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.     

Ultrastruktur der spinalen Liquorkontaktneurone beim Krallenfrosch (Xenopus laevis)

Zusammenfassung Zwischen und unter den Ependymzellen des Zentralkanals des Rückenmarkes von Xenopus laevis kommen Nervenzellen vor. Die intraependymalen Neurone sind rundlich und stehen mit dem Liquor cerebrospinalis durch eine breite Oberfläche in Berührung, von der sich längere und kürzere Fortsätze und ein Cilium (Typ 9+2) in das Lumen erheben. Die hypendymalen Neurone sind bipolar; ihr Dendrit verzweigt sich im Liquor ebenfalls in fingerförmige Fortsätze. Die Liquorkontaktfortsätze beider Zelltypen sind von feinen Filamenten ausgefüllt. Der Reissnersche Faden lagert sich manchen Fortsätzen an.In den intra- und hypendymalen Perikaryen findet man neben endoplasmatischem Retikulum, Golgi-Arealen und Mitochondrien kleine dense-core Vesikel (Durchmesser 600–900 Å). Der distale Fortsatz beider Neurontypen hat Neuritennatur. Axone, die synaptische und granulierte (Durchmesser 800–1200 Å) Vesikel enthalten, bilden relativ wenige Synapsen mit den Liquorkontaktneuronen. Im hypendymalen Neuropil findet man multipolare Nervenzellen, die 1000–1200 Å große granulierte Vesikel enthalten. Aufgrund des morphologischen Bildes wird die mögliche Rolle der Liquorkontaktfortsätze und des Ciliums bei der Funktion der Liquorkontaktneurone diskutiert.

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Ultrastructure of the spinal liquor contacting neurons in the clawed toad (Xenopus laevis)

Summary Nerve cells are situated between and below the ependymal cells of the central canal of the spinal cord of Xenopus laevis. The intraependymal neurons are round-shaped; they contact the cerebrospinal fluid by a large surface from which longer and shorter processes and a cilium (type 9+2) arise into the lumen. The hypendymal neurons are bipolar; their dendrite ramifies also into finger-like processes in the cerebrospinal fluid. The liquor contacting processes of both cell types contain fine filaments. The Reissner's fibre contacts some of the processes.In the intra- and hypendymal perikarya, small dense-core vesicles (diameter 600–900 Å) are found besides of endoplasmic reticulum, Golgi-areas and mitochondria. The distal process of both neuron types has neurite character. Axons containing synaptic and granulated (diameter 800–1200 Å) vesicles, form relatively few synapses with the liquor contacting neurons. In the hypendymal neuropile, multipolar nerve cells occur that contain granulated vesicles with a diameter of about 1000–1200 Å. On the basis of the morphological picture, the possible role of the liquor contacting processes and of the cilium in the function of the liquor contacting neurons are discussed.

     

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     

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     

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.     

Emigration of bilayered epidermal cell sheets from tadpole tails (Xenopus laevis)

Summary Migration of bilayered epidermal cell sheets out of explants of tadpole tails (Xenopus laevis) were investigated with time-lapse cinemicrography using reflection-contrast optics. Cell-sheet formation begins beneath the explant in a region where it is closely attached to the coverslip. A single basal cell extends a lamellipodium through the outer (surface) epidermal layer and starts moving in a direction free of attached cells. This cell remains connected to the following basal cell, which the also extends a lamellipodium onto the glass. The cell sheet develops as increasingly more adjacent basal cells start to migrate. Surface cells do not actively locomote but they remain attached to the basal cells and to adjacent surface cells. Thus, they are transported as an intact cell layer, and consequently the in situ arrangement of the tadpole epidermis is largely preserved in the cell sheet, i.e., basal cells adhere to the substratum and are covered by outer cells (surface cells) which face the culture medium. Basal cells extend lamellae beneath the rear end of the preceding cell, which is slightly fifted off the substratum. The direction of locomotion is determined by the frontal cells. Cell-sheet enlargement and locomotion cease when all the epidermal cells facing the coverslip have left the explant, and the cell sheet and epidermis covering the explant form a continuous layer.     

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.     

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.     

Changing complexity of endogenous lectin activities during juvenile development of Xenopus laevis

Galactoside-binding lectin has been isolated from whole Xenopus laevis embryos and tadpoles at four development stages: st. 24–26, 32, 41 and 47. The main lectin activity at st. 24–26 is -galactoside specific, producing a 34/35.5K doublet on SDS-PAGE. Later in development, lectin activities specific for a wide range of other sugars appear concommitant with the detection of a number of new protein bands on SDS-PAGE gels. The greatest variety of new lectin activities exists at st. 32 when lectins specific for all of the main sugar families found in nature are detected. After this stage and up to st. 47 (the beginning of metamorphosis), fewer different lectin activities are again detected. The results suggest that a complex, developmentally regulated battery of different lectins are present during early Xenopus development, perhaps with stage-specific roles to play in the control of tissue morphogenesis.     

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.     

Exogenous sphingosine enters Xenopus laevis embryos grown in petri dishes and it is metabolized

Xenopus embryos of different developmental stages were exposed to 0.1 M [1-³H]sphingosine. Labeled sphingosine was quickly absorbed by Xenopus embryos. The amount of radioactivity absorbed increased with embryo age and appeared to be linearly correlated (R=0.97) to the embryo surface area. About 45% of the total radioactivity associated to the embryos was found in the skin, 22% in the intestine, 15% in the heart, 12% in the liver and 6% in the brain.A portion of [1-³H]sphingosine entered very rapidly the biosynthetic pathway of sphingolipids; after 30 min of incubation, in fact, only a small amount of free radioactive sphingosine could be detected. Sphingomyelin was the main radioactive sphingolipid synthesized; radioactive ceramide, galactosylceramide and lactosylceramide could also be recognized and quantified. On the contrary, the amount of radioactive gangliosides was hardly detectable.A portion of [1-³H]sphinogosine absorbed by Xenopus embryos (30 to 60% depending on the developmental stage) entered the catabolic pathway producing radioactive phosphoethanolamine that was recycled for the biosynthesis of radioactive phosphatidylethanolamine. This phospholipid was produced mainly in the intestine and in the skin, while sphingomyelin was the main radioactive lipid in the heart, liver and brain.     

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.     

Calcium signalling during neural induction in Xenopus laevis embryos

In Xenopus, experiments performed with isolated ectoderm suggest that neural determination is a 'by default' mechanism, which occurs when bone morphogenetic proteins (BMPs) are antagonized by extracellular antagonists, BMP being responsible for the determination of epidermis. However, Ca(2+) imaging of intact Xenopus embryos reveals patterns of Ca(2+) transients which are generated via the activation of dihydropyridine-sensitive Ca(2+) channels in the dorsal ectoderm but not in the ventral ectoderm. These increases in the concentration of intracellular Ca(2+)([Ca(2+)]i) appear to be necessary and sufficient to orient the ectodermal cells towards a neural fate as increasing the [Ca(2+)]i artificially results in neuralization of the ectoderm. We constructed a subtractive cDNA library between untreated and caffeine-treated ectoderms (to increase [Ca(2+)]i) and then identified early Ca(2+)-sensitive target genes expressed in the neural territories. One of these genes, an arginine methyltransferase, controls the expression of the early proneural gene, Zic3. Here, we discuss the evidence for the existence of an alternative model to the 'by default' mechanism, where Ca(2+) plays a central regulatory role in the expression of Zic3, an early proneural gene, and in epidermal determination which only occurs when the Ca(2+)-dependent signalling pathways are inactive.     

The Activation Pathway of the Volume-Sensitive Organic Osmolyte Channel in Xenopus laevis Oocytes Expressing Skate Anion Exchanger 1 (AE1)

When swollen, skate red blood cells increase permeability and allow efflux of a number of solutes, including taurine. Hypoosmosis-induced taurine permeability appears to involve the red cell anion exchanger. However, three isoforms have been cloned from these cells. Therefore, to determine the ability of the individual isoform skate anion exchanger 1 (skAE1) to mediate hypoosmosis-induced taurine permeability as well as associated regulatory events, skAE1 was expressed in Xenopus oocytes. This study focused on investigating the role of tyrosine kinases and lipid rafts in the regulation of the channel. The results showed that tyrosine kinase inhibitors and lipid raft-disrupting agents inhibited the volume-sensitive organic osmolyte channel while protein tyrosine phosphatase inhibitors activated the channel in oocytes expressing skAE1. To study the role of lipid rafts in the activation of the volume-sensitive organic osmolyte channel, the cellular localization of skAE1 was investigated. Also, the role of tyrosine kinases was investigated by examining the tyrosine phosphorylation state of skAE1. Hypoosmotic stress induced mobilization of skAE1 into light membranes and the cell surface as well as tyrosine phosphorylation of skAE1. These events are involved in the activation of the volume-sensitive organic osmolyte channel in Xenopus oocytes expressing skAE1.     

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 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.     

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     

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. ¹⁵N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional ¹H–¹⁵N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein–protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

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.