Nerve-independence of limb regeneration in larval Xenopus laevis is related to the presence of mitogenic factors in early limb tissues.

Early limbs of larval Xenopus laevis can form a regeneration blastema in the absence of nerves. The nerve-independence could be due to the synthesis of neurotrophic-like factors by the limb bud cells. To test this hypothesis, two series of experiments were performed. Series A: the right hindlimbs of stage 57 larvae (acc. to Nieuwkoop and Faber. 1956. Normal table of Xenopus laevis [Daudin]. Amsterdam: North-Holland Pub. Co.), which are nerve-dependent for regeneration, were amputated through the tarsalia. The regenerating limbs were submitted to: sham denervation; denervation; denervation and implantation of a fragment of an early limb, or a late limb, or a spinal cord. Series B: froglets were subjected to amputation of both forelimbs. The cone blastemas were transplanted into denervated hindlimbs of stage 57 larvae, together with a fragment of an early or a late limb. The results in series A showed that the implantation of early limb tissue into the denervated blastema maintained cell proliferation at levels similar to those observed after the implantation of a spinal cord fragment or in sham denervated blastemas. However, the implantation of late limb tissues were ineffective. The results of series B showed that the implantation of early limb tissue, but not of late limb tissue prevented the inhibition of cell proliferation and the regression of denervated limb blastemas of juveniles. These results indicate that the nerve-independence is related to the synthesis of diffusible mitogenic neurotrophic-like factors in early limb tissues, and that nerve-dependence is established when differentiated cells of late limb tissues stop producing these factors.     

Hyperinnervation improves Xenopus laevis limb regeneration

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.     

Mesenchyme with fgf-10 expression is responsible for regenerative capacity in Xenopus limb buds

A young tadpole of an anuran amphibian can completely regenerate an amputated limb, and it exhibits an ontogenetic decline in the ability to regenerate its limbs. However, whether mesenchymal or epidermal tissue is responsible for this decrease of the capacity remains unclear. Moreover, little is known about the molecular interactions between these two tissues during regeneration. The results of this study showed that fgf-10 expression in the limb mesenchymal cells clearly corresponds to the regenerative capacity and that fgf-10 and fgf-8 are synergistically reexpressed in regenerating blastemas. However, neither fgf-10 nor fgf-8 is reexpressed after amputation of a nonregenerative limb. Nevertheless, nonregenerative epidermal tissue can reexpress fgf-8 under the influence of regenerative mesenchyme, as was demonstrated by experiments using a recombinant limb composed of regenerative limb mesenchyme and nonregenerative limb epidermis. Taken together, our data demonstrate that the regenerative capacity depends on mesenchymal tissue and suggest that fgf-10 is likely to be involved in this capacity.     

Transdifferentiation of ocular tissues in larval Xenopus laevis

Transdifferentiation phenomena offer a useful opportunity to study experimentally the mechanisms on which cell phenotypic stability depends. The capacities of vertebrate eye tissues to reprogram cell differentiation are well known in avian and mammalian embryos, and in larval and adult newt. From research into the capacity of anuran eye tissues to reprogram differentiation into a new pathway, considerable data have accumulated concerning the transdifferentiative capacities of eye tissues in larval Xenopus laevis. This work reviews the data concerning the transdifferentiative phenomena of eye tissues in that species and, based on these, aims to establish the extent of our knowledge about the mechanism controlling these processes. In larval Xenopus laevis the outer cornea can regenerate a lens by a lens-transdifferentiation process triggered and substained by a factor(s), probably of a protein nature, produced by the neural retina. In a normal eye phenotypic stability of the outer cornea is guaranteed by the presence of the inner cornea and lens, which prevent the spread of retinal factor(s). The stimulus for lens transdifferentiation of the outer cornea can be supplied by other tissues as well, but this capacity is not widely distributed. The iris and retinal pigmented epithelium can transdifferentiate into neural retina if isolated from the surrounding tissues and implanted in the vitreous chamber. As for lens transdifferentiation of the outer cornea, retinal transdifferentiation of the iris can be stimulated by certain nonocular tissues as well.     

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 novel Xenopus laevis larval keratin gene, xlk2: its gene structure and expression during regeneration and metamorphosis of limb and tail

A novel cytokeratin (CK) gene, xlk2, was cloned from a cDNA library prepared from regenerating limbs of Xenopus larvae. The deduced amino acid sequence indicated that its product, XLK2, is a 48 kDa type I (acidic) CK and has a high similarity to CK13, 15, and 19 with the highest homology (58%) to mouse CK15. The gene of xlk2 exclusively expressed in basal cells of the bi-layered larval epidermis, but not in other cells in larvae and not in other periods of life. Its expression was down-regulated during spontaneous and thyroid hormone-induced metamorphosis. The basal cells of the apical epidermal cap (AEC) formed on the regenerate of larval limbs terminated the expression of xlk2, whereas those of the adjacent normal epidermis continued to express it. The AEC-basal cells did not re-express the gene in the regenerate. In contrast, the basal cells of the tail regenerate also once terminated the expression of xlk2, but was able to re-express xlk2 later, supporting a notion that the "de-differentiated" basal cells of the tail epidermal regenerate re-differentiate into larval normal epidermal cells.     

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.     

Developmental changes in the pattern of larval beta-globin gene expression in Xenopus laevis. Identification of two early larval beta-globin mRNA sequences

We have analysed beta-globin mRNA sequences in total RNA extracted from embryos and tadpoles of Xenopus laevis at different stages of development and we have identified the most abundantly transcribed beta-globin mRNA (beta T1). The entire nucleotide sequence of a cDNA clone corresponding to this mRNA is known. We have now identified the gene corresponding to this mRNA and we have determined the nucleotide sequences of its immediate 5'-flanking region. Using a DNA fragment from within the coding region of the cloned beta T1 cDNA we show, by primer extension analysis, that beta T1 mRNA is first detectable at stage 28-32 of development. This is the time at which the first presumptive erythropoietic tissue, the ventral blood island, becomes observable histologically. We show that two minor beta-globin genes, distinct from beta T1, are expressed during early stages of development, and that their expression ceases shortly after the beginning of the feeding stage. We term these two early larval genes beta E1 and beta E2. A third minor beta-globin gene is expressed during early development but, unlike beta E1 and beta E2, it is also expressed throughout subsequent larval development. We term this gene beta T2 and show that it corresponds to a gene previously termed beta LII. Finally, using a primer derived from the major adult beta-globin gene (beta 1), we have analysed the accumulation of the major adult beta-globin mRNA during larval development, and we show that this sequence does not accumulate to any significant level before metamorphosis.     

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.     

Pigmented epithelium to retinal transdifferentiation and Pax6 expression in larval Xenopus laevis

This study examines the retinal transdifferentiation (TD) of retinal pigmented epithelium (RPE) fragments dissected from Xenopus laevis larvae and implanted into the vitreous chamber of non-lentectomized host eyes. In these experimental conditions, most RPE implants transformed into polarized vesicles in which the side adjacent to the lens maintained the RPE phenotype, while the side adjacent to the host retina transformed into a laminar retina with the photoreceptor layer facing the cavity of the vesicle and with the ganglionar cell layer facing the host retina. The formation of a new retina with a laminar organization is the result of depigmentation, proliferation and differentiation of progenitor cells under the influence of inductive factors from the host retina. The phases of the TD process were followed using BrdU labelling as a marker of the proliferation phase and using a monoclonal antibody (mAbHP1) as a definitive indicator of retina formation. Pigmented RPE cells do not express Pax6. In the early phase of RPE to retinal TD, all depigmented and proliferating progenitor cells expressed Pax6. Changes in the Pax6 expression pattern became apparent in the early phase of differentiation, when Pax6 expression decreased in the presumptive outer nuclear layer (ONL) of the new-forming retina. Finally, during the late differentiation phase, the ONL, which contains photoreceptors, no longer expressed Pax6, Pax6 expression being confined to the ganglion cell layer and the inner nuclear layer. These results indicate that Pax6 may have different roles during the different phases of RPE to retinal TD, acting as an early retinal determinant and later directing progenitor cell fate.     

Suppression of the allograft response by implants of mature lymphoid tissues in larval Xenopus laevis

One hundred and twenty-two larvae of Xenopus laevis, the South African clawed toad, at developmental stages 48, 50, 52 and 54, were implanted in the tail with two allografts from adult tissues. In each case, one allograft was from kidney, while the other was either from kidney, thymus, spleen, or liver. In any particular host the two implants were always from the same donor and the implants were all visually matched in size. The experimental period was a maximum of nine days, so as to minimize the large numbers of changes normally accompanying larval progress from stage to stage. We are concerned with the timing of allograft response initiation under the implant conditions of each experimental group at a particular point in development. An allograft response was defined as an infiltration and accumulation of small lymphocytes in the “test” kidney allograft. Larvae of all stages developed allograft responses within one week post-implantation when the variable implant was from kidney, but implants from spleen and thymus suppressed both the timing of initiation and the subsequent intensity of the response. Spleen was more effective in this regard than thymus and both were more effective in the earlier larval stages. Liver proved to be toxic to the larvae. The relationship between the maturation of the lymphomyeloid tissues and external morphological staging is also discussed.     

Developmental expression of Shisa-2 in Xenopus laevis

Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.     

Translation of human interleukin 2 mRNA in Xenopus laevis oocytes

Poly(A)-positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin-M and 12-o-tetradecanoyl phorbol 13-acetate was successfully translated into biologically active interleukin 2 (IL-2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)-negative RNA or buffer did not show any IL-2 activity. By sucrose density gradient centrifugation analysis, IL-2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10-11S.     

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.     

The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis. The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval-specific alpha-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes. Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.     

mRNA sequence of the Xenopus laevis paxillin gene and its expression.

Paxillin is a cytoskeletal protein found in structures of focal adhesions where cells adhere to the extracellular matrix. We isolated paxillin cDNA from the Xenopus laevis ovary. The cDNA sequence encodes a protein of 539 amino acids with four LIM and five LD motifs. 80% of the amino acids of frog paxillin are shared by human and chicken paxillins. Northern analysis showed that the frog gene is expressed in the spleen, kidney, testis and ovary. Immunocytochemistry showed that paxillin protein is accumulated in the nucleus as well as in the periphery of the cytoplasm of the A6 cell. This intriguing result shows that paxillin, which has been characterized as a cytoskeletal protein, is capable of translocating to the nucleus.     

Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF-2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)-expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all-trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.