Electric currents in Xenopus tadpole tail regeneration

Xenopus laevis tadpoles can regenerate tail, including spinal cord, after partial amputation, but lose this ability during a specific period around stage 45. They regain this ability after stage 45. What happens during this “refractory period” might hold the key to spinal cord regeneration. We hypothesize that electric currents at amputated stumps play significant roles in tail regeneration. We measured electric current at tail stumps following amputation at different developmental stages. Amputation induced large outward currents leaving the stump. In regenerating stumps of stage 40 tadpoles, a remarkable reversal of the current direction occurred around 12-24 h post-amputation, while non-regenerating stumps of stage 45 tadpole maintained outward currents. This reversal of electric current at tail stumps correlates with whether tails regenerate or not (regenerating stage 40—inward current; non-regenerating stage 45—outward current). Reduction of tail stump current using sodium-free solution decreased the rate of regeneration and percentage regeneration. Fin punch wounds healed normally at stages 45 and 48, and in sodium-free solution, suggesting that the absence of tail re-growth at stage 45 is regeneration-specific rather than a general inhibition of wound healing. These data suggest that electric signals might be one of the key players regulating regeneration.     

Acute phase response in amputated tail stumps and neural tissue‐preferential expression in tail bud embryos of the Xenopus neuronal pentraxin I gene

Regeneration of lost organs involves complex processes, including host defense from infection and rebuilding of lost tissues. We previously reported that Xenopus neuronal pentraxin I (xNP1) is expressed preferentially in regenerating Xenopus laevis tadpole tails. To evaluate xNP1 function in tail regeneration, and also in tail development, we analyzed xNP1 expression in tailbud embryos and regenerating/healing tails following tail amputation in the ‘regeneration’ period, as well as in the ‘refractory’ period, when tadpoles lose their tail regenerative ability. Within 10 h after tail amputation, xNP1 was induced at the amputation site regardless of the tail regenerative ability, suggesting that xNP1 functions in acute phase responses. xNP1 was widely expressed in regenerating tails, but not in the tail buds of tailbud embryos, suggesting its possible role in the immune response/healing after an injury. xNP1 expression was also observed in neural tissues/primordia in tailbud embryos and in the spinal cord in regenerating/healing tails in both periods, implying its possible roles in neural development or function. Moreover, during the first 48 h after amputation, xNP1 expression was sustained at the spinal cord of tails in the ‘regeneration’ period tadpoles, but not in the ‘refractory’ period tadpoles, suggesting that xNP1 expression at the spinal cord correlates with regeneration. Our findings suggest that xNP1 is involved in both acute phase responses and neural development/functions, which is unique compared to mammalian pentraxins whose family members are specialized in either acute phase responses or neural functions.     

Appendage-restricted gene induction using a heated agarose gel for studying regeneration in metamorphosed Xenopus laevis and Pleurodeles waltl

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage-specific gene induction using a simplified protocol named the “agarose-embedded heat shock (AeHS) method” involving the combination of a heat-shock-inducible system and insertion of an appendage in a temperature-controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord.     

Temporal distribution of 5BrdU-labelled cells suggests that most injured tissues contribute proliferating cells for the regeneration of the tail and limb in lizard

After tail and limb amputation in lizard, injection of 5BrdU for 6 days produces immunolabelled cells in most tissues of tail and limb stumps. After further 8 and 16 days, and 14 and 22 days of regeneration, numerous 5BrdU-labelled cells are detected in regenerating tail and limb, derived from most stump tissues. In tail blastema cone at 14 days, sparse-labelled cells remain in proximal dermis, muscles, cartilaginous tube and external layers of wound epidermis but are numerous in the blastema. In apical regions at 22 days of regeneration, labelled mesenchymal cells are sparse, while the apical wound epidermis contains numerous labelled cells in suprabasal and external layers, indicating cell accumulation from more proximal epidermis. Cell proliferation dilutes the label, and keratinocytes take 8 days to migrate into corneous layers. In healing limbs, labelled cells remain sparse from 14 to 22 days of regeneration in wound epidermis and repairing tissues and little labelling dilution occurs indicating low cell proliferation for local tissue repair but not distal growth. Labelled cells are present in epidermis, intermuscle and peri-nerve connectives, bone periosteum, cartilaginous callus and sparse fibroblasts, leading to the formation of a scarring outgrowth. Resident stem cells and dedifferentiation occur when stump tissues are damaged.     

Patterns of Mitosis and Activation of the Map-Kinase Cascade during Tadpole Tail Regeneration in the Refractory Period of <Emphasis Type="Italic">Xenopus laevis</Emphasis> Development

Patterns of mitotic cells’ distribution and activation of the MAP-kinase cascade during the regeneration of Xenopus laevis tadpole tails were studied before and during the refractory period. It is known that the tadpoles of Xenopus laevis are able to fully restore the full structure of the tail after amputation. However, in the refractory period (stage 45–47), the ability to regenerate is significantly reduced, until its complete absence. The mechanisms of this phenomenon are still poorly understood. We conducted a comparative analysis of the average number of mitotic cells on 0–4 days post amputation in normally regenerating tails and in tails amputated during the refractory period. A significant decrease in the number of proliferating cells throughout the surface of the tail in the refractory period compared with their sharp increase in the blastema area in normally regenerating tadpoles was shown. In addition, we detected activation of the MAP-kinase cascade (dpERK1/2) during normal regeneration and demonstrated its full inhibition during the refractory period. At the same time, in the distal part of the tail amputated in the refractory period, activation of the expression of the regenerative marker gene Fgf20 was not detected. Thus, we can conclude that the blocking of the regenerative capacity in tadpoles during the refractory period is accompanied by a sharp suppression of the mitotic activity of the cells and a misregulation of the activation of the Fgf–MAP-kinase cascade in the tail after amputation.     

Transient laminin beta 1a Induction Defines the Wound Epidermis during Zebrafish Fin Regeneration

The first critical stage in salamander or teleost appendage regeneration is creation of a specialized epidermis that instructs growth from underlying stump tissue. Here, we performed a forward genetic screen for mutations that impair this process in amputated zebrafish fins. Positional cloning and complementation assays identified a temperature-sensitive allele of the ECM component laminin beta 1a (lamb1a) that blocks fin regeneration. lamb1a, but not its paralog lamb1b, is sharply induced in a subset of epithelial cells after fin amputation, where it is required to establish and maintain a polarized basal epithelial cell layer. These events facilitate expression of the morphogenetic factors shha and lef1, basolateral positioning of phosphorylated Igf1r, patterning of new osteoblasts, and regeneration of bone. By contrast, lamb1a function is dispensable for juvenile body growth, homeostatic adult tissue maintenance, repair of split fins, or renewal of genetically ablated osteoblasts. fgf20a mutations or transgenic Fgf receptor inhibition disrupt lamb1a expression, linking a central growth factor to epithelial maturation during regeneration. Our findings reveal transient induction of lamb1a in epithelial cells as a key, growth factor-guided step in formation of a signaling-competent regeneration epidermis.     

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     

Methods of In Vivo Gene-Specific Knockdown Using Morpholino and Vivo-Morpholino Oligonucleotides

The functioning of the small GTPase gene, Ras-dva1, has been studied during regeneration processes of the tadpole tails of the clawed frog Xenopus laevis. For this purpose, we have developed two approaches for the gene knockdown using injections of sequence-specific antisense morpholino oligonucleotides (MO) or vivo-morpholino oligonucleotides (vivo-MO). It has been shown for the first time that intracellular Ras-dva-specific MO injected into Xenopus 4–16 of blastomere embryos or vivo-MO injected directly into the distal part of the tadpole tail at stages 40–41 efficiently inhibit the Ras-dva1 gene functioning and leads to the same morphological malformations of the tail regeneration. Using qRT-PCR, we detected the inhibition of expression of the regeneration marker genes (Fgf20a and Msx1b) on the first day after amputation in the tail tissues of tadpoles with the Ras-dva1 knockdown.     

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.     

Skin wound healing in different aged Xenopus laevis

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8‐ and 15‐month‐old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re‐epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti‐inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti‐tumor necrosis factor (TNF)‐α, iNOS, transforming growth factor (TGF)‐β1, and matrix metalloproteinase (MMP)‐9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti‐α‐SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar‐like pattern. The quantitative PCR analysis demonstrated an up‐regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar‐like tissue formation. J. Morphol. 274:956–964, 2013. © 2013 Wiley Periodicals, Inc.     

Retinoic acid enhances monoclonal antibody WE3 reactivity in the regenerate epithelium of the adult newt.

Monoclonal antibody (mAb) WE3 recognizes an antigen that is developmentally expressed in the wound epithelium during adult newt limb regeneration. Experiments were designed to determine whether retinoic acid (RA), dissolved in dimethyl sulfoxide (DMSO) and administered by intraperitoneal injection, would enhance the temporal appearance of the WE3 antigen. RA given on days 1 or 4 after amputation, when the WE3 antigen is not yet detectable, resulted in moderate reactivity to mAb 2 days after injection and strong reactivity throughout the wound epithelium 4 days after injection. DMSO alone had no enhancing effect. RA also caused limb skin epidermis to exhibit reactivity to mAb WE3, initially near the amputation level, but then also more proximally. By 4 and 6 days after RA injection, epidermis of the flank, eye lid, and unamputated hind limbs also became strongly reactive to mAb WE3. Outer layers of skin epidermis were shed, resulting in an epidermis only one or two cells thick. Epidermis of newts given DMSO alone remained non-reactive to mAb WE3. When RA was given on days 7 and 10 after amputation, when a low level of mAb WE3 reactivity is already present in the wound epithelium, a considerable enhancement of mAb WE3 reactivity occurred through the next few days. No such enhancement was seen with DMSO alone. RA also greatly increased mAb WE3 reactivity in the wound epithelium of denervated limbs, in which case the wound epithelial reactivity to mAb WE3 is normally low. Retinol palmitate also increased mAb WE3 reactivity. The results raise the possibility that the WE3 antigen is a component of most if not all retinoid target tissues in newts.     

Expression of FGF2 in the limb blastema of two Salamandridae correlates with their regenerative capability

Limb regenerative potential in urodeles seems to vary among different species. We observed that Triturus vulgaris meridionalis regenerate their limbs significantly faster than T. carnifex, where a long gap between the time of amputation and blastema formation occurs, and tried to identify cellular and molecular events that may underlie these differences in regenerative capability. Whereas wound healing is comparable in the two species, formation of an apical epidermal cap (AEC), which is required for blastema outgrowth, is delayed for approximately three weeks in T. carnifex. Furthermore, fewer nerve fibres are present distally early after amputation, consistent with the late onset of blastemal cell proliferation observed in T. carnifex. We investigated whether different expression of putative blastema mitogens, such as FGF1 and FGF2, in these species may underlie differences in the progression of regeneration. We found that whereas FGF1 is detected in the epidermis throughout the regenerative process, FGF2 onset of expression in the wound epidermis of both species coincides with AEC formation and initiation of blastemal cell proliferation, which is delayed in T. carnifex, and declines thereafter. In vitro studies showed that FGF2 activates MCM3, a factor essential for DNA replication licensing activity, and can be produced by blastemal cells themselves, indicating an autocrine action. These results suggest that FGF2 plays a key role in the initiation of blastema growth.     

Long-distance signals are required for morphogenesis of the regenerating Xenopus tadpole tail, as shown by femtosecond-laser ablation

Background

With the goal of learning to induce regeneration in human beings as a treatment for tissue loss, research is being conducted into the molecular and physiological details of the regeneration process. The tail of Xenopus laevis tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.

Methods and Results

Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.

Conclusions

We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches.     

Analysis of Gene Expressions during Xenopus Forelimb Regeneration

Xenopus laevis can regenerate an amputated limb completely at early limb bud stages, but the metamorphosed froglet gradually loses this capacity and can regenerate only a spike-like structure. We show that the spike formation in a Xenopus froglet is nerve dependent as is limb regeneration in urodeles, since denervation concomitant with amputation is sufficient to inhibit the initiation of blastema formation and fgf8 expression in the epidermis. Furthermore, in order to determine the cause of the reduction in regenerative capacity, we examined the expression patterns of several key genes for limb patterning during the spike-like structure formation, and we compared them with those in developing and regenerating limb buds that produce a complete limb structure. We cloned Xenopus HoxA13, a marker of the prospective autopodium region, and the expression pattern suggested that the spike-like structure in froglets is accompanied by elongation and patterning along the proximodistal (PD) axis. On the other hand, shh expression was not detected in the froglet blastema, which expresses fgf8 and msx1. Thus, although the wound epidermis probably induces outgrowth of the froglet blastema, the polarizing activity that organizes the anteroposterior (AP) axis formation is likely to be absent there. Our results demonstrate that the lost region in froglet limbs is regenerated along the PD axis and that the failure of organization of the AP pattern gives rise to a spike-like incomplete structure in the froglet, suggesting a relationship between regenerative capacity and AP patterning. These findings lead us to conclude that the spike formation in postometamorphic Xenopus limbs is epimorphic regeneration.     

Matrix metalloproteinases (MMPs) are required for wound closure and healing during larval leg regeneration in the flour beetle,Tribolium castaneum

Regenerative abilities are found ubiquitously among many metazoan taxa. To compare mechanisms underlying the initial stages of limb regeneration between insects and vertebrates, the roles of matrix metalloproteinases (MMPs) and fibroblast growth factor (FGF) signaling were investigated in the red flour beetle, Tribolium castaneum. RNA interference-mediated knockdown of MMP2 expression delayed wound healing and subsequent leg regeneration. Additionally, pairwise knockdown of MMP1/2 and MMP2/3, but not MMP1/3, resulted in inhibition of wound closure. Wound healing on the dorsal epidermis after injury was also delayed when MMPs were silenced. Our findings show that functionally redundant MMPs play key roles during limb regeneration and wound healing in Tribolium. This MMP-mediated wound healing is necessary for the subsequent formation of a blastema. In contrast, silencing of FGF receptor did not interfere with the initial stages of leg regeneration despite the alterations in tanning of the cuticle. Thus, insects and vertebrates appear to employ similar developmental processes for the initial stages of wound closure during limb regeneration, while the role of FGF in limb regeneration appears to be unique to vertebrates.