FGF-10 stimulates limb regeneration ability in Xenopus laevis

By reciprocal transplantation experiments with regenerative and nonregenerative Xenopus limbs, we recently demonstrated that the regenerative capacity of a Xenopus limb depends on mesenchymal tissue and we suggested that fgf-10 is likely to be involved in this capacity (Yokoyama et al., 2000, Dev. Biol. 219, 18-29). However, the data obtained in that study are not conclusive evidence that FGF-10 is responsible for the regenerative capacity. We therefore investigated the role of FGF-10 in regenerative capacity by directly introducing FGF-10 protein into nonregenerative Xenopus limb stumps. Exogenously applied FGF-10 successfully stimulated the regenerative capacity, resulting in the reinduction of all gene expressions (including shh, msx-1, and fgf-10) that we examined and the regeneration of well-patterned limb structures. We report here for the first time that a certain molecule activates the regenerative capacity of Xenopus limb, and this finding suggests that FGF-10 could be a key molecule in possible regeneration of nonregenerative limbs in higher vertebrates.     

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.     

Effect of denervation on hindlimb regeneration in Xenopus laevis larvae

Xenopus laevis larvae at stages 51-57, according to Nieuwkoop and Faber, were subjected to amputation of the right hindlimb or of both limbs at the thigh or the tarsal level, as well as to somatic denervation of the right limb. Larvae at the same stage having undergone amputation of the right limb or of both limbs and sham denervation of the right limb were used as controls. In experimental series I a single denervation of the right limb was performed at the time of amputation. In experimental series II repeated denervations were performed (before, during and after amputation). Results show that in larvae at stages 51-53 subjected to limb amputation at the proximal level (thigh) even repeated denervation of the right limb did not prevent regeneration, although giving rise to various degrees of hypotrophy. In stage-55 larvae partial inhibition of the regenerative process in the right limb was clearly visible only after repeated denervations and amputation at the proximal level. After amputation at the distal level (tarsalia) the regenerative process in the right limb underwent no significant delay with respect to the controls, although the regenerated right limb was hypotrophic. In stage-57 larvae even a single denervation at the time of amputation was enough to inhibit regeneration of the right limb after either proximal or distal amputation. Therefore, in Xenopus laevis larvae, nerve-dependence for hindlimb regeneration takes place proximodistally as the nerve fibers grow in the limb and it gradually undergoes a process of proximodistal differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in polyamine content during limb regeneration in adult Xenopus laevis

Polyamine contents in the regenerates were determined at various stages after amputation of the forelimbs of the adult female Xenopus laevis. Putrescine, spermidine, spermine, and sym-homospermidine were detected in all the specimens examined. Cadaverine was detected only in a limited number of samples. At 5 days after amputation of forelimbs, well before the formation of regenerates, the putrescine content in the stump tissues increased, followed by the increase in spermidine content. The putrescine level in the forelimb regenerates was highest between 30 and 50 days after amputation, and then decreased. The spermidine concentration in the regenerates was about 20 times greater than that in intact forelimbs all throughout the experiments. The concentration of spermine was initially lower than that of both putrescine and spermidine and further decreased soon after amputation. The concentration of sym-homospermidine was originally very low and increased slightly during regeneration. The significance of these results, with respect to the function of polyamines in forelimb regeneration of Xenopus laevis, is discussed.     

Protamine polymorphism in Xenopus laevis laevis

Protamines from individual frogs of the subspecies Xenopus laevis laevis were compared by electrophoresis on polyacrylamide gels containing acetic acid, urea, and Triton X-100 to determine if the expression of protamine genes differs among individuals. Two electrophoretic bands, SP2a and SP2b, appeared to be expressed as allelic variants. Of 33 frogs, 19 expressed only SP2a, 11 expressed both SP2a and SP2b, and three expressed only SP2b. Electrophoretic analysis of partial V8 protease digests could not distinguish the peptides released from SP2a and SP2b. Differences in sperm development between individuals were not detected by light or electron microscopy. The results suggest that protamine polymorphism can exist among individuals of a species without an apparent effect on sperm development or sperm function.     

Apoptosis is required during early stages of tail regeneration in Xenopus laevis

The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.     

Alternative housing for Xenopus laevis

At the authors' facility, housing arrangements for Xenopus laevis were cumbersome and labor-intensive, requiring technicians to wash frog tanks by hand several times a week. The authors describe an alternative housing solution they implemented by modifying a rack system that was originally used to maintain zebrafish. The rack's self-contained water circulation and filtration system saved technicians time and labor, and a commercial chiller attached to the mechanism efficiently controlled frogs' environmental temperature.     

The influence of denervation on grafted hindlimb regeneration of larval Xenopus laevis

The aim of the present research is to ascertain whether in larval Xenopus laevis nerve-independence for the regeneration of early stage limbs and nerve-dependence of late stage limbs observed in a previous work (Filoni and Paglialunga, '90) is related to extrinsic (systemic) factors or to intrinsic changes taking place in the limb cells themselves during development. In this paper the regenerative capacity of early and late stage hindlimbs under the same extrinsic conditions, insofar as both are grafted onto the denervated hindlimbs of host larvae at the same developmental stage, is studied. All the grafted limbs are amputated after the host larvae have reached stage 57-58 (according to Nieuwkoop and Faber, '56). In experiment I, the grafted limb is amputated at stage 52, at the thigh level; in experiment II, the grafted limb is amputated at stage 54-55, at the tarsalia level; in experiment III the grafted limb is amputated at stage 57, at the tarsalia level. In all three experiments, together with the grafted limb, also the host limb is amputated at the tarsalia level. The results show that while grafted limbs amputated at stages 52 and 54-55 regenerate in the absence of nerves, grafted limbs amputated at stage 57 cannot. The failure of late stage grafted limbs to regenerate cannot be explained in terms of an immune-type inhibiting reaction since it has been observed also in denervated autografted limbs and in the host limbs. Since all the grafted limbs are in the same environmental conditions, the results show that in larval Xenopus laevis nerve-independence for regeneration of early stage limbs and nerve-dependence of late stage limbs are not related to factors extrinsic to the limb but to intrinsic changes taking place in the limb cells themselves during development.     

非洲爪蟾眼的形态发生研究

详细观察和描述了非洲爪蟾Xenopus laevis眼的发生和发育变化过程,并分别对各发育时期视网膜的厚度进行了定量分析.非洲爪蟾眼的发牛开始于眼原基的形成,进而形成视泡;晶状体的发生是在视杯外壁增厚的同时诱导覆盖其上的胚胎外胚层内层增厚,形成预定晶状体板;在视网膜和晶状体共同诱导下,预定角膜上皮变为透明的角膜.在视杯出现之前,预定RPE的厚度由厚变薄,NR层不断地增厚直至结构功能完善.     

Telomere Variation in Xenopus laevis

Eukaryotic telomeres are variable at several levels, from the length of the simple sequence telomeric repeat tract in different cell types to the presence or number of telomere-adjacent DNA sequence elements in different strains or individuals. We have investigated the sequence organization of Xenopus laevis telomeres by use of the vertebrate telomeric repeat (TTAGGG)_n and blot hybridization analysis. The (TTAGGG)_n-hybridizing fragments, which ranged from less than 10 to over 50 kb with frequently cutting enzymes, defined a pattern that was polymorphic between individuals. BAL 31 exonuclease treatment confirmed that these fragments were telomeric. The polymorphic fragments analyzed did not hybridize to 5S RNA sequences, which are telomeric according to in situ hybridization. When telomeric fragments from offspring (whole embryos) were compared to those from the spleens of the parents, the inheritance pattern of some bands was found to be unusual. Furthermore, in one cross, the telomeres of the embryo were shorter than the telomeres of the parents’ spleen, and in another, the male’s testis telomeres were shorter than those of the male’s spleen. Our data are consistent with a model for chromosome behavior that involves a significant amount of DNA rearrangement at telomeres and suggest that length regulation of Xenopus telomeres is different from that observed for Mus spretus and human telomeres.     

Nerve-dependent expression of c-myc protein during forelimb regeneration of Xenopus laevis froglets.

The consequences of denervation on the expression of c-myc protein have been analyzed on the regenerating forelimb of young froglets of Xenopus laevis. The level of c-myc expression, low in control limbs and enhanced in the regenerate, is transiently increased after a three-hour total denervation. For this protein, the level of expression is not a function of the quantity of nerve in the regenerate. Four days after denervation, c-myc signal is back to its base level observed in the regenerate. A different pattern of expression is obtained for an S phase marker (PCNA protein) taken as a control in the same experimental conditions. The data presented here show that the nervous system normally exerts a negative control on the expression of c-myc and PCNA proteins in the limb regenerate of Xenopus.     

Gene-centromere mapping in Xenopus laevis

Gynogenesis was used to map eight loci to their centromeres in Xenopus laevis. Several loci remote from their centromeres were identified. This information may be useful in distinguishing gynogenetic diploid progeny produced by suppression of second polar bodies from gynogenetic diploid progeny homozygous at all loci produced by suppression of first cleavage of gynogenetic haploids.     

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.     

Mitogen-induced B-cell differentiation in Xenopus laevis

Abstract. Four genes are known to affect pigmentation in the Mexican axolotl. The purpose of this article is to review previous information pertinent to these genes and to reevaluate such information in light of new evidence that demonstrates (in a preliminary way) how pigments, and subsequently phenotypes, are affected by the various pigment genes. Each of the mutant phenotypes – m (melanoid), ax (axanthic), a (albino), and d (white) - is compared to the wild type (D). All of these genes are recessives, all of them affect phenotypic changes during development, and three of the four ( m, a , and d ) also affect specific biochemical (i.e., pigment) changes during development. In the axolotl, color patterns can be directly correlated to the presence(or absence) of a variety of pigments that are normally found in discrete pigment cells. Qualitative and quantitative analyses of the bright-colored pigments (pteridines and flavins in this case) present in axolotl skin demonstrate that these pigments vary significantly among the various phenotypes under consideration. These analyses raise some interesting questions with regard to how each of the pigment genes is believed to act, and numerous possibilities for continued experimentation are suggested.