Electron microscopic study on the early histogenesis of thymus in the toad,Xenopus laevis

Summary Sequential electron microscopic observations of thymic histogenesis in the toad, Xenopus laevis, reveal that the thymus arises as epithelial buddings of the visceral pouches at Nieuwkoop-Faber stage 40, and acquires its basic histological features at stages 48–49. In the rudiments and the surrounding mesenchyme at stages 43–45, there are non-epithelial cells with pseudopodia, abundant ribosomes, and marginated heterochromatin. These cells, possible precursor cells of thymic lymphocytes, are frequently observed to attach and pass through the basal lamina which coats the thymic rudiment. The proliferation and differentiation of large lymphocytes are evident at stage 47. During stages 48–49 the small lymphocytes, lymphoid cortex and epithelial medulla including the thymic cysts, differentiate, and vascularization occurs.The results provide an ultrastructural basis for recent experimental evidence that the thymus exerts its essential function at stages 47–48. The possibility of non-epithelial derivation of thymic lymphocytes is discussed.The author wishes to express his thanks to Asst. Prof. Ch. Katagiri for his helpful advice during the course of this study     

Biophysics of underwater hearing in the clawed frog,Xenopus laevis

Anesthetized clawed frogs (Xenopus laevis) were stimulated with underwater sound and the tympanic disk vibrations were studied using laser vibrometry. The tympanic disk velocities ranged from 0.01 to 0.5 mm/s (at a sound pressure of 2 Pa) in the frequency range of 0.4–4 kHz and were 20–40 dB higher than those of the surrounding tissue. The frequency response of the disk had two peaks, in the range of 0.6–1.1 kHz and 1.6–2.2 kHz, respectively. The first peak corresponded to the peak vibrations of the body wall overlying the lung. The second peak matched model predictions of the pulsations of the air bubble in the middle ear cavity. Filling the middle ear cavity with water lowered the disk vibrations by 10–30 dB in the frequency range of 0.5–3 kHz.Inflating the lungs shifted the low-frequency peak downwards, but did not change the high-frequency peak. Thus, the disk vibrations in the frequency range of the mating call (main energy at 1.7–1.9 kHz) were mainly caused by pulsations of the air in the middle ear cavity; sound transmission via the lungs was more important at low frequencies (below 1 kHz). Furthermore, the low-frequency peak could be reversibly reduced in amplitude by loading the larynx with metal or tissue glue. This shows that the sound-induced vibrations of the lungs are probably coupled to the middle ear cavities via the larynx. Also, anatomical observations show that the two middle ear cavities and the larynx are connected in an air-filled recess in submerged animals.This arrangement is unique to pipid frogs and may be a structural adaptation to connect all the air spaces of the frog and improve low-frequency underwater hearing. Another function of the recess may be to allow cross-talk between the two middle ear cavities. Thus, the ear might be directional. Our pilot experiments show up to 10 dB difference between ipsi- and contralateral stimulus directions in a narrow frequency range around 2 kHz.     

Prey-capture in the African clawed toad (Xenopus laevis): comparison of turning to visual and lateral line stimuli

Separately delivered visual and lateral line stimuli elicit similar but not identical orientation and approach by intact, sighted Xenopus. Response frequencies for visual stimuli declined sharply for distant or caudal stimuli while those for lateral line stimuli changed little. Turn angles correlated highly with stimulus angles but were smaller on average, so regression slopes were less than one. Regression slopes were smaller for visual than for lateral line stimuli, but this apparent difference was due to different distributions of stimulus distance interacting with the toad’s rotation center. Errors in final headings, most often under-rotations, did not differ by modality. Frequencies of lunges and arm capture movements were higher for visual stimuli both overall and especially for rostral proximal stimuli. The results demonstrate accurate orientation by sighted Xenopus to visual and lateral line stimuli; they are consistent with expectations based on in-register tectal maps. Orientation to lateral line stimuli is similar to previous results with blinded animals, revealing no heightened acuity in the latter. Modality differences indicate that the lateral line system is better for omnidirectional orientation and approach to distant stimuli whereas the visual system is more attuned to nearby rostral stimuli and more apt to mediate strikes.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The expression of creatine kinase isozymes in Xenopus tropicalis,Xenopus laevis laevis,and their viable hybrid

Starch gel electrophoresis of creatine kinase (CK) isozymes of Xenopus tropicalis shows that at least two different genes code for CK in this diploid (2n=20) species. These genes seem to be orthologous to the CK-A and CK-C genes of extant crossopterygian fish. Additional isozymes may be interpreted either as products of duplicate genes or, more probably, as epigenetically modified forms of the homodimers A^tA^t and C^tC^t, respectively. The originally tetraploid species X. laevis laevis (2n=36), which may have arisen by hybridization of diploid ancestors some 30–40 million years ago, has retained expression of all duplicate CK-A and CK-C genes. Differential expression during ontogenesis (CK-A genes) and in different adult tissues (CK-C genes) indicates that divergence occurred not only with respect to the primary sequence of these duplicate genes, but also with respect to the regulation of their expression. In the interspecific hybrid X. 1. laevis × X. tropicalis, all parental CK genes appear to be expressed simultaneously in the heart. However, several subunit combinations cannot be detected on the zymograms.This work was supported by Swiss National Foundation for Scientific Research Grant 3.775.0.80.     

LHRH-like system in the brain of Xenopus laevis daud

Summary Rabbit antiserum to synthetic LHRH was used with the immunofluorescence technique to identify the LHRH-secreting neurons and their axonal pathways in the brain of Xenopus laevis. Three groups of immunoreactive neurons were identified: the first, in the telencephalon, is a paired group of cells scattered near the two telencephalic ventricles; the second group lies near the preoptic recess; the third group occurs in the ventral wall of the infundibulum. Two principal neuronal pathways were observed: Fibres originating from the dorsally located telencephalic neurons converge on the cephalic median plane where they form a single bundle behind the telencephalic furrow. This bundle descends towards the anterior border of the preoptic recess where it divides into two nerve bundles which pass on either side of the preoptic recess, run above the optic chiasma then cross the infundibular floor and finally terminate in the median eminence. The second pathway is more direct. The more ventrally located telencephalic LHRH cells give rise to this second pathway. Their axons converge with the other LHRH fibres near the lateral border of the preoptic recess. Most of the LHRH nerve fibres terminate in the median eminence although some terminate near the paired pars tuberalis. No reaction was observed after the use of antiserum absorbed with synthetic antigen.Equipe de Recherche associée C.N.R.S. n 492. This work was financed by the D.G.R.S.T., Contract n 7470046     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

Immunolocalization of a mammalian aquaporin 3 homolog in water-transporting epithelial cells in several organs of the clawed toad Xenopus laevis

Nucleotide sequences of cDNA were used to construct antibodies against an aquaporin (AQP) expressed in the clawed toad, Xenopus laevis, viz., Xenopus AQP3, a homolog of mammalian AQP3. Xenopus AQP3 was immunolocalized in the basolateral membrane of the principal cells of the ventral skin, the urinary bladder, the collecting duct and late distal tubule of the kidney, the absorptive epithelial cells of the large intestine, and the ciliated epithelial cells of the oviducts. Therefore, we designated this AQP as basolateral Xenopus AQP3 (AQP-x3BL). The intensity of labeling for AQP-x3BL differed between the ventral and dorsal skin, with the basolateral membrane of the principal cells in the ventral skin showing intense labeling, whereas that in the dorsal skin was lightly labeled. AQP-x3BL was also immunolocalized in the basolateral membrane of secretory cells in the small granular and mucous glands of the skin. As AQP-x5, a homolog of mammalian AQP5, is localized in the apical membrane of these same cells, this provides a pathway for fluid secretion by the glands. Although Hyla AQP-h2 is translocated from the cytoplasm to the apical membrane of the Hyla urinary bladder in response to arginine vasotocin (AVT), AQP-h2 immunoreactivity in Xenopus bladder remains in the cytoplasm and barely moves to the apical membrane, regardless of AVT stimulation. AQP-x3 is localized in the basolateral membrane, even though the AVT-stimulated AQP-h2 does not translocate to the apical membrane. These findings provide new insights into AQP function in aquatic anurans.     

The reaction of the preoptic nucleus of Xenopus laevis tadpoles to osmotic stimulation

Summary The development of the preoptic nucleus of Xenopus laevis tadpoles during metamorphosis was studied and the effect of osmotic stimulation on this process investigated. The development of this region was not affected by treatment for one or more days in hypertonic media. It was found that at the end of metamorphosis the neurosecretory cells in the preoptic nucleus are localized in three regions: the rostro-dorsal, the caudo-dorsal and the ventral region. After osmotic stimulation only the neurosecretory cells of the caudo-dorsal region appeared to have reacted, as indicated by their loss of neurosecretory (PIC positive) material. It is concluded that the cells of this region may be involved in the synthesis of the posterior lobe hormones.The author thanks Prof. Dr. J. C. van de Kamer and Dr. F. C. G. van de Veerdonk for their interest and many helpful discussions, Dr. L. Boomgaart and Dr. A. P. van Overbeeke for correcting the English text and Miss C. M. G. van Bemmel for technical assistance.     

A mesoderm-inducing factor from a Xenopus laevis cell line

Summary We have compared the chemical properties and biological activities of the mesoderm-inducing factor that is secreted by the Xenopus XTC cell line with the vegetalizing factor from chicken embryos. The inducing activity of the factors was tested in different concentrations on totipotent ectoderm either by implantation into early gastrulae of Triturm alpestris or by application of solutions to isolated ectoderm of early gastrulae of Xenopus laevis. Both factors have similar properties. They are not irreversibly inactivated after treatment with 6 M urea or with phenol at 60° C. Reduction with thioglycolic acid inactivates the factors completely. The inducing activity of XTC-conditioned medium decreases only slightly after treatment with 50% formic acid. The apparent molecular mass and the isoelectric point of the factors are similar. The XTC factor was partially purified by size-exclusion and reversed-phase high-pressure liquid chromatography and by isoelectric focusing. The possible relationship of these factors to transforming growth factor is discussed.Dedicated to Prof. Dr. Sulo Toivonen on the occasion of his 80th birthday     

An SEM study of cellular morphology,contact, and arrangement,as related to gastrulation inXenopus laevis

Summary Cellular morphology, contact, and arrangement in the late blastula and in various stages of gastrulation ofXenopus were examined by SEM of specimens dissected after fixation or fractured in amyl acetate. The prospective ectoderm of the blastocoel roof consists of several layers of interdigitating cells connected by numerous small protrusions which may function in the decrease in number of cell layers observed during ectodermal epiboly. During gastrulation, prospective mesoderm is regionally differentiated by cellular morphology and arrangement into preinvolution mesoderm, the mesodermal involution zone, and involuted mesoderm. The involuted anterodorsal (head), lateral, and ventral mesoderm consists of a stream of loosely-packed, irregularly shaped cells having large extensions of the cell body attached locally to other cells by small protrusions. Involuted posterodorsal mesoderm (chordamesoderm) consists of elongated cells arranged in palisade fashion and connected by similar protrusions. Involuted mesodermal cells in all regions are attached to the overlying prospective ectodermal cells by numerous small protrusions along the entire interface between the two cell layers. Suprablastoporal endodermal cells involute as an epithelial sheet, changing in shape in the process, to form the roof of the archenteron. Bottle cell morphology, arrangement, and position with respect to the mesodermal cell stream is described. Evidence presented here and elsewhere suggests that involution of mesoderm and of the archenteron roof inXenopus is dependent primarily upon the relative movement of the mesodermal cell stream and of the overlying ectoderm.     

Ultrastructural observations on the germ line of Xenopus laevis

Summary The development of the male germ line in Xenopus laevis has been examined by electron microscopy. Findings have been compared to the parallel process in the female. Three structures unique to the germ line were found in both male and female cells: a fibrillar nuclear region free of DNA; largely proteinaceous masses of nuage material; and a chromatoid body. Germ plasm bodies of the egg and early embryo appear to represent a form of nuage material. The finding of a structure which can be identified as a chromatoid body in the female germ line is unique, as is its presence in sexually undifferentiated primordial germ cells. The chromatoid body in Xenopus, unlike that in mammals, does not persist in the spermatozoon. Instead, it dissociates into a series of coated vesicles during spermatogenesis. The chromosomal ultrastructure of meiotic prophase stages in Xenopus is similar in both sexes until diplotene, when male bivalents condense and enter meiotic metaphase instead of entering the extended lampbrush stage characteristic of the female. The multiple nucleoli present in gonia are lost at the onset of meiotic prophase, but no obvious mechanism for this process was observed.The author would like to thank Drs. Joseph Gall and Bernard Tandler for their helpful suggestions during the course of this investigation. The author is a postdoctoral fellow of the National Institutes of Health, U.S.A. This research was supported by N.I.H. Grants 51823 and 12427.     

Lateral line-mediated rheotactic behavior in tadpoles of the African clawed frog (<Emphasis Type=Italic>Xenopus laevis</Emphasis>)

Tadpoles (Xenopus laevis) have a lateral line system whose anatomical structure has been described, but whose functional significance has not been closely examined. These experiments tested the hypothesis that the lateral line system is involved in rheotaxis. Tadpoles in developmental stages 47–56 oriented toward the source of a water current. Orientation was less precise after treatment with cobalt chloride or streptomycin, but was similar to that of untreated animals after exposure to gentamicin. In no current conditions, tadpoles exhibited a characteristic head-down posture by which they held themselves in the water column at an angle around 45°. This body posture became significantly less tilted in the presence of water current. Treatment with cobalt chloride or streptomycin increased the angle of tilt close to that seen in no current conditions, while gentamicin treatment tended to decrease tilt angle. The data are consistent with anatomical and physiological findings that tadpole neuromasts are similar to superficial, but not canal, neuromasts in fishes, and they suggest that the lateral line system is involved in both directional current detection and current-related postural adjustments in Xenopus.     

Dynamic distribution of region-specific maternal protein during oogenesis and early embryogenesis of Xenopus laevis

Summary For analysing spatial distribution of maternal proteins in an amphibian egg, monoclonal antibodies specific to certain regions were raised. One monoclonal antibody was found (MoAB Xa5B6) which reacted specifically with the animal hemisphere of the mature Xenopus laevis egg. The maternal protein that reacted with the MoAb Xa5B6 was shown to be distributed asymmetrically along the dorso-ventral axis in the upper region of the equatorial zone of the fertilized egg. At late blastula stage, the antigen protein could be observed clearly in both the marginal zone and animal cap. It was localized predominantly in mesodermal and ectodermal cells of late neurula embryos. The Xa5B6 antigen accumulated during oogenesis. The distribution pattern of maternal protein was remarkably different in the developmental stages of the oocyte. The pattern in the mature oocyte was completely different from that of the immature egg in which the antigen was located in the radial striations of the oocyte cytoplasm. After maturation, the distribution pattern changed drastically to an animal-vegetal polarization and the striation labellings were no longer observed. By Western blot examination, it was confirmed that the amounts of antigen protein were constant during early embryogenesis and the mesoectoderm contained a greater amount of antigens than the endoderm at late blastula. The antibody detected two bands of approximately 70 × 10³ and 30 × 10³ Mr by Western blot analysis. The latter molecule may possibly be a degrading moiety of the former. The results were discussed in relation to establishment of animal-vegetal (A/V) and dorso-ventral (D/V) polarization at the molecular level.Offprint requests to: A.S. Suzuki     

Immunohistochemical demonstration of TSR-, LH- and ACTH-cells in the hypophysis of tadpoles of Xenopus laevis D.

Summary By use of the immunofluorescence technique TSH-, LH- and ACTH-cells were localized in the hypophysis of tadpoles of Xenopus laevis. The first signs of the activity of these cells were observed in early stages of the development, i.e., stage 39 for ACTH, and stage 42 for TSH and LH.     

Sexual differences as adaptation to the different gender roles in the frogXenopus laevis Daudin

1. Various physiological parameters were determined in fed, adult, male and female Xenopus laevis acclimated to 20 degrees C and with a light: dark cycle of 12:12. The results were compared for sex differences. 2. There were significant differences in food intake, oxygen consumption, and motor activity with lower values for each parameter in males than in females (Table 1). 3. Further significant differences were found in the plasma concentrations of calcium, total lipids, and aldosterone (Table 3), in the somatic indices of fat body and gonads, in the glycogen and protein content of the liver (Table 4), and in the activities of glucose-6-phosphate dehydrogenase, fructose-1,6-diphosphatase, and phosphoenolpyruvate carboxykinase in the liver (Table 5). 4. It is assumed that the observed differences are essentially a result of differences in body growth and gametogenesis between the sexes. The lack of capacity of males to store glycogen and lipids in the male gonads is a further explanation for the differences.     

Prion protein from Xenopus laevis: overexpression in Escherichia coli of the His-tagged protein and production of polyclonal antibodies

Prion protein (PrP) and PrP-related proteins have been identified in reptiles, amphibians, and fishes by means of cDNA cloning, genome database searching and comparative genomics. However, no studies have been reported so far on the expression of PrP at the protein level in those animals. This report presents a procedure to obtain and purify recombinant PrP from Xenopus laevis expressed in Escherichia coli as a fusion protein in which mature PrP (residues 21-194) is linked to a 35-amino acid N-terminal extension containing a hexahistidine stretch. The protein was used to raise and purify by affinity chromatography anti-Xenopus PrP polyclonal antibodies which were suitable to detect the presence of PrP in Xenopus brain by Western blot. This is the first report of a positive identification of PrP in amphibian at the protein level. Anti-Xenopus PrP antibodies do not cross react with PrP from different sources (human, bovine, sheep, and turtle). Similarly, Xenopus PrP do not react with anti-turtle PrP(143-248) antibodies.