Assembly of correct kinetochore architecture in Xenopus laevis egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Attachment of DNA Loops to an Artificial Matrix Does Not Affect the Replication Origin Specificity in Early Development of Xenopus laevis

Replication initiation proceeds in a random fashion in early development of Xenopus laevis. The replication origins become fixed only at later stages of development after the mid-blastula transition. Specification of replication origins occurs at the same time with the specification of the DNA attachment to the nuclear matrix. Replication origins of many species coincide or are located in the vicinity of sites of DNA attachment to the nuclear matrix. The present work was dedicated to development of an experimental system where DNA loops were specifically attached to an artificial matrix and a study of an effect of this attachment on specificity of DNA replication initiation in extracts of Xenopus laevis oocytes. We have found that DNA attachment to the artificial matrix increases the efficacy of DNA replication as compared to the control, but does not affect the replication specificity. It is likely that the transition from non-specific to specific replication is determined by a combination of several factors, and specificity of DNA attachment to a matrix alone is not sufficient for specification of a replication origin.     

Repair of psoralen interstrand cross-links in Xenopus laevis egg extracts is highly mutagenic

The recognition and removal of interstrand cross-links is perhaps the least understood of all repair pathways in eukaryotic cells. We have shown previously that uncoupling of cross-links occurs in mammalian cell extracts and have identified a number of factors that mediate this process. However, we have not observed complete repair of the substrate in this system. Here, we show that uncoupling of interstrand cross-links also occurs in Xenopus laevis egg extracts, and that the initial products of this reaction are identical to the products observed in mammalian cell extracts suggesting a common mechanism. However in contrast to mammalian cell extracts, we observe repair of the cross-linked substrate in the Xenopus extracts presumably by a translesion bypass mechanism that allows replication past the uncoupled monoadduct, and its likely subsequent removal by nucleotide excision repair. This repair process is shown to be highly mutagenic consistent with bypass synthesis.     

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.     

Evidence that DNA replication is not regulated by ubiquitin-dependent proteolysis in Xenopus egg extract

The Xenopus early embryonic cell cycle consists of rapid oscillations between mitosis and DNA synthesis. We used ubiquitin (Ub)-dependent proteolysis inhibitors to determine whether Ub-mediated proteolysis regulates the initiation of DNA replication in Xenopus egg extract. Methylated Ub, a chemically modified Ub that cannot form chains, and S5a, a Ub chain-binding subunit of the 26S proteasome, were added to extract at concentrations known to inhibit cyclin B proteolysis and their effects on cell cycle progression and DNA replication were examined. DNA replication initiated concomitant with controls and proceeded in a semiconservative fashion in the presence of both methylated Ub and S5a. However, mitotic progression was halted, showing that the inhibitors were functional. We conclude that initiation of DNA replication is not regulated by Ub-dependent proteolysis in the early Xenopus cell cycle.     

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     

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.     

The development of a medium supplemented with egg extract for the maintenance ofDrosophila cell lines

Summary A saline extract was prepared fromDrosophila eggs. When diluted to a concentration of 1% withDrosophila tissue culture medium, it did not support growth of cells from theDrosophila line D1 during the first few days of subculture as well as medium containing serum. When cells reached a stationary phase, however, the cell density in medium containing extract was greater than in medium containing serum. By altering the concentrations of the extract, and by adding bovine albumin, a medium was obtained in which D1 cells survived initial culturing, and which supported cell growth by day 4 as well as medium plus serum. The initial retardation of growth in medium containing egg extract might be due to the need of the cells to adapt to the new medium. At the present time fourDrosophila cell lines have been maintained in this medium for more than 16 passages. Preliminary experiments with primary embryonicDrosophila cells indicate that medium containing 2% extract and bovine albumin retards the differentiation of these cells. This work was supported by a grant from the Science Research Council of Great Britain.     

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.     

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)⁺ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).     

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.     

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.     

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     

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     

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.     

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.     

Glider and Vision: Two New Families of Miniature Inverted-Repeat Transposable Elements in Xenopus Laevis Genome

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the α-tropomyosin (α-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated α-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284 bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the laevis genome. Every MITEs elements but two described in our study are found either in 5′ or in 3′ regulatory regions of genes suggesting a potential role in gene regulation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nuclear reconstitution of demembranated Orychophragmus violaceus sperm in Xenopus laevis egg extracts

The cell-free extracts from animal Xenopus laevis egg could induce chromatin decon-densation and pronuclear formation from demembranated plant (Orychophragmus violaceus) sperm. The demembranated Orychophragmus violaceus sperm began to swell in 30 min incubation, and then were gradually decondensed. The reassembly of nuclear envelope in the reconstituted nuclei had been visualized by means of electron microscopy and fluorescent microscopy. Membrane vesicles fused to form the double envelope around the periphery of the decondensed chromatin. The morphology of the newly formed nucleus, with a double membrane, was similar to those nuclei after fertilization. Transmission electron microscope micrograph of the whole mount prepared nuclear matrix-lamina showed the reconstituted nucleus to be filled with a dense network.