transcription factor XER81 is a target of the FGF signaling pathway.

We report the cloning of a cDNA encoding a Xenopus laevis Ets-type transcription factor. This new Xenopus gene belongs to the PEA3 subfamily of Ets proteins and shows the highest degree of sequence similarity to the mouse and human ER81 genes. The Xenopus ER81 gene (XER81) is transcribed in the embryo after mid blastula transition (MBT) and three transcripts of 3, 4 and 6 kb are detected throughout embryogenesis. XER81 mRNA is localized in the animal pole of the late blastula stage and higher levels of XER81 transcripts are detected in the marginal zone at the onset of gastrulation. In later embryogenesis XER81 transcripts are found in neural crest cells, eyes, otic vesicles and pronephros. The transcription of XER81 can be stimulated by bFGF and eFGF in animal and vegetal cap explants. Expression of the dominant negative FGF receptor mutant in animal caps and embryos blocks XER81 transcription, arguing that the expression of this Ets gene requires active FGF signaling. The spatial overlap of eFGF and XER81 expression domains supports the idea that XER81 transcription could be a marker for regions with active FGF signaling in the embryo.

Amino Acid Sequence↗

The RNA-binding protein gene, hermes, is expressed at high levels in the developing heart.

In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif) family. The gene has been named hermes (for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and contains a single RRM domain. In situ hybridization analysis indicates that hermes is expressed at highest levels in the myocardium of the heart and to a lesser extent in the ganglion layer of the retina, the pronephros and epiphysis. Expression of hermes in each of these tissues begins at approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of the hermes orthologues from chicken and mouse reveals that, like Xenopus, the most prominent tissue of expression is the developing heart. The sequence and expression pattern of hermes suggests a role in post-transcriptional regulation of heart development.

Amino Acid Sequence↗

Expression pattern of insulin receptor mRNA during Xenopus laevis embryogenesis.

We have identified a member of the insulin receptor (InsR)/insulin-like growth factor-1 receptor (IGF-1R) family. The Xenopus insulin receptor (Xe-InsR) is present as a maternal 6.6 kb transcript. Northern blot analysis reveals the presence of this transcript until the mid-blastula transition (MBT), when levels decrease. At neurulation, two distinct transcripts of 6.6 and 7.7 kb are detected, both of which persist throughout embryogenesis. In situ hybridization analysis shows that InsR expression is restricted to regions of ectodermal and mesodermal origin, notably the encephalon, otic vesicles, optic vesicles, gills, somites and the pronephros.

Animals↗

Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development.

We have analyzed the expression and intracellular distribution, during oogenesis and embryogenesis, of Vg1 RBP, a protein implicated in the intracellular localization of Vg1 mRNA to the vegetal cortex of Xenopus oocytes. Vg1 RBP (protein) colocalizes with Vg1 RNA at all stages of oogenesis. Vg1 RBP RNA, however, localizes to the animal pole during late oogenesis, and remains in the animal blastomeres and ectodermal precursors until its zygotic transcription is activated, around stage 12. Vg1 RBP mRNA then becomes expressed throughout the neural epithelium. Vg1 RBP mRNA expression is also detected in what appears to be neural crest cells undergoing delamination and lateral migration. By tailbud stages, Vg1 RBP expression is present in the branchial arches, otic vesicle, pronephros, and along the neural tube. To examine the expression pattern in different species, we cloned the zebrafish homolog of Vg1 RBP by using a highly homologous EST clone to screen an embryonic cDNA library. In situ hybridization reveals that Vg1 RBP RNA localizes early in oogenesis to the animal pole. Although Vg1 RBP RNA is detected in all blastomeres of the early embryo, the expression pattern in the one day old zebrafish embryo is almost identical to that of the equivalent stage Xenopus embryo. These results indicate that the zygotic expression pattern is similar in frogs and fish, and that there is a conserved zygotic expression of Vg1 RBP distinct from its expression in the oocyte.

Amino Acid Sequence↗

Distinct promoter elements mediate endodermal and mesodermal expression of the HNF1alpha promoter in transgenic Xenopus.

The gene encoding the tissue specific transcription factor HNF1alpha is expressed in vertebrates in tissues of endodermal origin such as the liver and the gut as well as in the kidney, a mesoderm derived organ. Using a 6 kb HNF1alpha promoter fragment linked to GFP we observed green fluorescence in transgenic embryos restricted to the liver and gut as well as to the pronephros, the embryonic kidney. By deletion and mutation analysis of the HNF1alpha promoter we succeeded in dissecting the HNF1alpha promoter into two entities that are either active in the endoderm or the mesoderm. In conclusion, our data establish that the generation of transgenic Xenopus allows the functional dissection of promoters in the context of the entire organism.

Animals↗

Sequence and embryonic expression of deltaC in the zebrafish.

Four genes - deltaA, deltaB, deltaC and deltaD - coding for homologues of the Notch ligand Delta have been discovered in zebrafish (Haddon et al., 1998b). We report here the cDNA sequence and expression pattern of deltaC. Its closest relatives are deltaB and Xenopus X-Delta-2. Unlike deltaA, deltaB, and deltaD, deltaC is not expressed in the majority of nascent primary neurons; but it is strongly expressed in the early retina, where it precedes other delta genes. It is also expressed in cranial ganglia, in sensory epithelia including ear and lateral line, and in scattered epidermal cells. In the mesoderm, expression is visible by 50% epiboly; it is seen subsequently in the tail bud, in stripes in the presomitic mesoderm and in the posterior half of each somite. There is expression also in notochord, blood vessels and pronephros.

Animals↗

Differential expression of the Groucho-related genes 4 and 5 during early development of Xenopus laevis.

Recently, we demonstrated that the Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors (Roose et al., 1998. Nature 395, 608-612). A long form of the Groucho-related genes, XGrg-4, was shown to repress axis formation in the Xenopus embryo, whereas a short form, XGrg-5, acted as a potentiator. In this study, the temporal and spatial expression of XGrg-4 and XGrg-5 is described in Xenopus laevis embryos. Both genes are maternally expressed. In the gastrula, transcripts of both genes are present in the animal as well as the vegetal region. At later stages, XGrg-4 and XGrg-5 show specific patterns of expression in the central nervous system (CNS), cranial ganglia, eyes, otic vesicles, stomodeal-hypophyseal anlage, cement gland, head mesenchyme, branchial arches, neural crest and derivatives, somites, pronephros, pronephric duct, heart and tailbud. Differences in the expression of XGrg-4 and XGrg-5 were found in the CNS, cranial ganglia, olfactory placodes, stomodeal-pharyngeal anlage, cement gland, head mesenchyme and ectoderm.

Animals↗

Identification and developmental expression of par-6 gene in Xenopus laevis.

The par genes (partitioning defective) are required to establish polarity in the Caenorhabditis elegans embryo. We have identified the Xenopus homologue of C. elegans PAR-6 (XPAR-6). XPAR-6 is a protein of 377 amino acids with one PDZ domain which is involved in mediating protein-protein interactions. It shares 59% and 58% amino acid identity with the mouse and Drosophila PAR-6, respectively, and 54% overall identity with C. elegans PAR-6. Xpar-6 is expressed both maternally and zygotically. Xpar-6 is first detected in the animal half of the egg, and this pattern of expression persists into the cleavage and blastula stages. At the gastrula stage, the message is detected in animal pole area and in a broad domain of ventral region, but is excluded from dorsal region. With the onset of neurulation, the localized expression of Xpar-6 becomes more obvious, leading to it being enriched in the dorsolateral region along the lateral edges of neural plate and anterior presumptive head region surrounding the anterior border of neural plate. At late tailbud stage, Xpar-6 transcripts show localized expression throughout the head, labeling the branchial arches, eyes, otic vesicles and brain, while more posteriorly Xpar-6 labels the somites, pronephros, tail tip and proctodeum. Therefore, this analysis suggests that Xpar-6 has a regionalized pattern of expression during Xenopus early embryogenesis.

Amino Acid Sequence↗

Kidney morphogenesis: cellular and molecular regulation.

Development of an organ is directed by cell and tissue interactions and these also occur during the formation of functional kidney. During vertebrate development inductive signalling between mesenchyme and epithelium controls the organogenesis of all three kinds of kidneys: pronephros, mesonephros and metanephros. In higher animals the metanephros differentiates into the permanent kidney and in this review we will mainly concentrate on its development. Molecular interactions currently known to function during nephrogenesis have primarily been based on the use of knockout techniques. These studies have highlighted the role for transcription factors, signalling molecules, growth factors and their receptors and also for extracellular matrix components in kidney development. Finally in this review we will represent our own model for kidney development according to the knowledge of the genes involved in the development of the functional excretory organ, kidney.

Animals↗

Tissue generation from amphibian animal caps.

Formation of three germ layers is the most important event in early vertebrate development. Animal cap assays can be used to reproduce the in vivo induction of amphibian tissues in order to investigate the differentiation processes that occur in normal embryonic development. Activin treatment strongly and dose-dependently induces various types of mesodermal and endodermal tissue in cultured animal caps. Beating heart, pronephros, pancreas and cartilage can be induced by microsurgical manipulation and simultaneous treatment with activin and other factors. These in vitro induction systems will be helpful for elucidating the mechanisms of tissue induction and organ formation in vertebrate development.

Activins↗

Making a zebrafish kidney: a tale of two tubes.

The kidney can be thought of as the pairing of two tubes: an epithelial tube (the nephron), carrying filtered blood and engaged in ion and water transport; and endothelial tubes (the blood vessels), delivering blood and carrying away recovered solute. The development of the nephron presents several interesting questions. How does an epithelial tube form and how is it patterned into functionally distinct components and segments? What guides the interaction between the vasculature and kidney epithelia? How are epithelial cell shape and lumen diameter maintained, and what goes wrong when kidney tubules balloon into cysts? Here, I outline the progress that has been made in answering these questions using the zebrafish pronephros as a simple, accessible model of nephron development.

Animals↗

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry.

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM). Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.

Animals↗

Hormonal status and phagocytic activity in sea bream infected with vibriosis.

Serum taken from female, sexually mature, silver sea bream (Sparus sarba) displaying either no symptoms of vibriosis, mild infection, severe infection or moribundity were assayed for a number of key hormones. Serum cortisol levels were not significantly different among symptomless, mildly- and severely-infected groups, whereas moribund fish displayed hypercortisolemia with a 14-fold increase in serum cortisol in comparison to symptomless fish. Serum estradiol levels were significantly reduced 19-fold in mildly-infected fish and remained at a low level as infection progressed, whereas serum testosterone increased gradually during vibriosis with a 1.8-fold increase in moribund groups in comparison to symptomless groups. Both serum thyroxine (T(4)) and triiodothyronine (T(3)) gradually decreased during vibriosis being 26- and 2.8-fold lower, respectively, in moribund fish in comparison to symptomless fish. The non-specific immune response, as determined by phagocytic activity, was also assessed using macrophages isolated from the pronephros and spleen of infected fish. Phagocytic indices significantly increased in mildly- and severely-infected fish and then decreased from these stimulated levels in moribund fish.

Animals↗

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes.

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.

Acyltransferases↗