A stepwise model system for limb regeneration

The amphibian limb is a model that has provided numerous insights into the principles and mechanisms of tissue and organ regeneration. While later stages of limb regeneration share mechanisms of growth control and patterning with limb development, the formation of a regeneration blastema is controlled by early events that are unique to regeneration. In this study, we present a stepwise experimental system based on induction of limb regeneration from skin wounds that will allow the identification and functional analysis of the molecules controlling this early, critical stage of regeneration. If a nerve is deviated to a skin wound on the side of a limb, an ectopic blastema is induced. If a piece of skin is grafted from the contralateral side of the limb to the wound site concomitantly with nerve deviation, the ectopic blastema continues to grow and forms an ectopic limb. Our analysis of dermal cell migration, contribution, and proliferation indicates that ectopic blastemas are equivalent to blastemas that form in response to limb amputation. Signals from nerves are required to induce formation of both ectopic and normal blastemas, and the diversity of positional information provided by blastema cells derived from opposite sides of the limb induces outgrowth and pattern formation. Hence, this novel and convenient stepwise model allows for the discovery of necessary and sufficient signals and conditions that control blastema formation, growth, and pattern formation during limb regeneration.     

Nerve-induced ectopic limb blastemas in the Axolotl are equivalent to amputation-induced blastemas

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.     

Combining classical and molecular approaches elaborates on the complexity of mechanisms underpinning anterior regeneration

The current model of planarian anterior regeneration evokes the establishment of low levels of Wnt signalling at anterior wounds, promoting anterior polarity and subsequent elaboration of anterior fate through the action of the TALE class homeodomain PREP. The classical observation that decapitations positioned anteriorly will regenerate heads more rapidly than posteriorly positioned decapitations was among the first to lead to the proposal of gradients along an anteroposterior (AP) axis in a developmental context. An explicit understanding of this phenomenon is not included in the current model of anterior regeneration. This raises the question what the underlying molecular and cellular basis of this temporal gradient is, whether it can be explained by current models and whether understanding the gradient will shed light on regenerative events. Differences in anterior regeneration rate are established very early after amputation and this gradient is dependent on the activity of Hedgehog (Hh) signalling. Animals induced to produce two tails by either Smed-APC-1(RNAi) or Smed-ptc(RNAi) lose anterior fate but form previously described ectopic anterior brain structures. Later these animals form peri-pharyngeal brain structures, which in Smed-ptc(RNAi) grow out of the body establishing a new A/P axis. Combining double amputation and hydroxyurea treatment with RNAi experiments indicates that early ectopic brain structures are formed by uncommitted stem cells that have progressed through S-phase of the cell cycle at the time of amputation. Our results elaborate on the current simplistic model of both AP axis and brain regeneration. We find evidence of a gradient of hedgehog signalling that promotes posterior fate and temporarily inhibits anterior regeneration. Our data supports a model for anterior brain regeneration with distinct early and later phases of regeneration. Together these insights start to delineate the interplay between discrete existing, new, and then later homeostatic signals in AP axis regeneration.     

activin-2 is required for regeneration of polarity on the planarian anterior-posterior axis

Planarians are flatworms and can perform whole-body regeneration. This ability involves a mechanism to distinguish between anterior-facing wounds that require head regeneration and posterior-facing wounds that require tail regeneration. How this head-tail regeneration polarity decision is made is studied to identify principles underlying tissue-identity specification in regeneration. We report that inhibition of activin-2, which encodes an Activin-like signaling ligand, resulted in the regeneration of ectopic posterior-facing heads following amputation. During tissue turnover in uninjured planarians, positional information is constitutively expressed in muscle to maintain proper patterning. Positional information includes Wnts expressed in the posterior and Wnt antagonists expressed in the anterior. Upon amputation, several wound-induced genes promote re-establishment of positional information. The head-versus-tail regeneration decision involves preferential wound induction of the Wnt antagonist notum at anterior-facing over posterior-facing wounds. Asymmetric activation of notum represents the earliest known molecular distinction between head and tail regeneration, yet how it occurs is unknown. activin-2 RNAi animals displayed symmetric wound-induced activation of notum at anterior- and posterior-facing wounds, providing a molecular explanation for their ectopic posterior-head phenotype. activin-2 RNAi animals also displayed anterior-posterior (AP) axis splitting, with two heads appearing in anterior blastemas, and various combinations of heads and tails appearing in posterior blastemas. This was associated with ectopic nucleation of anterior poles, which are head-tip muscle cells that facilitate AP and medial-lateral (ML) pattern at posterior-facing wounds. These findings reveal a role for Activin signaling in determining the outcome of AP-axis-patterning events that are specific to regeneration.     

Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians

The process by which the proper pattern is restored to newly formed tissues during metazoan regeneration remains an open question. Here, we provide evidence that the nervous system plays a role in regulating morphogenesis during anterior regeneration in the planarian Schmidtea mediterranea. RNA interference (RNAi) knockdown of a planarian ortholog of the axon-guidance receptor roundabout (robo) leads to unexpected phenotypes during anterior regeneration, including the development of a supernumerary pharynx (the feeding organ of the animal) and the production of ectopic, dorsal outgrowths with cephalic identity. We show that Smed-roboA RNAi knockdown disrupts nervous system structure during cephalic regeneration: the newly regenerated brain and ventral nerve cords do not re-establish proper connections. These neural defects precede, and are correlated with, the development of ectopic structures. We propose that, in the absence of proper connectivity between the cephalic ganglia and the ventral nerve cords, neurally derived signals promote the differentiation of pharyngeal and cephalic structures. Together with previous studies on regeneration in annelids and amphibians, these results suggest a conserved role of the nervous system in pattern formation during blastema-based regeneration.     

The TALE Class Homeobox Gene Smed-prep Defines the Anterior Compartment for Head Regeneration

Planaria continue to blossom as a model system for understanding all aspects of regeneration. They provide an opportunity to understand how the replacement of missing tissues from preexisting adult tissue is orchestrated at the molecular level. When amputated along any plane, planaria are capable of regenerating all missing tissue and rescaling all structures to the new size of the animal. Recently, rapid progress has been made in understanding the developmental pathways that control planarian regeneration. In particular Wnt/beta-catenin signaling is central in promoting posterior fates and inhibiting anterior identity. Currently the mechanisms that actively promote anterior identity remain unknown. Here, Smed-prep, encoding a TALE class homeodomain, is described as the first gene necessary for correct anterior fate and patterning during planarian regeneration. Smed-prep is expressed at high levels in the anterior portion of whole animals, and Smed-prep(RNAi) leads to loss of the whole brain during anterior regeneration, but not during lateral regeneration or homeostasis in intact worms. Expression of markers of different anterior fated cells are greatly reduced or lost in Smed-prep(RNAi) animals. We find that the ectopic anterior structures induced by abrogation of Wnt signaling also require Smed-prep to form. We use double knockdown experiments with the S. mediterranea ortholog of nou-darake (that when knocked down induces ectopic brain formation) to show that Smed-prep defines an anterior fated compartment within which stem cells are permitted to assume brain fate, but is not required directly for this differentiation process. Smed-prep is the first gene clearly implicated as being necessary for promoting anterior fate and the first homeobox gene implicated in establishing positional identity during regeneration. Together our results suggest that Smed-prep is required in stem cell progeny as they form the anterior regenerative blastema and is required for specifying anterior cell fates and correct patterning.     

A novel member of the Xenopus Zic family, Zic5, mediates neural crest development

We characterized Xenopus Zic5 which belongs to a novel class of the Zic family. Zic5 is more specifically expressed in the prospective neural crest than other Zic genes. Overexpression of Zic5 in embryos led to ectopic expression of the early neural crest markers, Xsna and Xslu, with the loss of epidermal marker expression. In Zic5-overexpressing animal cap explants, there was marked induction of neural crest markers, without mesodermal and anterior neural markers. This was in contrast to other Xenopus Zic genes, which induce both anterior and the neural crest markers in the same assay. Injection of a dominant-negative form of Zic5 can block neural crest formation in vivo. These results indicate that Zic5 expression converts cells from an epidermal fate to a neural crest cell fate. This is the first evidence for neural crest tissue inductive activity separate from anterior neural tissue inductive activity in a Zic family gene.     

Dermal fibroblasts contribute to multiple tissues in the accessory limb model

The accessory limb model has become an alternative model for performing investigations of limb regeneration in an amputated limb. In the accessory limb model, a complete patterned limb can be induced as a result of an interaction between the wound epithelium, a nerve and dermal fibroblasts in the skin. Studies should therefore focus on examining these tissues. To date, however, a study of cellular contributions in the accessory limb model has not been reported. By using green fluorescent protein (GFP) transgenic axolotl tissues, we can trace cell fate at the tissue level. Therefore, in the present study, we transgrafted GFP skin onto the limb of a non‐GFP host and induced an accessory limb to investigate cellular contributions. Previous studies of cell contribution to amputation‐induced blastemas have demonstrated that dermal cells are the progenitors of many of the early blastema cells, and that these cells contribute to regeneration of the connective tissues, including cartilage. In the present study, we have determined that this same population of progenitor cells responds to signaling from the nerve and wound epithelium in the absence of limb amputation to form an ectopic blastema and regenerate the connective tissues of an ectopic limb. Blastema cells from dermal fibroblasts, however, did not differentiate into either muscle or neural cells, and we conclude that dermal fibroblasts are dedifferentiated along its developmental lineage.     

Morphogenesis and organogenesis in the regenerating planktotrophic larvae of asteroids and echinoids

In a previous study, we described complete body regeneration (with organogenesis) following surgical bisection in the planktotrophic larvae of the asteroids Luidia foliolata and Pisaster ochraceus. Here we present further detailed observations of these unique regenerative processes not presented in the previous paper. Furthermore, we describe for the first time complete regeneration following surgical bisection of planktotrophic larvae of the regular echinoid Lytechinus variegatus and the irregular echinoid Dendraster excentricus. Larvae of both asteroids and echinoids displayed a capacity for rapid regeneration regardless of their developmental stage. Within 48 h after bisection, aggregations of mesenchyme cells with pseudopodia were observed at the site of surgical bisection. These cellular aggregations were similar in appearance to the mesenchymal blastemas that form in adult echinoderms prior to their arm regeneration, and to those described in other deuterostomes that undergo regeneration. When asteroid larvae were surgically bisected in the early stages of their development, clusters of mesenchyme cells developed into completely new pairs of coelomic pouches located anterior to the newly regenerated digestive tract. This indicates that cell fate in regenerating asteroid larvae remains indeterminate during early development. In the larvae of P. ochraceus, regardless of the developmental stage at the time of bisection, both the anterior and posterior portions regenerated all their missing organs and tissues. However, the larvae of L. foliolata displayed differential regenerative capacity in bisected larval halves at the late bipinnaria stage. The differences observed may be due to differences in larval development (L. foliolata has no brachiolaria stage), and may have evolutionary implications. In the regular echinoid L. variegatus, both larval portions regenerated into morphologically and functionally normal larvae that were indistinguishable from non-bisected control larvae. The regenerative processes were similar to those we observed in planktotrophic asteroid larvae. Regenerating larvae readily metamorphosed into normal juveniles. In the irregular echinoid D. excentricus, posterior portions of larvae completed regeneration and metamorphosis, but anterior portions regenerated only partially during the 2-week study. Our observations confirm that asteroid and echinoid larvae provide excellent models for studies of regeneration in deuterostomes.     

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.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.     

Transferrin and the trophic effect of neural tissue on amphibian limb regeneration blastemas

Nerves promote regeneration of amputated urodele limbs, but the chemical basis of the effect is not known. We have examined the possible involvement of the iron-transport factor transferrin, which is important for cell proliferation and is present in vertebrate nervous tissue. Newt brain extract stimulated incorporation of [3H]thymidine in cultured blastemas from regenerating newt forelimbs, showing a biphasic dose-response similar to that of heterologous transferrin. As shown previously for transferrin, the inhibitory effect of brain extract at high concentrations was relieved by the addition of iron. Activity of brain extract was reduced by treatment with an iron-chelating agent and fully restored by the readdition of iron. Double immunodiffusion of newt tissue extracts and antibodies against newt plasma transferrin demonstrated the presence of transferrin-like factors in brain, spinal cord, and peripheral nerve. These results indicate that activity of transferrin may be part of the trophic effect of brain extract on cultured blastemas.     

Differences in blastema-associated proteins according to position along body axis in regenerating planarians

To test the hypothesis that neoblasts in different positions in regenerating pieces of planarians may be differentially responsive to diffusible stimuli through differentially expressed membrane receptors, we compared membrane surface proteins in blastemas induced at various positions along an anterior-posterior axis in Dugesia gonocephala (Dugès). The proteins were biotinylated and identified by molecular weight in SDS-PAGE (sodium-dodecylsulfate polyacrilamide gel electrophoresis). This SDS-PAGE pattern was then compared with that of N-glycosylated proteins incorporating ³H-mannose. One 20 kDa glycoprotein present in all blastemas at 3 d was absent from more anterior blastemas at 6 d irrespective of whether those blastemas were at the cephalic or the caudal end of the regenerating piece. The expression of this protein appears to be determined by the position of the blastema along the body axis rather than by its prospective fate.     

Epimorphic vs. tissue regeneration in Xenopus forelimbs.

Postmetamorphic froglets of Xenopus laevis regenerate hypomorphic unbranched spikes from amputated arm stumps. These are composed primarily of cartilage, produced from blastemalike structures sparsely populated with cells and rich in connective tissue. Some consider these outgrowths to be an example of epimorphic regeneration produced from blastemas, albeit deficient ones. Others interpret them as a case of tissue regeneration derived from fibroblastemas augmented by chondrocytes and periosteal and perichondrial fibroblasts. To resolve these alternatives, forelimbs were amputated proximal to the wrist, skinned, and inserted through the body wall into the abdominal cavity. In the absence of skin, epidermal wound healing failed to occur and blastemas could not develop. After 2 months, by which time controls had regenerated spikes averaging 3.38 mm long, the denuded stumps had not given rise to outgrowths. They typically developed cartilaginous caps on the severed ends of the radius-ulna, and in rare cases formed amorphous growths of cartilage. If blastema formation is considered diagnostic of epimorphic regeneration and tissue regeneration can proceed in the absence of epidermal wound healing and blastema formation, these findings lead to the conclusion that Xenopus limb regeneration is epimorphic.     

Proposal of a model of mammalian neural induction

How does the vertebrate embryo make a nervous system? This complex question has been at the center of developmental biology for many years. The earliest step in this process - the induction of neural tissue - is intimately linked to patterning of the entire early embryo, and the molecular and embryological of basis these processes are beginning to emerge. Here, we analyze classic and cutting-edge findings on neural induction in the mouse. We find that data from genetics, tissue explants, tissue grafting, and molecular marker expression support a coherent framework for mammalian neural induction. In this model, the gastrula organizer of the mouse embryo inhibits BMP signaling to allow neural tissue to form as a default fate-in the absence of instructive signals. The first neural tissue induced is anterior and subsequent neural tissue is posteriorized to form the midbrain, hindbrain, and spinal cord. The anterior visceral endoderm protects the pre-specified anterior neural fate from similar posteriorization, allowing formation of forebrain. This model is very similar to the default model of neural induction in the frog, thus bridging the evolutionary gap between amphibians and mammals.     

Conversion of ectoderm into a neural fate by ATH-3, a vertebrate basic helix-loop-helix gene homologous to Drosophila proneural gene atonal.

We have isolated a novel basic helix-loop-helix (bHLH) gene homologous to the Drosophila proneural gene atonal, termed ATH-3, from Xenopus and mouse. ATH-3 is expressed in the developing nervous system, with high levels of expression in the brain, retina and cranial ganglions. Injection of ATH-3 RNA into Xenopus embryos dramatically expands the neural tube and induces ectopic neural tissues in the epidermis but inhibits non-neural development. This ATH-3-induced neural hyperplasia does not require cell division, indicating that surrounding cells which are normally non-neural types adopt a neural fate. In a Xenopus animal cap assay, ATH-3 is able to convert ectodermal cells into neurons expressing anterior markers without inducing mesoderm. Interestingly, a single amino acid change from Ser to Asp in the basic region, which mimics phosphorylation of Ser, severely impairs the anterior marker-inducing ability without affecting general neurogenic activities. These results provide evidence that ATH-3 can directly convert non-neural or undetermined cells into a neural fate, and suggest that the Ser residue in the basic region may be critical for the regulation of ATH-3 activity by phosphorylation.     

Transferrin is necessary and sufficient for the neural effect on growth in amphibian limb regeneration blastemas

Cell proliferation during the early phase of growth in regenerating amphibian limbs requires a permissive influence of nerves. Based on analyses of proliferative activity in denervated blastemas, it was proposed that nerves provide factors important for cells to complete the proliferative cycle rather than for mitogenesis itself. One such factor, the iron-transport protein transferrin (Tf), is abundant in regenerating peripheral nerves where it is axonally transported and released at growth cones. Using blastemas in organ culture, which have been widely used in previous investigations of the neural effect on growth, it was shown here that the growth-promoting activity of neural extract was completely removed by immuno-absorption with antiserum against Tf and restored by addition of Tf. Purified Tf or a low molecular weight ferric ionophore were as active as the neural extract in this assay, indicating that the trophic effect of Tf involves its capacity for iron delivery. Both Tf and ferric ionophore also maintained DNA synthesis in denervated blastemas in vivo . A dose-response assay indicated that purified axolotl Tf stimulates growth of cultured blastemal cells at concentrations as low as 100 ng/mL. The Tf mRNA in axolotl nervous tissue was shown by northern analysis to be similar in size to that of liver. These results are discussed together with those from previous in vitro studies of blastemal growth and support the hypothesis that cell division in the blastema depends on axonally released Tf during the early, nerve-dependent phase of limb regeneration.