Aroclor 1254 impairs the hearing ability of Xenopus laevis

In this study we assessed the effects of chronic, dietary exposure of Aroclor 1254 (A1254) on the hearing of Xenopus frogs. We used the auditory brainstem response (ABR) to assay changes in hearing physiology; ABR thresholds, as well as latency-intensity and amplitude-intensity profiles of the initial positive (P1) and negative (N1) peaks were measured. Two groups of animals that received 50 ppm and 100 ppm of A1254 in their diet from 5 days post-fertilization through metamorphosis were compared to a control group that received untreated chow. The results showed significant threshold elevations in the 3–4 kHz range and significantly delayed peak latencies and reduced amplitudes at these frequencies in A1254 treated animals as compared to control animals. These findings indicate that A1254 selectively damages the high-frequency sensorineural hearing system associated with the basilar papilla of frogs. This preferential damage may be related to inherent differences in the vulnerability of the basilar versus amphibian papilla in the frog. The overall results of this study are also consistent with the reported A1254-induced auditory deficits in mammals indicating that the basilar papilla of the Xenopus frog may serve as an effective model for studying the effects of A1254 on the auditory system.     

Diel activity and thermoregulatory behavior of a fully aquatic frog: Xenopus laevis

Ten adult Xenopus laevis were tested individually for 48-hr periods, following an initial 24-hr introductory period, in electronic shuttleboxes which allowed them to control water temperatures without operant conditioning. Locomotor activity was recorded via photocell-monitored light beams. The frogs were nocturnal, being nearly twice as active at night as during the day. The mean preferred temperature was 22.4°C, with no significant difference between night (22.5°C) and day (22.3°C), although the modal preferendum shifted from 24°C by day to 22°C at night, with a corresponding change in skewness. The range of voluntarily occupied temperatures was 14–32°C by day and 14–29°C at night. The median thermal preferendum was 22°C both day and night.     

Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.     

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.     

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     

Structure of the main ganglioside from the brain of Xenopus laevis

The main component of the ganglioside¹ mixture from the brain of the adult amphibian Xenopus laevis accounts for 35% of the total, as lipid bound sialic acid. This ganglioside has been purified and characterized by thin layer chromatography, partial and exhaustive enzymatic hydrolysis with sialidase, TLC-overlay procedures with anti-Gg₄Cer and anti-Neu5Ac6GalNAc specific monoclonal antibodies and mass spectrometry. All together the results suggest the following structure:Neu5Ac8Neu5Ac3Gal3(Neu5Ac8Neu5Ac6)GalNAc4Gal4Glc1Ceror, IV³--Neu5Ac₂,III⁶--Neu5Ac₂-Gg₄Cer.     

The relationship of innervation and differentiation to regenerative capacity in the reamputated hindlimb of larval Xenopus laevis

Regenerated hindlimbs of larval Xenopus laevis were reamputated at critical larval stages and levels, viz when amputation of the control limb at the same larval stage and level is followed by reduced regeneration. Reamputations were performed at the level of (1) the original plane of amputation, (2) the early regenerate (cone/palette stage), (3) the late regenerate (digit stage). Reamputation increased both the percentage rate of regeneration and the morphological complexity of the regenerates in all experimental series. Cell counts in lateral motor columns and spinal ganglia innervating the hindlimb, together with histological observations and mitotic index and labelling index determinations in reamputated and control limbs showed that improved regeneration in the reamputated limb was related to an increase in undifferentiated and proliferating cells in the stump. We did not find any evidence suggesting that renewed regeneration in reamputated anuran limbs results from an increase in innervation, as has previously been hypothesized. We support our conclusions by demonstrating an improvement in regenerationen in the reamputated and denervated hindlimbs.     

Developmental immunohistology of melanotrophs in Xenopus laevis tadpoles

Summary The adenohypophysial primordium of Xenopus laevis tadpoles at stages 33/34 to 46 (Nieuwkoop and Faber, 1956) were examined immuno-histologically for -MSH, -MSH and ACTH. -MSH was demonstrated from stage 37/38 onwards, and -MSH from stage 39. No signs of ACTH production were detected. -MSH and -MSH occurred in the same cells. No differences were found in the intensity of immunofluorescence between tadpoles which were kept on a black and a white background. The present study lends no support to the hypothesis concerning the derivation of -MSH from ACTH. The observations made suggest that the morphological formation of the pars intermedia is accomplished during stages 37/38 to 39. Acknowledgement. The authors express warm thanks to Dr. M.P. Dubois (Laboratoire de Physiologie de la Reproduction, INRA, Nouzilly, France), who prepared and verified the antibodies. Grants from Swedish Natural Science Research Council and Landshovding Per Westlings minnesfond, Lund, Sweden are gratefully acknowledged     

Ultrastructural study on hepatic melanin in Xenopus laevis

Summary The livers of Xenopus laevis, grouped by chronological age (0.5,2 and 3 yrs), were studied electron microscopically. Ultrastructurally most of the melanin granules in the mature female liver showed an-internal structure similar to the melanin granules of the oocytes. The hepatic melanin granules of immature females and of all males were pleomorphic and failed to show the characteristic internal structure similar to those of the oocytes. The oocyte is the probable source of most of the hepatic melanin of the mature female.     

Fission yeast tmsl protein abrogates normal development in Xenopus laevis embryos

Recently we cloned tms1 (a putative dehydrogenase) by complementation of a human tumour-derived mutant p53 induced growth arrest in fission yeast. Microinjection of purified tmsl protein into Xenopus laevis embryos abrogated normal embryo development by causing cleavage retardation or cleavage arrest of injected blastomeres in a concentration dependant manner, whereas injection of specific affinity purified tms1 antiserum showed no significant morphological defects. Microinjection of tms1 protein together with affinity purified tms1 antibody resulted in a significantly reduced number of cleavage arrested embryos.     

Some morphogenetic features of the adenohypophysial primordium of early Xenopus laevis tadpoles

Summary The adenohypophyses of Xenopus laevis tadpoles at developmental stages 20 to 46 (Nieuwkoop and Faber, 1956) were studied. From its first appearance at about stage 20 to 21, the adenohypophysial primordium passes through four morphogenetic phases, each characterized by internal events. The first phase (stages 20 to about 33/34) is characterized by extensive proliferation of the primordium. During the second phase (stages 33/34 to about 37/38), the growth of the primordium is arrested. This arrest coincides with the attainment of secretory function. The primordium is claviform in shape at these stages. The third phase, roughly stage 39, is characterized by a thorough reorganization of the adenohypophysial cells, leading to the formation of the pars distalis and pars intermedia. The shape of the primordium changes, and its volume temporarily increases. The last phase is characterized by the organization of the pars distalis cells into cell cords which possibly demonstrate a functional relation to a specialized region (the hilus) of the adenohypo-physis-brain interspace. Acknowledgements. Grants from the Faculty of Mathematics and Science, University of Lund, the Royal Physiographic Society, Lund, and the Swedish Natural Sience Research Council are gratefully acknowledged     

Generation of trangenic Xenopus laevis using the Sleeping Beauty transposon system

Using the Sleeping Beauty (SB) transposon system, we have developed a simple method for the generation of Xenopus laevis transgenic lines. The transgenesis protocol is based on the co-injection of the SB transposase mRNA and a GFP-reporter transposon into one-cell stage embryos. Transposase-dependent reporter gene expression was observed in cell clones and in hemi-transgenic animals. We determined an optimal ratio of transposase mRNA versus transposon-carrying plasmid DNA that enhanced the proportion of hemi-transgenic tadpoles. The transgene is integrated into the genome and may be transmitted to the F1 offspring depending on the germline mosaicism. Although the transposase is necessary for efficient generation of transgenic Xenopus, the integration of the transgene occurred by an non-canonical transposition process. This was observed for two transgenic lines analysed. The transposon-based technique leads to a high transgenesis rate and is simple to handle. For these reasons, it could present an attractive alternative to the classical Restriction Enzyme Mediated Integration (REMI) procedure.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

The area octavo-lateralis in Xenopus laevis

Summary Central projections of afferents from the lateral line nerves and from the individual branches of the VIIIth cranial nerve in Xenopus laevis and Xenopus mülleri were studied by the application of HRP to the cut end of the nerves.Upon entering the rhombencephalon, the lateral line afferents form a longitudinal fascicle of ascending and descending branches in the ventro-lateral part of the lateral line neuropile. The fascicle exhibits a topographic organization, that is not reflected in the terminal field of the side branches. The terminal field can be subdivided into a rostral, a medial and a caudal part, each of which shows specific branching and terminal pattern of the lateral line afferents. These different patterns within the terminal field are interpreted as the reflection of functional subdivisions of the lateral line area. The study did not reveal a simple topographic relationship between peripheral neuromasts and their central projections.Two nuclei of the alar plate with significant lateral line input were delineated: the lateral line nucleus (LLN) and the medial part of the anterior nucleus (AN). An additional cell group, the intermediate nucleus (IN), is a zone of lateral line and eighth nerve overlap, although such zones also exist within the ventral part of the LLN and the dorsal part of the caudal nucleus (CN). Six nuclei which receive significant VIIIth nerve input are recognized: the cerebellar nucleus (CbN), the lateral part of the anterior nucleus, the dorsal medullary nucleus (DMN), the lateral octavus nucleus (LON), the medial vestibular nucleus (MVN) and the caudal nucleus (CN).All inner ear organs have more than one projection field. All organs project to the dorsal part of the LON and the lateral part of the AN. Lagena, amphibian papilla and basilar papilla project to separate regions of the dorsal medullary nucleus (DMN). There is evidence for a topographic relation between the hair cells of the amphibian papilla (AP) and the central projections of AP fibers. The sacculus projects extensively to a region between the DMN and the LON. Fibers from the sacculus and the lagena project directly to the superior olive. Fibers from the utriculus and the three crista organs terminate predominantly in the medial vestibular nucleus (MVN) and in the adjacent parts of the reticular formation, and their terminal structures appear to be organotopically organised. Octavus fiber projections to the cerebellum and to the spinal cord are also described.     

Nerve-independent DNA synthesis and mitosis in regenerating hindlimbs of larval Xenopus laevis

Summary Xenopus laevis larvae at stage 52–53 (according to Nieuwkoop and Faber 1956) were subjected to amputation of both limbs at the thigh level as well as to repeated denervations of the right limb. Results obtained in larvae sacrificed during wound healing (1 after amputation), blastema formation (3 days) and blastema growth (5 and 7 days) showed that denervated right limbs have undergone the same histological modifications observed in innervated left limbs and have formed a regeneration blastema consisting of mesenchymal cells with a pattern of DNA synthesis and mitosis very similar to that in presence of nerves. Also, the patterns of cellular density in regenerating right and left limbs were very similar. On the whole, the data here reported show a highly remarkable degree of nerve-independence for regeneration in hindlimbs of larval Xenopus laevis at stage 52–53 and lend some substance to the hypothesis that, in early limbs, there would exist trophic factors capable of replacing those released by nerves, promoting DNA synthesis and mitosis in blastemal cells. Offprint requests to: S. Filoni     

The distribution of nucleoplasmin in early development and organogenesis of Xenopus laevis

Summary The fate of the germinal vesicle-derived protein, nucleoplasmin, was followed in embryos and tadpoles of Xenopus using monoclonal antibodies and indirect immunofluorescent staining. Nucleoplasmin was found in all nuclei up to feeding tadpole stages. Thereafter its level decreased in all nuclei. It was not detected in nuclei of advanced tadpoles or of adults. Contrasting with another protein, N₁, that was previously monitored in the nuclei of dividing gonia of both sexes, nucleoplasmin was only detected in the nuclei of ovarian oocytes starting at diplotene. Traces of nucleoplasmin have also been found in a rapidly-dividing fibroblastic cell-line by immunohistology and protein blotting.     

Xenopus laevis tadpole limb regeneration in vivo and in vitro: thyroxine directly promotes blastemal cell proliferation and morphogenesis

Regeneration in hindlimbs of Xenopus laevis larvae which were amputated at stage 53 and 55 through the tarsalia region is promoted by thyroxine (T4), while propyl-thiouracil (PTU) inhibits regeneration when compared to controls. In this paper, by in vivo and in vitro experiments, we demonstrate that the promoting effect of T4 on the regenerative processes of larval X. laevis hindlimbs is a direct effect of this hormone on the blastemal cells. By contrast, the inhibitory effect of PTU on the regenerative process is not due to a direct effect on blastemal cells or to a general toxic effect on the treated larvae, but is related to hypothyroidism induced by the drug. We find that: (i) an increase in blastemal cell proliferation is observed not only in blastemata of T4-treated larvae, but also in blastemata cultured in vitro in a medium supplemented with T4; (ii) the renegerative process is accelerated not only in larvae reared in T4 but also in larvae submitted to a combined treatment of T4 and PTU; (iii) inhibition of cell proliferation is observed in blastemata of PTU-reared larvae but not in blastemata cultured in vitro in a medium supplemented with PTU. Experiments on thyroidless larvae (which were submitted to transplantation of hindlimbs from larvae at stages 53 and 55 followed by amputation of their own right hindlimb and the transplanted limbs) have shown that without thyroid hormone the regenerative process is arrested at cone stage and the promoting effect of T4 treatment is dependent on limb stage and amputation level.     

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.     

Lens formation from cornea implanted into amputated hindlimbs of Xenopus laevis larvae requires innervation or proliferating cell populations in the stump

The capacity of amputated early and late limbs of larval Xenopus laevis to promote lens-forming transformations of corneal implants in the absence of a limb regeneration blastema has been tested by implanting outer cornea fragments from donor larvae at stage 48 (according to Nieuwkoop and Faber 1956), into limb stumps of larvae at stage 52 and 57. Blastema formation has been prevented either by covering the amputation surface with the skin or by reconnecting the amputated part to the limb stump. Results show that stage 52 non-regenerating limbs could promote lens formation from corneal implants not only when innervated but also when denervated. A similar result was observed in stage 57 limbs where blastema formation was prevented by reconnecting the amputated part to the stump. In this case, relevant tissue dedifferentiation was observed in the boundary region between the stump and the autografted part of the limb. However, stage 57 limbs, where blastema formation was prevented by covering the amputation surface with skin, could promote lens formation from the outer cornea only when innervated. In this case, no relevant dedifferentiation of the stump tissues was observed. These results indicate that blastema formation is not a prerequisite for lens-forming transformations of corneal fragments implanted into amputated hindlimbs of larval X. laevis and that lens formation can be promoted by factors delivered by the nerve fibres or produced by populations of undifferentiated or dedifferentiated limb cells.     

The homeobox leucine zipper gene Homez plays a role in Xenopus laevis neurogenesis

The Homez gene encodes a protein with three atypical homeodomains and two leucine zipper motifs of unknown function. Here we show that during neurula stages, Xenopus Homez is broadly expressed throughout the neural plate, the strongest expression being detected in the domains where primary neurons arise. At later stages, Homez is maintained throughout the central nervous system in differentiating progenitors. In accordance with this expression, Homez is positively regulated by neural inducers and by Ngnr1 and negatively by Notch signaling. Interference with Homez function in embryos by injection of an antisense morpholino oligonucleotide results in the specific disruption of the expression of late neuronal markers, without affecting the expression of earlier neuronal and early neurectodermal markers. Consistent with this finding, Homez inhibition also interferes with the expression of late neuronal markers in Ngnr1 overexpressing animal cap explants and in Notch inhibited embryos. In gain of function experiments, Homez inhibits the expression of late neuronal markers but has no effect on earlier ones. These data suggest a role for Homez in neuronal development downstream of proneural/neurogenic genes.