Nuclear transplantations between two subspecies of Xenopus laevis, (Xenopus laevis laevis and Xenopus laevis petersi).
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Factors affecting oogenesis in the South African clawed frog (Xenopus laevis).
Xenopus laevis, commonly known as the South African Clawed frog, is a hardy adaptable species that is relatively easy to maintain as a laboratory animal. Gametogenesis in wild Xenopus laevis is continuous and under ideal conditions, reproduction can occur year round. This unique aspect of amphibian reproduction offers an advantage over mammalian model systems: the eggs and oocytes collected from laboratory maintained Xenopus laevis provide an abundant and readily obtainable supply of material for cellular and biological research. However, many investigators report that laboratory Xenopus laevis go through periods of unexplained inefficient or complete failure of oocyte production or the production of poor quality oocytes. This results in experimental delays, inability to reproduce data, and ultimately the use of more animals. There is a lack of evidenced based information regarding the housing conditions that are necessary to optimize the health and fecundity of this species in captivity, but studies of wild Xenopus laevis have shown that temperature, age of the female, and nutrition are of key importance. The objective of this report is to review oogenesis with a special emphasis on these factors as they pertain to laboratory Xenopus laevis maintained for the purpose of providing a steady supply of eggs and oocytes. Harvesting methods and other experimental techniques that affect the quality of eggs and oocytes are also discussed.
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RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis.
Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.
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Oestrogen control of the sexual dimorphism in the Harderian gland of Xenopus laevis.
Xenopus laevis shows a sexual dimorphism of the electrophoretic pattern of Harderian gland (HG) proteins. The male pattern displays three protein fractions whose molecular sizes are approx. 205, 180 and 78 kDa, respectively, and which are absent in the female pattern. Conversely, the female pattern displays two protein fractions of approx. 190 and 76 kDa, respectively. This sexual dimorphism led us to hypothesize a sex steroid control of the HG. Administration of 17beta-oestradiol to male Xenopus converts the male protein pattern into the female one, while the administration of testosterone to the female has no effect. In this respect neither Northern analysis nor the RNase-protection assay performed using a 213 bp encoding for the androgen-binding domain reveals the presence of an androgen receptor mRNA in Xenopus HG. Conversely, Northern analysis has shown an oestrogen receptor mRNA whose size is approx. 6.5 kb and the RNase-protection assay performed by using a 197 bp encoding for the oestrogen-binding domain has also displayed the presence of an oestrogen receptor mRNA in the female HG but not in the male one. In addition, the oestrogen administration to male Xenopus induces the appearance of an oestrogen receptor mRNA. Androgen administration to female toad is ineffective. Taken together, all these findings suggest that in Xenopus laevis oestrogens are involved into the HG physiology. The appearance of an oestrogen receptor mRNA in the oestradiol treated males supports the hypothesis of the occurrence of autoinduction of oestrogen receptor mRNA expression in the HG.
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Expression patterns of glycoconjugates in the three distinctive olfactory pathways of the clawed frog, Xenopus laevis.
Xenopus laevis has three distinctive olfactory neuroepithelia. We examined the axonal projection from each of these epithelia to the olfactory bulb by Di-I labeling, and confirmed that the Xenopus primary olfactory pathways involve the dorsal pathway from the olfactory epithelium to the dorsal region of the main olfactory bulb, the ventral pathway from the middle chamber epithelium to the ventral region of the main olfactory bulb, and the vomeronasal pathway from the vomeronasal epithelium to the accessory olfactory bulb. We next examined expression patterns of glycoconjugates in the three olfactory pathways by lectin-histochemistry using 21 biotinylated lectins. Fourteen out of 21 lectins stained the Xenopus primary olfactory system. RCA-I stained the three olfactory pathways uniformly. PHA-E stained only the dorsal pathway. LEL, STL, PNA, ECL and UEA-I stained the dorsal pathway more intensely than the ventral pathway, and among them, only UEA-I stained the vomeronasal pathway. In contrast, s-WGA, DBA, SBA, BSL-I VVA, SJA and PHA-L showed intense stainings in the ventral pathway and moderate stainings in the vomeronasal pathway, but faint or weak stainings in the dorsal pathway. These observations suggest that the ventral pathway expresses glycoconjugates shared commonly with either the dorsal or the vomeronasal pathway. In addition, from the binding patterns of the lectins with a binding specificity for N-acetylgalactosamine, glycoconjugates containing this saccharide seem to play an important role for the organization of the olfactory pathways.
Affinity Labels↗
Development of the interphotoreceptor matrix in Xenopus laevis.
Xenopus laevis interphotoreceptor matrix (IPM) contains a relatively aqueous insoluble wheat germ agglutinin (WGA)-binding component containing unidentified sialoglycoconjugates (Wood et al [1984] J. Comp. Neurol. 228:299-307). The appearance of WGA-binding macromolecules in the IPM was assessed during late embryonic stages (32-45) and in retinal rudiment cultures, using lectin cytochemistry and Western blotting techniques. Metabolic labeling of the neural retina versus retinal pigment epithelium (RPE)-choroid of juvenile Xenopus with 35S-MET was also evaluated in vivo and in vitro. Lectin cytochemistry of eyes from developmental stages 32-42 demonstrated distinct WGA-ferritin-binding sites on the developing outer segment membranes and in the IPM compartment. At stages 44-46 extensive WGA-binding domains were present as an extracellular network with other randomly scattered domains near the retinal pigment epithelium. Retinal rudiments from stage 32-33 were isolated and allowed to differentiate in hanging drop culture (Hollyfield and Witkowsky [1974] J. Exp. Zool. 189:357-377) with or without an investing pigment epithelium. Cultures developing with RPE exhibited an elaborate IPM with an anastomosing meshwork of WGA-ferritin binding sites. In the absence of RPE only limited amounts of binding restricted to the immediate vicinity of the developing photoreceptor outer segment membranes was observed. When Western blots were probed with WGA-HRP, stage 32-45 retinas demonstrated a major WGA-binding band of 126 kD. Similar amounts of WGA-binding macromolecules were synthesized in preparations cultured in the presence or absence of the investing RPE. During development the major WGA-binding component is a 126-kD protein. Equivalent synthesis of this protein in the presence and absence of RPE suggests that the PE is not required for synthesis of this 126-kD component. These results suggest that the retina is the primary site of synthesis of the WGA-binding components of the Xenopus IPM, whereas the PE plays a principal role in their assembly and organization.
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Confocal imaging of early heart development in Xenopus laevis.
Xenopus laevis provides a number of advantages to studies on cardiovascular development. The embryos are fairly large, are easy to obtain, and can develop at ambient temperature in simple buffer solutions. Although classic descriptions of heart development exist, the ability to use whole-mount immunohistochemical methods and confocal microscopy may enhance the ability to understand both normal and experimentally perturbed cardiovascular development. We have started to examine the early stages of cardiac development in Xenopus, seeking to identify antibodies and fixatives that allow easy examination of the developing heart. We have used monoclonal antibodies (mAbs) raised against bovine cardiac troponin T and chicken tropomyosin to visualize cardiac muscle, a goat antibody recognizing bovine type VI collagen to stain the lining of vessels, and the JB3 mAb raised against chicken fibrillin, which allows the visualization of a variety of cardiovascular tissues during early development. Results from embryonic stages 24-46 are presented.
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Aerial respiration facilitates growth in suspension-feeding anuran larvae (Xenopus laevis).
Xenopus laevis tadpoles are midwater suspension-feeders that use buccopharyngeal surfaces for both food capture and aquatic respiration, but also have functioning lungs well before metamorphosis. To examine the effect of aerial respiration on premetamorphic growth and development in this species, tadpoles were raised in normoxic water for from one to three weeks at three different concentrations of food (yeast cells in suspension). At each food concentration the larvae were either allowed or denied access to air. Xenopus larvae reached greater snout-vent lengths, weighed more and developed more rapidly when allowed to breathe air. The difference in growth and development between those tadpoles with and without access to air was proportional to the concentration of particulate matter. In the extreme, tadpoles denied access to air at the highest food concentration all asphyxiated shortly after the start of the experiment. Xenopus tadpoles faced with the functional conflict of using buccopharyngeal surfaces for both feeding and respiration retard ingestion. They do this, however, not by ceasing mucus outflow to their branchial food traps, as has been speculated previously, but rather by capturing food particles in mucus and then expectorating it. Lungs appear to be advantageous to aquatic organisms even in normoxic water in that they allow buccopharyngeal surfaces to be dedicated fully to feeding rather than respiration.
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Ontogeny of the alloimmune response against a transplanted tumor in Xenopus laevis.
Xenopus laevis lymphoid tumor cells of the ff genotype grow after transplantation in inbred ff tadpoles or young post-metamorphic animals, but do not grow in fully grown ff adults. The ability to grow is lost progressively after metamorphosis and is apparently due to an immune response of the adult host against minor histocompatibility antigens (non-MHC encoded) expressed by the tumor cells. The difference in alloimmune responses between the larval and the adult immune system of the amphibian Xenopus has been subsequently investigated with this new in vivo model. The resistance of the host against transplanted tumor cells rises during the post-metamorphic development in parallel with the second histogenesis observed in the thymus, the expression of MHC class II by peripheral T cells and the recovery of T cell effector functions such as MLR, and can be abrogated by sub-lethal irradiation. Pre-immunization of ff adults with irradiated ff-2 cells specifically accelerates subsequent ff skin graft rejection, which implies the generation of memory against antigenic determinants common between the ff skin and the tumor cells. Similarly, both anti-ff alloserum and anti-ff-2 serum contain antibodies specifically precipitating two surface proteins (180-200 kDa) from ff-2 cells. One of these proteins is also detected on normal ff thymocytes and splenic T cells. On the other hands, ff-2 tumor cells (MHC I+II-) are not rejected by class I-negative tadpoles (class I expression on the tumor cell surface is even increased), and no anti-tumor antibody response can be detected. However, tumor growth has been reduced in tadpoles following priming with irradiated ff-2 cells, although immunization is not sufficient to prevent ultimate tumor development and tadpole death. Moreover, priming with irradiated ff-2 cells at larval stages does interfere with tumor growth in transplanted young post-metamorphic adults, suggesting that long-lived memory has been generated and has been maintained through metamorphosis. These results suggest that the lack of tumor rejection by larvae results from an incomplete effector function rather than an absence of recognition. Full responsiveness against minor H antigens cannot be elicited before adulthood.
Age Factors↗
Coordinate transcriptional regulation of the three fibrinogen subunit genes by glucocorticoids in cultured primary liver cells from Xenopus laevis.
Xenopus laevis primary hepatocytes in culture are induced by glucocorticoid hormones to synthesize and secrete fibrinogen. The increase in production of the protein is preceded by a 10-to 30-fold elevation of the mRNAs coding for the three fibrinogen subunits, A alpha, B beta, and gamma. To analyze the mechanisms underlying this coordinate control of independent genes in a common regulatory network, we show here that the steroid hormone induced simultaneous activation of transcription of the three fibrinogen subunit genes. Using an optimized transcription run-on assay for nuclei from Xenopus primary liver cells, we demonstrate that glucocorticoids rapidly stimulated transcription of the A alpha fibrinogen subunit gene by 15- to 20-fold, the B beta gene by 5- to 10-fold, and the gamma gene by 5- to 15-fold. The three genes exhibited a highly concerted response to the hormone, in which maximal stimulation occurred by 30 min and was maintained for at least 16 h. Blocking new protein synthesis before hormone treatment reduced total transcription by 45% and partially inhibited specific hormonal induction of all three fibrinogen subunit genes. The effect of glucocorticoids on fibrinogen transcription, therefore, was dependent in part on ongoing protein synthesis, suggesting that hormonal stimulation uses already synthesized stable factors, but also requires labile or newly synthesized factors for the full effect.
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Electroporation-mediated gene transfer in free-swimming embryonic Xenopus laevis.
Xenopus laevis are a rich resource for vertebrate embryology and cell biology. Transplantation and transgenesis have provided much information about the developmental mechanisms of embryogenesis and molecule function, however existing methods have faced limitations regarding either the precise localization of gene expression or flexibility in the timing of gene transfer. Here we have found that electroporation of tailbud (stage 29/30) embryos is a rapid and efficient method of combining cell-specific expression with variation in temporal delivery. At the low voltages required for electroporation, embryos resumed normal swimming behavior and development. We conclude that electroporation has wide experimental application to Xenopus developmental and cell biology.
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Presence of high molecular weight glycans on the surface of germ and embryonic cells of Xenopus laevis.
Xenopus laevis male germ cells, fertilized eggs and gastrula cells were labelled with 3H labelled sodium borohydride reduction after galactose oxidase treatment. After pronase digestion, the bulk of the label is carried by high molecular weight glycans (greater than or equal to 6,000 D). The high molecular weight of these labelled glycans and their susceptibility to degradation by endo-beta-galactosidase suggest that they may be related to the polylactosaminoglycans.
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Effect of caffeine on induced damage in cells of Xenopus laevis.
Xenopus laevis cells were treated with mitomycin C and subsequently treated with caffeine during either the S- or G2-phase of the cell cycle. The frequency of induced chromatid exchanges was especially increased by caffeine posttreatment during the S-phase. This increase took place at the expense of the frequency of isochromatid breaks, which was reduced, both when the caffeine posttreatment was given during the S- and G2-phase.
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Analysis of scleraxis and dermo-1 genes in a regenerating limb of Xenopus laevis.
Xenopus laevis larvae can regenerate an exact replica of the missing part of a limb after amputation at an early limb bud stage. However, this regenerative capacity gradually decreases during metamorphosis, and a froglet is only able to regenerate hypomorphic cartilage, resulting in a spike-like structure (spike). It has been reported that the spike has tissue deformities, e.g., a muscleless structure. However, our previous study demonstrated that the muscleless feature of the spike can be improved. The existence of other kinds of tissue, such as tendon, has not been clarified. In this study, we focused on the tendon and dermis, and we isolated the scleraxis and dermo-1 genes, which are known to be marker genes for the tendon and dermis, respectively. The expressions of these genes were investigated in both the developmental and regenerating processes of a Xenopus limb. Although muscle was needed to maintain scleraxis expression, scleraxis transcription was detectable in the muscleless spike. Additionally, although grafting of matured skin, including dermal tissue, inhibited limb regeneration, the expression of dermo-1, a dermal marker gene, was detected from the early stage of the froglet blastema. These results indicate that tendon precursor cells and dermal cells exist in the regenerating froglet blastema. Our results support the idea that spike formation in postmetamorphic Xenopus limbs is epimorphic regeneration.
Amino Acid Sequence↗
Lipid metabolism during embryonic and early postembryonic development of Xenopus laevis.
Xenopus laevis embryos were analyzed for their lipid content from fertilization to feeding stage. The amounts of the major lipids did not change until after hatching, but at the feeding stage the amounts of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sterol esters (SE) increased approximately twofold. Prior to hatching, [14C]acetate label was incorporated primarily into the fatty acyl chains of PE. After hatching, increasing amounts of label were found in other phospholipids, in their glycerol backbones, and in nonglycerol lipids. The most marked changes occurred just before the onset of feeding, and pulse-chase experiments suggest that nonlipid reserves are mobilized at this stage for de novo lipid synthesis.
Acetates↗
Analysis of the RNA structural elements involved in the binding of the transcription factor III A from Xenopus laevis.
Xenopus laevis 5S rRNA isolated from 7S particles or transcribed in vitro is found to adopt two alternative conformations. These two conformers contain different structural elements within the major TF III A binding domain, which, when isolated as RNA fragments, still interact with the transcription factor. Chemical modification of easily accessible adenines in their N-1 position does not have any measurable effect on the binding of 5S rRNA to TF III A. These observations are in support of the idea that only a small amount of conserved sequence information is required for the binding of the transcription factor, whereas specific secondary structure features seem to be essential.
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Two classes of olfactory receptors in Xenopus laevis.
Xenopus laevis possess a gene repertoire encoding two distinct classes of olfactory receptors: one class related to receptors of fish and one class similar to receptors of mammals. Sequence comparison indicates that the fish-like receptors represent closely related members of only two subfamilies, whereas mammalian-like receptors are more distantly related, most of them representing a different subfamily. The fish-like receptor genes are exclusively expressed in the lateral diverticulum of the frog's nose, specialized for detecting water-soluble odorants, whereas mammalian-like receptors are expressed in sensory neurons of the main diverticulum, responsible for the reception of volatile odors.
Amino Acid Sequence↗
Wasting disease associated with cutaneous and renal nematodes, in commercially obtained Xenopus laevis.
Xenopus laevis, the South African clawed frog or toad, is a member of the family Pipidae. Now in high demand for research purposes, they are available commercially. Its reported lifespan is up to fifteen years. Investigators and caretakers are frustrated when commercially obtained, young frogs (four years or younger), not subjected to any studies, "waste" and die. The wasting syndrome is characterized by anorexia, color change, and "flaky skin." Often the first sign of this syndrome is the presence of large fragments of "flakes" of desquamated epithelium in the water. At necropsy, these frogs are thin and have rough skin instead of smooth slimy skin. Histologic examination reveals tortuous intraepithelial cavities or tunnels that contain nematodes, and associated mild to moderate granulomatous inflammation. Nematodes are also found in the kidneys of some of these frogs, usually in Bowman's space, wrapped around the glomerulus. The cutaneous capillarid nematode is identified as Pseudocapillaroides xenopi. Successful treatment with ivermectin and thiabendizole has been reported. The renal glomerular nematode has not been identified.
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