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Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development.

The presence of immunocompetent cells was studied in the larval and adult stages of the spotted wolffish, Anarhichas minor. In situ hybridization with a probe complementary to the secretory Igmu-chain was used to localize immunoglobulin producing cells or plasma cells in organs from adult fish and the appearance of these cells in lymphoid tissues during juvenile development. Plasma cells were located in pronephros, spleen, gut, gills and skin of adult wolffish. In juveniles, the first plasma cells were detected in the kidney 1 week post-hatching and the appearance in other lymphoid organs was in the order spleen, gut and thymus. No plasma cells were detected in skin and gills during the sampling period of juveniles (<10 cm). Our study confirmed that plasma cells are present in both the systemic and mucosal compartments of adult fish but during ontogeny there is an earlier appearance of plasma cells in the gut compared to gill and skin compartments.

Animals↗

Damselfish with neurofibromatosis exhibit cytotoxicity toward tumor targets.

Damselfish neurofibromatosis (DNF) is a malignant transmissible disease affecting Schwann cells, and is the only naturally occurring animal model of human neurofibromatosis type 1. The current study was designed to determine whether fish in the early stages of disease have measurable immune responses toward DNF tumor cells. Three DNF tumor cell lines were used as targets in standard 51Cr cytotoxic assays. In addition, Lutjanus griseus erythrocytes served as nonspecific targets, and concanavalon A (Con A) blasts from healthy animals served as normal target cells. Results of this study show that tumor-bearing damselfish have cells capable of destroying tumor targets but healthy animals display minimal, if any, reactivity toward the DNF tumor lines. The majority of antitumor activity resides in the spleen; the pronephros appears to contain the majority of nonspecific activity. Data also show that some of the effector cells are analogous to the nonspecific cytotoxic cells of catfish. No lysis of healthy damselfish targets was observed. Thus damselfish have cytotoxic cells capable of interacting with tumor targets, but in the majority of animals this response is not adequate to circumvent the process of tumorigenesis.

Animals↗

Characterization of a T cell lineage marker in carp (Cyprinus carpio L.).

A monoclonal antibody against thymocytes (WCL9; of IgG1 class) was produced by immunization of mice with isolated membrane molecules of carp thymocytes. Flow cytometric and fluorescence microscopic analysis showed that WCL9 was reactive with 30-50% of thymocytes and not with lymphoid cells from blood, pronephros, spleen and intestine. Cryo-sections of thymus showed a WCL9+ and WCL9- region in 3-month-old fish and only WCL9+ cells in 1-week-old fish. Because the WCL9- region is more medulla-like, the WCL9+ cells can be considered as cortical thymocytes. The majority of WCL9+ thymocytes appeared to have a higher density (1.06-1.07 g/mL) than the WCL9- cells (1.02-1.06 g/mL). Immunogold labelling or comparison of both density fractions did not show clear ultrastructural differences between WCL9+ and WCL9- thymocytes. The WCL9- fraction could be stimulated much better with PHA than the WCL9+ fraction. Removal of adherent cells or adding adherent accessory cells did not influence this result. Immunochemical analysis showed that WCL9 reacted with a protein determinant present on two molecules (M(r): 200 and 155 kDa) under reduced and non-reduced conditions. These results, together with the absence of WCL9+ cells in other lymphoid organs, strongly suggest that WCL9 is a specific marker for early thymocytes.

Animals↗

Expression of MhcCyca class I and class II molecules in the early life history of the common carp (Cyprinus carpio L.)

In this study transcription of class I alpha chain (Cyca-UA), beta2-microglobulin (Cyca-B2m) and class II alpha (Cyca-DXA) and beta (Cyca-DAB) during the early stages of embryo development was investigated by semiquantitative PCR. No transcripts of the genes under investigation were detected in the unfertilized egg. The expression of the genes encoding for the class II molecules revealed to be synchronized starting at day 1, unlike those for the class I molecules. Transcription of Cyca-B2m was first detected at day 7, whereas Cyca-UA was already present on day 1. This discrepancy would suggest absence of class I molecules during early development. The transcription of the Mhc genes in lymphoid organs was well established on day 21, with the exception of the spleen. In later stages of ontogeny cell surface expression of class I molecules was studied using polyclonal antibodies to Cyca-UA and Cyca-B2m in conjunction with detection of surface Ig. In week 3-10 Cyca-B2m was found on a higher percentage of cells from pronephros, spleen and thymus compared to Cyca-UA, suggesting the use of an alternative class I alpha chain. In the thymus, unlike the other organs, this difference remained present in the adult stage. The most likely candidates are alpha chains encoded by non-classical class I genes.

Animals↗

Isolation and characterization of Xenopus laevis aldolase B cDNA and expression patterns of aldolase A, B and C genes in adult tissues, oocytes and embryos of Xenopus laevis.

Following previous cloning and expression studies of Xenopus aldolase C (brain-type) and A (muscle-type) cDNAs, we cloned here two Xenopus aldolase B (liver-type) cDNAs (XALDB1 and XALDB2, 2447 and 1490 bp, respectively) using two different liver libraries. These cDNAs had very similar ORF with only one conservative amino acid substitution, but 3'-UTR of XALDB1 contained ca. 1 kb of unrelated reiterated sequence probably ligated during library construction as shown by genomic Southern blot analysis. In adult, aldolase B mRNA (ca. 1.8 kb) was expressed strongly in kidney, liver, stomach, intestine, moderately strongly in skin, and very weakly in all the other tissues including muscles and brain, which strongly express aldolase A and C mRNAs, respectively. In oocytes and early embryos, aldolase A and C mRNAs occurred abundantly as maternal mRNAs, but aldolase B mRNA occurred only at a residual level, and its strong expression started only after the late neurula stage, mainly in liver rudiment, pronephros, epidermis and proctodeum. Thus, active expression of the gene for aldolase B, involved in dietary fructose metabolism, starts only later during development (but before the feeding stage), albeit genes for aldolases A and C, involved in glycolysis, are expressed abundantly from early stages of embryogenesis, during which embryos develop depending on yolk as the only energy source.

Amino Acid Sequence↗

In vitro control of organogenesis and body patterning by activin during early amphibian development.

In the process of amphibian development, an embryonic body plan is established through cell division, sequential gene expression, morphogenesis and cell differentiation. The mechanism of body patterning is complex and includes multiple induction events. Activin, a TGF-beta family protein, can induce several kinds of mesodermal and endodermal tissues in animal cap explants in a dose-dependent manner. In a recent study of the role of activin in organogenesis, we succeeded in raising a beating heart by treating animal caps with a high concentration of activin. Activin also participates in kidney organogenesis in combination with retinoic acid. An embryonic kidney induced by activin and retinoic acid in vitro can function in vivo when it is transplanted into a larva in which pronephros rudiments have already been removed. Further, the activin-treated animal caps clearly show organizer actions that are closely related to body patterning along the anteroposterior axis. These experiments will help to serve as a model system for understanding organogenesis and body patterning at the cellular and molecular levels.

Activins↗

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes.

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19 degrees C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5 degrees C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A2 is involved in the heat shock response.

Animals↗

Sequence and expression of zebrafish foxc1a and foxc1b, encoding conserved forkhead/winged helix transcription factors.

Mouse Foxc1 (previously Mf1) is a member of the conserved forkhead/winged helix transcription factor gene family. It is expressed in many mesodermal tissues including paraxial mesoderm of the trunk and head, prechondrogenic mesenchyme, branchial arches and developing kidney. Homozygous mutants die perinatally with hydrocephalus and skeletal, cardiovascular, ocular and genitourinary defects. Here, we report the cloning and expression of two zebrafish foxc1 homologues, foxc1a and foxc1b. During gastrulation and somitogenesis both genes have similar expression patterns in the hypoblast, paraxial and presomitic mesoderm, somites and trunk adaxial cells. Expression in the somites is downregulated as they differentiate, but is maintained in the sclerotome. Later, some differences in expression pattern emerge. For example, only foxc1a transcripts are detected in the pronephros primodia and in the head mesoderm around the eyes, while only foxc1b is expressed in the pharyngeal arches and pectoral fins. Early expression of foxc1a in the paraxial mesoderm is modified in chordino, swirl, somitabun, and spadetail mutants.

Amino Acid Sequence↗

Molecular cloning and embryonic expression of Xenopus Six homeobox genes.

Six genes are vertebrate homologues of the homeobox-containing gene sine oculis, which plays an essential role in controlling Drosophila compound eye development. Here we report the identification and expression patterns of all three subfamilies of Xenopus Six genes. Two Six2 subfamily genes (Six1, Six2) showed very similar expression patterns in cranial ganglia, otic placodes and the eyes. Non-neural expression of Six1 and Six2 was observed with mesodermal head mesenchyme, somites and their derivatives, the muscle anlagen of the embryonic trunk. In addition, Six2 expression was also found with mesenchyme associated with the developing stomach and pronephros. Expression of Six3 subfamily genes (Six3.1, Six3.2, Six6.1, and Six6.2) was restricted to the developing head, where expression was especially observed in derivatives of the forebrain (eyes, optic stalks, the hypothalamus and pituitary gland). Interestingly, expression of all Six3 subfamily members but Six6.2 was also found with the pineal gland primordium and the tegmentum. Expression of Six4 subfamily genes (Six4.1, Six4.2) was present in the developing visceral arches, placodal derivatives (otic vesicle, olfactory system), head mesenchyme and the eye. The observed dynamic expression patterns are largely conserved between lower and higher vertebrates and imply important roles of Six family genes not only in eye formation and myogenesis, but also in the development of the gut, the kidney and of placode-derived structures.

Alleles↗

Quantitative expression studies of aldolase A, B and C genes in developing embryos and adult tissues of Xenopus laevis.

We previously cloned cDNAs for all the members (A, B and C) of Xenopus aldolase gene family, and using in vitro transcribed RNAs as references, performed quantitative studies of the expression of three aldolase mRNAs in embryos and adult tissues. A Xenopus egg contains ca. 60 pg aldolase A mRNA and ca. 45 pg aldolase C mRNA, but contains only ca. 1.5 pg aldolase B mRNA. The percent composition of three aldolase mRNAs (A:B:C) changes from 56:1.5:42.5 (fertilized egg) to 54:10:36 (gastrula), to 71:14.5:14.5 (neurula) and to 73:20:7 (tadpole) during development. These results are compatible with the previous results of zymogram analysis that aldolases A and C are the major aldolases in early embryos, whose development proceeds depending on yolk as the only energy source. Aldolase B mRNA is expressed only late in development in tissues such as pronephros, liver rudiment and proctodeum which are necessary for the future dietary fructose metabolism, and the expression pattern is consistent to that in adult tissues. We also show that three aldolase genes are localized on different chromosomes as single copy genes.

Animals↗

Xenopus Eya1 demarcates all neurogenic placodes as well as migrating hypaxial muscle precursors.

We cloned two isoforms of the Xenopus Eya1 orthologue. They show identical patterns of expression that closely resemble the previously described expression of XSix1, but partly differ from the expression of Eya1 in other vertebrates. XEya1 is expressed in the somites and hypaxial muscle precursors, but not in the pronephros. Moreover, all ectodermal placodes except the lens placode strongly express XEya1. At neural plate stages, ectodermal XEya1 expression starts in two domains, the anterior neural folds and a domain lateral to the neural folds. At tailbud stages, XEya1 expression continues in the adenohypophysis, all neurogenic placodes and placodally-derived structures including cranial ganglia, the otic vesicle and lateral line primordia.

Amino Acid Sequence↗

Molecular cloning and embryonic expression of the Xenopus Arnt gene.

In this paper, we report the cloning of a Xenopus bHLH/PAS factor homologous to the mammalian aryl hydrocarbon receptor nuclear translocator (Arnt) or Drosophila Tango gene. Sequence data analysis indicates that protein domains organization in xArnt is strongly conserved and that xArnt is highly related to the mammalian Arnt1 isoform. As revealed by reverse transcriptase polymerase chain reaction and whole-mount in situ hybridization, xArnt gene is expressed during early and late development. At early stages, xArnt transcripts are restricted to the ectoderm and extends to the marginal zone at gastrula stage. In tail bud embryo, xArnt is strongly expressed in branchial arches, optical and optical vesicles, and pronephros and pronephritic duct.

Amino Acid Sequence↗

Tissue-specific expression of the Ets gene Xsap-1 during Xenopus laevis development.

We report the cloning of Xenopus laevis Xsap-1 cDNA, encoding a member of the ternary complex factor subfamily of ETS transcription factors. The expression pattern of Xsap-1 was examined during Xenopus embryogenesis using whole-mount in situ hybridization. Spatial expression of Xsap-1 mRNA is first detected at the animal pole at the mid-blastula stage. During neurulation Xsap-1 is expressed in cells participating in neural tube formation, in the sensorial layer of the epidermal ectoderm, and in an anterior region of the ventral mesoderm. Later, Xsap-1 expression is observed in the eye, ear vesicle, branchial arches, heart, pronephros, in the somites, and the developing nervous system, such as fore-, mid-, and hindbrain as well as in the cranial ganglion X.

Amino Acid Sequence↗

Degradation of hyaluronan by a Hyal2-type hyaluronidase affects pattern formation of vitelline vessels during embryogenesis of Xenopus laevis.

A Hyal2-type hyaluronidase of Xenopus laevis (Xhyal2) was characterized by molecular cloning, biochemical analysis and ectopic overexpression in embryos. When expressed in Xenopus oocytes, Xhyal2 exists as a soluble protein in the extracellular space and in intercellular compartments as well as being attached to the cell surface through a glycosyl-phosphatidyl-inositol anchor. This enzyme specifically degrades hyaluronan not only at acidic pH values but more slowly also under physiological conditions. Xhyal2 is differentially expressed during embryogenesis. Particularly striking is the high level of expression in the developing brain, the head mesenchyme and the pronephros. Elevated levels of mRNA were also found in endothelial cells which will later form vascular structures. Ectopic overexpression of Xhyal2 in frog embryos causes loss of hyaluronan in the cellular environment. This causes severe defects in the assembly of the highly structured plexus of the vitelline vessels from prevascular endothelial cells. Our data support the notion that the level of Xhyal2 expression determines the organization of the extracellular environment so that cells can merge and/or migrate within an originally impenetrable matrix.

Amino Acid Sequence↗

Isolation of three zebrafish dachshund homologues and their expression in sensory organs, the central nervous system and pectoral fin buds.

Drosophila dachshund (dac) interacts with sine oculis (so), eyes absent (eya) and eyeless (ey) to control compound eye development. We have cloned three zebrafish dac homologues, dachA, dachB and dachC, which are expressed widely, in distinct but overlapping patterns. Expression of all three is found in sensory organs, the central nervous system and pectoral fin buds. dachA is also expressed strongly in the somites and dachC in the neural crest and pronephros. These expression domains overlap extensively with those of zebrafish pax, eya and six family members, the homologues of Drosophila ey, eya and so, respectively. This is consistent with the proposal that Dach, Eya, Six and Pax family members may form networks, similar to that found in the fly eye, in the development of many vertebrate organs.

Amino Acid Sequence↗

CAP1 expression is developmentally regulated in Xenopus.

We have cloned and characterized a Xenopus member of the cyclase associated protein (CAP) gene family. xCAP1 is expressed as a maternal transcript, but is up-regulated prior to gastrulation and subsequently localizes to head mesenchyme, lens, otic vesicle, and trunk mesoderm including the pronephros. At different stages, the gene also appears to differentiate surface from deep (sensorial) ectoderm. As in Drosophila, Xenopus CAP1 is expressed in the developing eye, specifically in the differentiating lens. However, in distinction to Drosophila, Xenopus CAP1 does not express in periodically arrayed neural bands.

Animals↗

Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos.

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

Amino Acid Sequence↗

Gene expression pattern analysis of the tight junction protein, Claudin, in the early morphogenesis of Xenopus embryos.

To study how epithelial layers are formed during early development in Xenopus embryos, we have focused on Claudin, the major component of the tight junction. So far, 19 claudin genes have been found in the mouse, expressed in different epithelial tissues. However, though a number of cytological studies have been done for the roles of Claudins, their expression patterns and functions during early embryogenesis are largely unknown. We found three novel Xenopus claudin genes, which are referred to as claudin-4L1, -4L2, and -7L1. At the early gastrula stage, claudin-4L1, -4L2, and -7L1 mRNAs were detected in the ectoderm and in the mesoderm. At the late gastrula stage, claudin mRNAs were detected in the ectoderm through the involuting archenteron roof. At the neurula stage, claudin-4L1/4L2 and -7L1 mRNAs were differentially expressed in the neural groove and the epidermal ectoderm. At the tailbud stage, the claudin mRNAs were found in the branchial arches, the otic vesicles, the sensorial layer of the epidermis, and along the dorsal midline of the neural tube. In addition, claudin-4L1/4L2 mRNAs were detected in the pronephros and the endoderm, whereas claudin-7L1 mRNA was observed in the epithelial layer of the epidermis.

Amino Acid Sequence↗