Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate

The tail of the frog tadpole, comprising spinal cord, muscle, and notochord, regenerates following partial amputation. We show that, in Xenopus, this occurs throughout development, except for a "refractory period" between stages 45 and 47, when tails heal over without regeneration. Regeneration can be enabled during this refractory period by activation of either the BMP or Notch signaling pathways. Conversely, regeneration can be prevented during the later, regenerative, stages by inhibition of either pathway. BMP signaling will cause regeneration of all tissues, whereas Notch signaling activates regeneration of spinal cord and notochord, but not muscle. An activated form of Msx1 can promote regeneration in the same way as BMP signaling. Epistasis experiments suggest that BMP signaling is upstream of Notch signaling but exerts an independent effect on muscle regeneration. The results demonstrate that regenerative capability can be enabled by genetic modifications that reactivate specific components of the developmental program.     

Cell lineage tracing during Xenopus tail regeneration

The tail of the Xenopus tadpole will regenerate following amputation, and all three of the main axial structures - the spinal cord, the notochord and the segmented myotomes - are found in the regenerated tail. We have investigated the cellular origin of each of these three tissue types during regeneration. We produced Xenopus laevis embryos transgenic for the CMV (Simian Cytomegalovirus) promoter driving GFP (Green Fluorescent Protein) ubiquitously throughout the embryo. Single tissues were then specifically labelled by making grafts at the neurula stage from transgenic donors to unlabelled hosts. When the hosts have developed to tadpoles, they carry a region of the appropriate tissue labelled with GFP. These tails were amputated through the labelled region and the distribution of labelled cells in the regenerate was followed. We also labelled myofibres using the Cre-lox method. The results show that the spinal cord and the notochord regenerate from the same tissue type in the stump, with no labelling of other tissues. In the case of the muscle, we show that the myofibres of the regenerate arise from satellite cells and not from the pre-existing myofibres. This shows that metaplasia between differentiated cell types does not occur, and that the process of Xenopus tail regeneration is more akin to tissue renewal in mammals than to urodele tail regeneration.     

Control of muscle regeneration in the Xenopus tadpole tail by Pax7

The tail of the Xenopus tadpole will regenerate completely after transection. Much of the mass of the regenerate is composed of skeletal muscle, but there has been some uncertainty about the source of the new myofibres. Here, we show that the growing tail contains many muscle satellite cells. They are active in DNA replication, whereas the myonuclei are not. As in mammals, the satellite cells express pax7. We show that a domain-swapped construct, pax7EnR, can antagonize pax7 function. Transgenic tadpoles were prepared containing pax7EnR driven by a heat-inducible promoter. When induced, this reduces the proportion of satellite cells formed in the regenerate. A second amputation of the resulting tails yielded second regenerates containing notochord and spinal cord but little or no muscle. This shows that inhibition of pax7 action does not prevent differentiation of satellite cells to myofibres, but it does prevent their maintenance as a stem cell population.     

Evidence that regenerative ability is an intrinsic property of limb cells in Xenopus

Xenopus laevis exhibits an ontogenetic decline in the ability to regenerate its limbs: Young tadpoles can completely regenerate an amputated limb, whereas post metamorphic froglets regenerate at most a cartilagenous "spike." We have tested the regenerative competence of normally regenerating limb buds of stage 52-53 Xenopus tadpoles grafted onto limb stumps of postmetamorphic froglets. The limb buds become vascularized and innervated by the host and, when amputated, regenerate limbs with normal or slightly less than normal numbers of tadpole hindlimb digits. Reciprocal grafts of froglet forelimb blastemas onto tadpole hindlimb stumps resulted in either autonomous development of tadpole hindlimb structures and/or formation of a cartilaginous spike typical of froglet forelimb regeneration. Our results suggest that the Xenopus froglet host environment is completely permissive for regeneration and that the ability to regenerate a complete limb pattern is an intrinsic property of young tadpole limb cells, a property that is lost during ontogenesis.     

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.     

Tail regeneration in the Xenopus tadpole

The tail of the Xenopus tadpole contains major axial structures, including a spinal cord, notochord and myotomes, and regenerates within 2 weeks following amputation. The tail regeneration in Xenopus can provide insights into the molecular basis of the regeneration mechanism. The regenerated tail has some differences from the normal tail, including an immature spinal cord and incomplete segmentation of the muscle masses. Lineage analyses have suggested that the tail tissues are reconstructed with lineage-restricted stem cells derived from their own tissues in clear contrast to urodele regeneration, in which multipotent blastema cells derived from differentiated cells play a major role. Comprehensive gene expression analyses resulted in the identification of a panel of genes involved in sequential steps of the regeneration. Manipulation of genes' activities suggested that the tail regeneration is regulated through several major signaling pathways.     

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.     

Spinal cord is required for proper regeneration of the tail in Xenopus tadpoles

Tail regeneration in urodeles is dependent on the spinal cord (SC), but it is believed that anuran larvae regenerate normal tails without the SC. To evaluate the precise role of the SC in anuran tail regeneration, we developed a simple operation method to ablate the SC completely and minimize the damage to the tadpole using Xenopus laevis . The SC-ablated tadpole regenerated a twisted and smaller tail. These morphological abnormalities were attributed to defects in the notochord (NC), as the regenerated NC in the SC-ablated tail was short, slim and twisted. The SC ablation never affected the early steps of the regeneration, including closure of the amputated surface with epidermis and accumulation of the NC precursor cells. The proliferation rate of the NC precursor cells, however, was reduced, and NC cell maturation was retarded in the SC-ablated tail. These results show that the SC has an essential role in the normal tail regeneration of Xenopus larvae, especially in the proliferation and differentiation of the NC cells. Gene expression analysis and implantation of a bead soaked with growth factor showed that fibroblast growth factor-2 and -10 were involved in the signaling molecules, which were expressed in the SC and stimulated growth of the NC cells.     

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.     

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.     

Developmental aspects of spinal cord and limb regeneration

The ability of birds and mammals to regenerate tissues is limited. By contrast, urodele amphibians can regenerate a variety of injured tissues such as intestine, cardiac muscle, lens and neural retina, as well as entire structures such as limbs, tail and lower jaw. This regenerative capacity is associated with the ability to form masses of mesenchyme cells (blastemas) that differentiate into the missing tissues or parts. Understanding the mechanisms that underlie blastema formation in urodeles will provide valuable tools with which to achieve the goal of stimulating regeneration in mammalian tissues that do not naturally regenerate. Here we discuss an example of tissue regeneration (spinal cord) and an example of epimorphic appendage regeneration (limb) in the axolotl Ambystoma mexicanum , emphasizing analysis of the processes that produce the regeneration blastema and of the tissue interactions and blastemal products that contribute to the regeneration-promoting environment.     

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Background

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results

Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions

Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.     

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.     

Early gene expression during natural spinal cord regeneration in the salamander Ambystoma mexicanum

In contrast to mammals, salamanders have a remarkable ability to regenerate their spinal cord and recover full movement and function after tail amputation. To identify genes that may be associated with this greater regenerative ability, we designed an oligonucleotide microarray and profiled early gene expression during natural spinal cord regeneration in Ambystoma mexicanum. We sampled tissue at five early time points after tail amputation and identified genes that registered significant changes in mRNA abundance during the first 7 days of regeneration. A list of 1036 statistically significant genes was identified. Additional statistical and fold change criteria were applied to identify a smaller list of 360 genes that were used to describe predominant expression patterns and gene functions. Our results show that a diverse injury response is activated in concert with extracellular matrix remodeling mechanisms during the early acute phase of natural spinal cord regeneration. We also report gene expression similarities and differences between our study and studies that have profiled gene expression after spinal cord injury in rat. Our study illustrates the utility of a salamander model for identifying genes and gene functions that may enhance regenerative ability in mammals.     

Regeneration of neural crest derivatives in the <Emphasis Type="Italic">Xenopus</Emphasis>tadpole tail

Background  

After amputation of the Xenopus tadpole tail, a functionally competent new tail is regenerated. It contains spinal cord, notochord and muscle, each of which has previously been shown to derive from the corresponding tissue in the stump. The regeneration of the neural crest derivatives has not previously been examined and is described in this paper.     

Identification of genes associated with regenerative success of <Emphasis Type="Italic">Xenopus laevis</Emphasis>hindlimbs

Background  

Epimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.