Sergio Filoni

Relationships between Presence of the Eye Cup and Maintenance of Lens‐Forming Capacity in Larval Xenopus laevis

The lentectomized eye of larval Xenopus laevis can regenerate a lens by a process of lens‐transdifferentiation of the cornea and pericorneal epidermis. These tissues can form the lens only when they become in direct communication with the environment of the vitreous chamber (neural retina) indicating that the eye cup plays a fundamental role in this process.

The lens-regenerating competence in the outer cornea and epidermis of larval Xenopus laevis is related to pax6 expression

After lentectomy, larval Xenopus laevis can regenerate a new lens by transdifferentiation of the outer cornea and pericorneal epidermis (lentogenic area). This process is promoted by retinal factor(s) accumulated into the vitreous chamber. To understand the molecular basis of the lens‐regenerating competence (i.e. the capacity to respond to the retinal factor forming a new lens) in the outer cornea and epidermis, we analysed the expression of otx2, pax6, sox3, pitx3, prox1, βB1‐cry (genes all involved in lens development) by Real‐time RT‐PCR in the cornea and epidermis fragments dissected from donor larvae. The same fragments were also implanted into the vitreous chamber of host larvae to ascertain their lens‐regenerating competence using specific anti‐lens antibodies. The results demonstrate that there is a tight correlation between lens‐regenerating competence and pax6 expression. In fact, (1) pax6 is the only one of the aforesaid genes to be expressed in the lentogenic area; (2) pax6 expression is absent in head epidermis outside the lentogenic area and in flank epidermis, both incapable of transdifferentiating into lens after implantation into the vitreous chamber; (3) in larvae that have undergone eye transplantation under the head or flank epidermis, pax6 re‐expression was observed only in the head epidermis covering the transplanted eye. This is consistent with the fact that only the head epidermis reacquires the lens‐regenerating competence after eye transplantation, forming a lens following implantation into the vitreous chamber; and (4) in larvae that have undergone removal of the eye, the epidermis covering the orbit maintained pax6 expression. This is consistent with the fact that after the eye enucleation the lentogenic area maintains the lens‐regenerating competence, giving rise to a lens after implantation into the vitreous chamber. Moreover, we observed that misexpression of pax6 is sufficient to promote the acquisition of the lens‐regenerating competence in flank epidermis. In fact, flank epidermis fragments dissected from pax6 RNA injected embryos could form lenses when implanted into the vitreous chamber. The data indicate for the first time that pax6 is a pivotal factor of lens‐regenerating competence in the outer cornea and epidermis of larval X. laevis.

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.

The optic vesicle promotes cornea to lens transdifferentiation in larval Xenopus laevis.

The outer cornea and pericorneal epidermis (lentogenic area) of larval Xenopus laevis are the only epidermal regions competent to regenerate a lens under the influence of the retinal inducer. However, the head epidermis of the lentogenic area can acquire the lens‐regenerating competence following transplantation of an eye beneath it. In this paper we demonstrate that both the outer cornea and the head epidermis covering a transplanted eye are capable of responding not only to the retinal inducer of the larval eye but also to the inductive action of the embryonic optic vesicle by synthesizing crystallins. As the optic vesicle is a very weak lens inductor, which promotes crystallin synthesis only on the lens biased ectoderm of the embryo, these results indicate that the lens‐forming competence in the outer cornea and epidermis of larval X. laevis corresponds to the persistence and acquisition of a condition similar to that of the embryonic biased ectoderm.

Effect of ammodytin L from the venom of Vipera ammodytes on Xenopus laevis differentiated muscle fibres and regenerating limbs.

Ammodytin L is a non-catalytic, phospholipase-like snake venom toxin from Vipera ammodytes, which shows a cytotoxic activity on differentiated myotubes when tested in vitro. In the range of concentrations in which ammodytin L induced necrosis of myogenic cells in culture, other cell types (erythrocytes, platelets, fibroblasts) did not appear to be affected. To test the in vivo toxicity and the effective cytolytic specificity of ammodytin L we have followed the morphological changes in muscle tissue of Xenopus laevis limbs after intramuscular toxin injection. Only muscular cells were affected by ammodytin L, and the toxin did not induce any morphological change in other cell types. Further evidence of the muscle-specific action of the toxin was obtained from experiments carried out using the Xenopus kidney cell line B3.2 in culture. Ammodytin L was unable to affect parameters of cell viability such as lactate dehydrogenase leakage, [3H]thymidine incorporation, growth curves and morphological changes. Moreover, direct ammodytin L application to cultured regenerative limbs did not provoke alterations in undifferentiated myoblasts. These data suggest that ammodytin L, like other phospholipase-like toxins, exerts its toxicity by selectively damaging differentiated muscle fibres.

Effects of thyroxine and propyl-thiouracil on hindlimb regeneration of larvalXenopus laevis

Xenopus laevis larvae at stage 53 and 55 (according to Nieuwkoop and Faber 1956) were subjected to amputation of one or both hindlimbs and reared either in thyroxine (T4) 2.5 to 10 μg/l or in propyl-thiouracil (PTU) 0.01%. Results have shown that when the limb was amputated through a nearly undifferentiated region (tarsalia level, at stage 53) or through a differentiating region (tarsalia level, at stage 55), T4 accelerated the regenerative process and enhanced the mitotic and labelling indices of blastemal cells, when compared with controls. However, PTU delayed the regenerative process and lowered the mitotic and labelling indices. When the limb was amputated through an almost differentiated region (mid-thigh level, at stage 55), T4 inhibited the conic blastema formation, while PTU did not significatively influence limb regeneration. T4 did not modify the morphogenetic properties of the regenerative blastemata, which are characteristic of the developmental stage and the degree of differentiation of the limb tissues at the amputation level. On the whole, the data show that T4, besides being indirectly responsible for the decline of the limb regenerative capacity in a proximodistal direction by promoting limb differentiation, also exerts a direct effect on the regenerative process.

Transdifferentiation of larval Xenopus laevis iris under the influence of the pituitary

Fragments of larvalXenopus laevis dorsal iris implanted together with the pituitary into the tail fin transdifferentiate into neural retina. On the contrary, in the control experiments the implanted tissues, dorsal iris alone, pituitary, or dorsal iris with liver fragments, do not undergo any retinal transformation.

Neurogenesis during optic tectum regeneration in Xenopus laevis

The regenerative neurogenesis of the optic tectum of larval Xenopus laevis has been studied analyzing the proliferative and morphogenetic phases of the regeneration process after removal of one optic lobe. To this end, short‐term and long‐term pulses were carried out using the thymidine analog BrdU, selectively incorporated into cells during the S phase of the cell cycle. Results indicate that while in early larvae (stage 49/50, according to Nieuwkoop & Faber 1967 ) regeneration occurs mainly at the expense of the stem cells present in extensive proliferation zones (“matrix areas”) of the midbrain, in late larvae (stage 55/56) regeneration occurs at the expense of stem cells present in very limited matrix areas of the brain and of quiescent cells, which re‐enter the cell cycle following trauma. Moreover, in early larvae, morphogenesis of the optic tectum is carried out according to a precise spatio‐temporal order from rostro‐caudal to latero‐medial. By contrast, in late larvae, the topographical order of the regenerative morphogenesis of the optic lobe is completely altered. As a consequence, the regenerated optic tectum in early larvae has an apparently normal structure, while the regenerated optic tectum in late larvae lacks stratification.

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.

Dachshund expression during embryonic and larval development of Xenopus laevis

Abstract Two Xenopus Dachshund genes, XDachA and XDachB, were isolated, whose cDNAs were 2273 and 2117 bp long, respectively. They contained full‐length open reading frames of 610 and 558 amino acids and showed an amino acid sequence highly similar to murine Dachl (70%) and chick Dachl (60%). The two Xenopus proteins presented a marked similarity (about 95%) at the level of the Dachbox‐N of other Dach proteins and a low level of similarity (about 30%) at that of the Dachbox‐C. DachA and DachB expression was analyzed by “whole mount”; in situ hybridization and RT‐PCR on embryos, at stages between 12 and 34, larval brain, eye and limb buds. The two genes are expressed in overlapping patterns during eye, ear, brain and limb development and show a significant expression similarity to mouse and chick Dachl.