Two isoforms of peroxiredoxin 6 of <Emphasis Type="Italic">Xenopus laevis</Emphasis>

The genome of Xenopus laevis codes for two genes of peroxiredoxin 6, i.e., xen1 (Acc. no. EMBL Data Bank-BCO54278) and xen2 (Acc. no. EMBL Data Bank-BCO54309). Both the genes were cloned and expressed in Escherichia coli. The amino acid sequences of Xen1 and Xen2 enzymes are identical by 95%, and they possess the same peroxidase activity as well as similar optimums of temperature, pH, and thermostability. The genes of peroxiredoxin 6 of Xenopus laevis considerably differ in the period of expression during ontogenesis; i.e., xen2 is expressed during every stage of development, somewhat more intensively after stages 0–5; the expression of xen1 is initiated later, i.e., during the developmental stages of 47–48 h. Expression of xen2 increases after the incubation of embryos in a medium with hydrogen peroxide. Comparison of the amino acid sequences of Xen1 and Xen2 proteins shows that only Xen2 can possess phospholipase activity because its amino acid sequence contain residues of the phospholipase A2 active center: Ser31, His25, and Asp139.     

Cement gland as the adhesion organ in Xenopus laevis embryos

The cement gland in batrachians is a temporal ectodermic organ which is necessary for an embryo's attachment to the substrate. In this review, some notions about the origin of the cement gland of Xenopus laevis frogs, its functioning, genes being expressed in it, and regulation of its formation and development are provided. The role of some homologies of agrgenes of the cement gland in Xenopus laevis is noted at different conditions of other animals and man.     

Studies on <Emphasis Type="Italic">Xenopus laevis</Emphasis>intestine reveal biological pathways underlying vertebrate gut adaptation from embryo to adult

Background  

To adapt to its changing dietary environment, the digestive tract is extensively remodeled from the embryo to the adult during vertebrate development. Xenopus laevis metamorphosis is an excellent model system for studying mammalian gastrointestinal development and is used to determine the genes and signaling programs essential for intestinal development and maturation.     

Duplication of the MHC-linked Xenopus complement factor B gene

We have previously reported the molecular cloning of the mammalian major histocompatibility complex (MHC) class III gene, complement factor B (Bf) from Xenopus laevis, and linkage of the gene to the frog MHC. Here, we estimated the copy number of the Xenopus Bf gene by genomic Southern blotting analysis and demonstrated that Xenopus laevis has two copies of the Bf gene. Both genes co-segregated with the MHC-linked HSP70 genes among 19 offspring of an f/r × f/r cross, indicating a close linkage of the two Bf genes to the frog MHC. Both genes are transcribed and contain open reading frames. When compared with the previously determined cDNA sequence (Xenopus Bf A), the predicted amino acid sequence of the second cDNA species (Xenopus Bf B) shows 82% overall identity. Polymerase chain reaction analysis indicated that all of the partially inbred frogs with the f, r, g, and j MHC haplotypes, as well as 12 outbred frogs tested have both Bf genes, suggesting that the duplicated Bf genes are stable genetic traits in Xenopus laevis.     

Histological development of the cement gland in Xenopus laevis: a light microscopic study

The differentiation and degeneration of the cement gland in Xenopus laevis is described. The gland is first observed histologically at stage 19 (neural tube stage) as a packed group of apical ectoderm cells heavily laden with oocyte pigment granules, lying ventral to the cranial neural fold. By tailbud stage 35/36, the gland cells have increased in height and are approximately ten times taller than nonglandular apical ectoderm cells. The nuclei divide the gland cells into an apical region that is eosinophilic and contains oocyte pigment granules, and a basal region that contains clear droplets. The cells are decreasing in height by stage 40 (early tadpole) and begin to lose their pigment granules. Between stages 45 and 48, the pigment is extruded and the clear basal droplets diminish in number. From stage 48 to 49 the cells become vacuolated and the histotypic characteristics of the functional gland are lost. The gland is not vascularized, nor do phagocytic cells appear in its vicinity during any stage of its development. It remains bordered at its base by subjacent basal ectoderm during its entire life cycle of 10 to 12 days at room temperature.     

Protein synthesis in interspecies hybrid embryos of the amphibian Xenopus

The newly synthesized abundant proteins of early Xenopus laevis and Xenopus borealis embryos have been examined by two-dimensional electrophoresis after labelling with [³⁵S]methionine. Six prominent polypeptides specific to Xenopus laevis embryos and a further six specific to Xenopus borealis have been identified. Overall, embryos of the two species are estimated to differ by approx. 15% in their protein synthetic patterns from blastula to tailbud stage. Interspecific hybrid embryos (Xenopus laevis (♀)/Xenopus borealis (♂)) synthesise only the maternally specified set of proteins until gastrulation after which they produce the full complement of both Xenopus laevis and Xenopus borealis specific proteins. The possible use of such molecular markers of parental genome activity in facilitating further embryological study is discussed.     

An immunocytochemical method for the visualization of tubulin-containing structures in the egg of Xenopus laevis

Summary Tubulin can be isolated and purified from Xenopus laevis egges through modification of Olmstedt's (1970) tubulin isolation method, viz. by repeating the vinblastin precipitation step after resuspension of the sediment in a detergent-containing stabilizing medium. By this we overcome the deleterious influence of the yolk granules in the isolation procedure. From 1 l of Xenopus laevis eggs 25 mg VB-paracrystals can be obtained. The apparent molecular weight of the purified tubulin is 52,800. Antiserum against the purified Xenopus VB-paracrystals, raised in 2 Chinchilla rabbits, cross-reacts in immunodiffusion tests in agar gels with rat brain tubulin and with tubulin isolated from Xenopus laevis eggs by the described procedure. Specific indirect fluorescence staining and appropriate control reactions reveal that cilia of Tetrahymena pyriformis, cytoplasmic networks in cultured mouse Leydig cells, as well as mitotic spindles and nuclear regions in paraffin sections of Xenopus laevis blastulae, react with the antibodies against Xenopus laevis egg tubulin as well as with monoclonal antibodies against pig brain tubulin.These results provide additional evidence for the view that tubulin antibodies are neither species nor tissue specific and show that under appropriate conditions tubulin containing structures can be visualized in paraffin sections.     

An ontology for <Emphasis Type="Italic">Xenopus</Emphasis> anatomy and development

Background  

The frogs Xenopus laevis and Xenopus (Silurana) tropicalis are model systems that have produced a wealth of genetic, genomic, and developmental information. Xenbase is a model organism database that provides centralized access to this information, including gene function data from high-throughput screens and the scientific literature. A controlled, structured vocabulary for Xenopus anatomy and development is essential for organizing these data.     

Differential regulation of two histidine ammonia-lyase genes during <Emphasis Type="Italic">Xenopus</Emphasis> development implicates distinct functions during thyroid hormone-induced formation of adult stem cells

Background

Organ-specific, adult stem cells are essential for organ-homeostasis and tissue repair and regeneration. The formation of such stem cells during vertebrate development remains to be investigated. Frog metamorphosis offers an excellent opportunity to study the formation of adult stem cells as this process involves essentially the transformations of all larval tissues/organs into the adult form. Of particular interest is the remodeling of the intestine. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. A major advantage of this metamorphosis model is its total dependence on thyroid hormone (T3). In an effort to identify genes that are important for stem cell development, we have previously carried out tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis.

Results

We report the detailed characterization of one of the genes thus identified, the histidine ammonia-lyase (HAL) gene, which encodes an enzyme known as histidase or histidinase. We show that there are two duplicated HAL genes, HAL1 and HAL2, in both Xenopus laevis and Xenopus tropicalis, a highly related but diploid species. Interestingly, only HAL2 is highly upregulated by T3 and appears to be specifically expressed in the adult intestinal progenitor/stem cells while HAL1 is not expressed in the intestine during metamorphosis. Furthermore, when analyzed in whole animals, HAL1 appears to be expressed only during embryogenesis but not metamorphosis while the opposite appears to be true for HAL2.

Conclusions

Our results suggest that the duplicated HAL genes have distinct functions with HAL2 likely involved in the formation and/or proliferation of the adult stem cells during metamorphosis.

     

Bromodeoxyuridine-immunohistochemistry on cellular differentiation and migration in the fundic gland of Xenopus laevis during development

Summary Cellular differentiation and migration in the fundic glands of adult and larval Xenopus laevis have been examined using bromodeoxyuridine-immunohistochemistry. In the adult fundic gland, cumulative labeling with bromodeoxyuridine revealed a proliferative cell zone between the surface mucous cells and mucous neck cells, in what is referred to as the neck portion of the gland. The labeling-index of mucous neck cells had rapidly increased by week-5. The labeling-index of oxynticopeptic cells showed a more delayed increase until week-7, coincident with the decrease in the labeling of mucous neck cells. In the immature fundic glands of larvae, the labeled proliferating cells were randomly distributed throughout the developing gastric mucosa. During metamorphosis, the labeling-index of immature epithelial cells was highest at stage 63. Following administration of bromodeoxyurdine at this, stage, there was no significant loss of labeled epithelial cells during the metamorphosing period. Furthermore, there was no significant difference in the labeling-indices among the epithelial cells, such as surface mucous cells/generative cells, mucous neck cells, and oxynticopeptic cells, 7 days after administration. Cellular differentiation and migration pathways of epithelial cells in the fundic gland of adult X. laevis and its larvae are discussed.     

Spatial pattern of constitutive and heat shock‐induced expression of the small heat shock protein gene family,hsp30, in Xenopus laevis tailbud embryos

We employed whole‐mount in situ hybridization and immunohistochemistry to study the spatial pattern of hsp30 gene expression in normal and heatshocked embryos during Xenopus laevis development. Our findings revealed that hsp30 mRNA accumulation was present constitutively only in the cement gland of early and midtailbud embryos, while hsp30 protein was detected until at least the early tadpole stage. Heat shock‐induced accumulation of hsp30 mRNA and protein was first observed in early and midtailbud embryos with preferential enrichment in the cement gland, somitic region, lens placode, and proctodeum. In contrast, cytoskeletal actin mRNA displayed a more generalized pattern of accumulation which did not change following heat shock. In heat shocked midtailbud embryos the enrichment of hsp30 mRNA in lens placode and somitic region was first detectable after 15 min of a 33°C heatshock. The lowest temperature capable of inducing this pattern was 30°C. Placement of embryos at 22°C following a 1‐h 33°C heat shock resulted in decreased hsp30 mRNA in all regions with time, although enhanced hsp30 mRNA accumulation still persisted in the cement gland after 11 h compared to control. In late tailbud embryos the basic midtailbud pattern of hsp30 mRNA accumulation was enhanced with additional localization to the spinal cord as well as enrichment across the embryo surface. These studies demonstrate that hsp30 gene expression can be detected constitutively in the cement gland of tailbud embryos and that heat shock results in a preferential accumulation of hsp30 mRNA and protein in certain tissues. Dev. Genet. 25:365–374, 1999. © 1999 Wiley‐Liss, Inc.     

Specific cleavage of beta-amyloid peptides by a metallopeptidase from Xenopus laevis skin secretions

Dactylysin (EC 3.5.24.60) is a metalloendopeptidase first isolated from the skin granular gland secretions of Xenopus laevis. This peptidase hydrolyzes bonds on the amino-terminus of singlets and between doublets of hydrophobic amino acids and was considered to play a role in the in vivo inactivation of biologically active regulatory peptides. Here, we show that dactylysin has also the ability to cleave human β[1-40]-amyloid peptide and related peptides. Cleavage of the wild type β[1-40]-amyloid peptide form, and to a lesser extent Flemish and Dutch mutants, occurred predominantly at the His¹⁴-Glu¹⁵ bond. We demonstrate that frog skin exudate contains a full-length amyloid protein precursor detected by immunochemical cross-reactivity with monoclonal antibody against C-terminal human amyloid protein precursor. The possibility that dactylysin, might be involved in normal catabolism of β amyloid peptide of Xenopus laevis is discussed.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

The Xenopus FcR family demonstrates continually high diversification of paired receptors in vertebrate evolution

Background  

Recent studies have revealed an unexpected diversity of domain architecture among FcR-like receptors that presumably fulfill regulatory functions in the immune system. Different species of mammals, as well as chicken and catfish have been found to possess strikingly different sets of these receptors. To better understand the evolutionary history of paired receptors, we extended the study of FcR-like genes in amphibian representatives Xenopus tropicalis and Xenopus laevis.     

Xenopus Xotx2 and Drosophila otd share similar activities in anterior patterning of the frog embryo

Despite the obvious anatomical differences between the fly and the vertebrate body plans, several genes involved in their development are largely conserved. In this work we provide evidence that overexpression of the Drosophila orthodenticle (otd) gene in Xenopus laevis has a similar effect to that of its homolog Xotx2. Injections of otd mRNA in whole embryos lead to posterior truncations and to induction of ectopic cement glands, similar to Xotx2 injections. In animal cap assays, otd, like Xotx2, is able to activate the cement gland marker XAG and to suppress the expression of the epidermal marker XK81. Finally, as assayed by Einsteck transplantation assays, otd, like Xotx2, is able to respecify a tail/trunk organizer to a head organizer. In this work we also show that Xotx2 and otd share molecular functions that regulate early regional specification of the Xenopus anterior neural plate. Gain-of-function experiment targeting low doses of either otd or Xotx2 mRNAs in the neural plate promote reduction of Xrx1 and Xbf1 expression domain; no changes are observed for the anterior mesodermal marker Xgsc, the dorsal diencephalic marker Xbh1, and the midbrain/hindbrain marker Xen2. otd/Xotx2 inhibition activity of Xrx1 and Xbf1 expression is consistent with the strong inhibition of Xfgf8 expression in the anterior neural ridge observed upon otd/Xotx2 mRNA injection.     

Distinct axonal projections from two types of olfactory receptor neurons in the middle chamber epithelium of <Emphasis Type="BoldItalic">Xenopus laevis</Emphasis>

Most vertebrates have two olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ. African clawed frog, Xenopus laevis, which spends their entire life in water, have three types of olfactory sensory epithelia: the OE, the middle chamber epithelium (MCE) and the vomeronasal epithelium (VNE). The axons from these epithelia project to the dorsal part of the main olfactory bulb (d-MOB), the ventral part of the MOB (v-MOB) and the accessory olfactory bulb, respectively. In the MCE, which is thought to function in water, two types of receptor neurons (RNs) are intermingled and express one of two types of G-proteins, Golf and Go, respectively. However, axonal projections from these RNs to the v-MOB are not fully understood. In this study, we examined the expression of G-proteins by immunohistochemistry to reveal the projection pattern of olfactory RNs of Xenopus laevis, especially those in the MCE. The somata of Golf- and Go-positive RNs were separately situated in the upper and lower layers of the MCE. The former were equipped with cilia and the latter with microvilli on their apical surface. These RNs are suggested to project to the rostromedial and the caudolateral regions of the v-MOB, respectively. Such segregation patterns observed in the MCE and v-MOB are also present in the OE and olfactory bulbs of most bony fish. Thus, Xenopus laevis is a very interesting model to understand the evolution of vertebrate olfactory systems because they have a primitive, fish-type olfactory system in addition to the mammalian-type olfactory system.