Involvement of canonical Wnt/Wingless signaling in the determination of the positional values within the leg segment of the cricket Gryllus bimaculatus

The cricket Gryllus bimaculatus is a hemimetabolous insect whose nymphs possess the ability to regenerate amputated legs. Previously, we showed that Gryllus orthologues of Drosophila hedgehog (Gb'hh), wingless (Gb'wg) and decapentaplegic (Gb'dpp) are expressed during leg regeneration and play essential roles in the establishment of the proximal-distal axis. Here, we examined their roles during intercalary regeneration: when a distally amputated tibia with disparate positional values is placed next to a proximally amputated host, intercalary growth occurs in order to regenerate the missing part. In this process, we examined expression patterns of Gb'hh and Gb'wg. We found that expressions of Gb'hh and Gb'wg were induced in a regenerate and the host proximal to the amputated region, but not in the grafted donor distal to the regenerate. This directional induction occurs even in the reversed intercalation. Because these results are consistent with a distal-to-proximal respecification of the regenerate, Gb'wg may be involved in the re-establishment of the positional values in the regenerate. Furthermore, we found that no regeneration occurs when Gb'armadillo (the orthologue of beta-catenin) was knocked down by RNA interference. These results indicate that the canonical Wnt/Wingless signaling pathway is involved in the process of leg regeneration and determination of positional information in the leg segment.     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Retinoic acid proximalizes level-specific properties responsible for intercalary regeneration in axolotl limbs

The objective of this study was to determine whether retinoic acid (RA) coordinately proximalizes positional memory and the cellular recognition system that detects pattern discontinuity in regenerating amphibian limbs. The strategy was to test the capacity of RA-treated blastemas to evoke intercalary regeneration when grafted to an amputation level proximal to their level of origin. Control wrist and ankle, or elbow and knee blastemas treated with the retinoid solvent, dimethylsulphoxide, evoked intercalary regeneration as effectively as untreated blastemas, when grafted to the midstylopodial amputation surface of host limbs. RA-treated wrist and ankle or elbow and knee blastemas were proximalized and formed complete limbs that were at an angle to, or continuous with, the midstylopodium of the host limb. No intercalary regeneration, from either graft or host, was observed in these cases. The results indicate that the cellular mechanism that recognizes disparities between non-neighbouring cells and initiates intercalary regeneration is coordinately proximalized with positional memory. Thus the recognition mechanism and positional memory are directly related. Intercalary regeneration and corrective displacement (affinophoresis), both of which restore a pattern of normal cell neighbours by different means in regenerating axolotl limbs, appear to use the same mechanism to recognize pattern discontinuity.     

Leg regeneration in insects. An experimental analysis in Drosophila and a new interpretation.

Fragments from prospective distal regions of Drosophila male foreleg imaginal discs failed to undergo proximal intercalary regeneration across leg segment borders when mechanically intermixed and cultured for 8 days with various fragments from prospective proximal disc regions. The failure of the distal cells to regenerate proximal leg segments was not due to a general restriction in their developmental potentials: Distal fragments, when deprived of their distal-most tips, regenerated in the distal direction at a high frequency. It is concluded that there exist in Drosophila leg discs the same restrictions with respect to regeneration along the proximodistal leg axis as had been previously observed in legs of several hemimetabolous insect species: Intersegmental discontinuities between grafted tissue pieces are not eliminated by intercalation. Based on the available evidence in hemimetabolous insects and in Drosophila, a new interpretation of the different aspects of regeneration in insect legs is offered. It is proposed that the two categories of regulative fields observed in insect legs, the leg segment fields and the whole leg field, represent the units of regulation for two fundamentally different regulative pathways that a cell at a wound edge can follow, the intercalative pathway and the terminal pathway, respectively. It is suggested that the criterion used by cells at healing wounds to choose between the two pathways is the difference in circumferential positional information between juxtaposed cells. The intercalative regulative pathway is switched on when cells with disparities in their axial positional information, or cells with less than maximal disparities in their circumferential information, contact one another. The terminal regulative pathway is triggered whenever cells with maximal circumferential disparities come into contact.     

Regulative interactions between cells of wing discs from different dipteran species

The mechanism by which patterns are produced appears to be repeated in each segment of an animal, and it has been proposed that it may even have been conserved in evolution so that different species would have the same system of positional information. This idea has been tested by mixing cells of a defined fragment of the wing disc of Drosophila melanogaster with wing disc fragments of five other dipteran species to assay the ability of these disc fragments to stimulate intercalary regeneration of the D. melanogaster cells. The genetically marked (y; mwh) D. melanogaster fragment was mechanically mixed with wing discs or wing disc fragments of four drosophilids (D. melanogaster as a control, D. virilis, D. hydei, Zaprionus vittiger), of Musca domestica, and of Piophila casei. The mixed aggregates were cultured in vivo for 7 days, then metamorphosed in D. melanogaster larval hosts. The D. melanogaster fragments were only stimulated to regenerate when combined with complementary fragments from D. melanogaster or D. virilis wing discs. In the combination between D. melanogaster and D. hydei, the tissue formed integrated mosaic patterns, but no regeneration ensued. The one positive result (D. melanogaster mixed with D. virilis) shows that positional cues can be exchanged and correctly interpreted between cells of different species. The negative results do not prove that the mechanism for establishing patterns is different in the tested species, but may be due to incompatibilities that are not related to pattern formation.     

Leg Regeneration in Insects: Cell Interactions and Lineage

SYNOPSIS. Developing insect legs have positional informationspecified down the length and around the leg circumference.After grafting or amputation of larval cockroach or cricketlegs healing confronts epidermal cells with different positionalvalues. This stimulates growth, the intercalary regenerationof intervening tissue, the regeneration of all more distal tissuefrom a complete leg circumference and often the formation ofan incomplete distal regenerate from a symmetrical part-circumference.These processes will lead to regeneration of missing structures,duplication of structures, or the formation of branched supernumerarylegs, depending on the situation. During regeneration, cellscannot cross lineage restrictions which divide the leg intoanterior and posterior compartments.     

Intercalary regeneration and level interactions in the fresh-water planarianDugesia lugubris

Summary The aim of the present experiments was the analysis of possible dorso-ventral regulation in planarians. The experiments employ half-thickness pieces obtained by a horizontal cut between the ventral and dorsal faces. When the dorsal face of an anterior part was superposed onto the ventral face of a posterior part (or vice versa) with its antero-posterior polarity reversed, both faces maintained their orgginal organization. When half-thickness pieces of the same face (dorsal or ventral) were fused by their transverse cut surfaces and cultured in vitro, intercalary regeneration ensued as it does in full-thickness pieces combined in the same manner. When a half-thickness piece was grafted to a distant site on the same face, the ensuing remodelling of tissues proceeded in the same manner as in similar experiments carried out earlier with full-thickness grafts. When full-thickness pieces representing different antero-posterior levels were joined so that the dorsal face of one fused with the ventral face of the other, intercalary regenerates were formed as if the pieces had been joined by their homologous faces-unless and terminal blastema appeared early on the suture. These experiments show that (1) there is no dorso-ventral self-regulating system: (2) the antero-posterior system resides in the periphery of the worm.     

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.     

Constitutive gene expression and the specification of tissue identity in adult planarian biology

Planarians are flatworms that constitutively maintain adult tissues through cell turnover and can regenerate entire organisms from tiny body fragments. In addition to requiring new cells (from neoblasts), these feats require mechanisms that specify tissue identity in the adult. Crucial roles for Wnt and BMP signaling in the regeneration and maintenance of the body axes have been uncovered, among other regulatory factors. Available data indicate that genes involved in positional identity regulation at key embryonic stages in other animals display persisting regionalized expression in adult planarians. These expression patterns suggest that a constitutively active gene expression map exists for the maintenance of the planarian body. Planarians thus present a fertile ground for the identification of factors regulating the regionalization of the metazoan body plan and for the study of the attributes of these factors that can lead to the maintenance and regeneration of adult tissues.     

The regeneration capacity of the flatworm Macrostomum lignano—on repeated regeneration, rejuvenation, and the minimal size needed for regeneration

The lion’s share of studies on regeneration in Plathelminthes (flatworms) has been so far carried out on a derived taxon of rhabditophorans, the freshwater planarians (Tricladida), and has shown this group’s outstanding regeneration capabilities in detail. Sharing a likely totipotent stem cell system, many other flatworm taxa are capable of regeneration as well. In this paper, we present the regeneration capacity of Macrostomum lignano, a representative of the Macrostomorpha, the basal-most taxon of rhabditophoran flatworms and one of the most basal extant bilaterian protostomes. Amputated or incised transversally, obliquely, and longitudinally at various cutting levels, M. lignano is able to regenerate the anterior-most body part (the rostrum) and any part posterior of the pharynx, but cannot regenerate a head. Repeated regeneration was observed for 29 successive amputations over a period of almost 12 months. Besides adults, also first-day hatchlings and older juveniles were shown to regenerate after transversal cutting. The minimum number of cells required for regeneration in adults (with a total of 25,000 cells) is 4,000, including 160 neoblasts. In hatchlings only 1,500 cells, including 50 neoblasts, are needed for regeneration. The life span of untreated M. lignano was determined to be about 10 months.An erratum to this article can be found at     

Stem cell-based growth, regeneration, and remodeling of the planarian intestine

Although some animals are capable of regenerating organs, the mechanisms by which this is achieved are poorly understood. In planarians, pluripotent somatic stem cells called neoblasts supply new cells for growth, replenish tissues in response to cellular turnover, and regenerate tissues after injury. For most tissues and organs, however, the spatiotemporal dynamics of stem cell differentiation and the fate of tissue that existed prior to injury have not been characterized systematically. Utilizing in vivo imaging and bromodeoxyuridine pulse-chase experiments, we have analyzed growth and regeneration of the planarian intestine, the organ responsible for digestion and nutrient distribution. During growth, we observe that new gut branches are added along the entire anteroposterior axis. We find that new enterocytes differentiate throughout the intestine rather than in specific growth zones, suggesting that branching morphogenesis is achieved primarily by remodeling of differentiated intestinal tissues. During regeneration, we also demonstrate a previously unappreciated degree of intestinal remodeling, in which pre-existing posterior gut tissue contributes extensively to the newly formed anterior gut, and vice versa. By contrast to growing animals, differentiation of new intestinal cells occurs at preferential locations, including within newly generated tissue (the blastema), and along pre-existing intestinal branches undergoing remodeling. Our results indicate that growth and regeneration of the planarian intestine are achieved by co-ordinated differentiation of stem cells and the remodeling of pre-existing tissues. Elucidation of the mechanisms by which these processes are integrated will be critical for understanding organogenesis in a post-embryonic context.     

Epimorphic regeneration of the distal part of the planarian pharynx

The totipotent stem cells called neoblasts seem to be concerned with the remarkable regeneration ability of planarians. However, the pharynx is able to regenerate after the amputation of its distal part, in spite of a lack of neoblasts in the pharynx. The process of regeneration has been referred to as morphallaxis, based on conventional histochemical observations. We examined it again immuno-histochemically using anti-Dugesia japonica proliferating cell nuclear antigen (DjPCNA) antibody for neoblasts and anti-D. japonica myosin heavy chain-A (DjMHC-A) antibody for pharynx muscle fibers. This immuno-histochemical study, together with observations of the regeneration process of planarians irradiated with X-rays in particular regions, revealed that after the amputation, neoblasts from outside the pharynx entered that organ, moved through the mesenchyme of the pharynx to the wounded area, and differentiated into the cells that had been lost there. We show here that the regeneration after amputation of the distal part of the pharynx is an 'epimorphic' process.     

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.     

Distal transformation in Drosophila leg imaginal disc fragments.

For an appendage to regenerate distal elements, it has been thought that the stump must contain a full set of circumferential positional information. We have shown that this rule is not binding for tarsus regeneration in the male foreleg imaginal disc of Drosophila melanogaster. Distal transformation was not restricted to fragments containing complete proximal segments, but was also observed in pieces with small or even substantial deficiencies that were not regenerated in their proximal segments.     

Regulation after proximal or distal transposition of limb regeneration blastemas and determination of the proximal boundary of the regenerate.

When blastemas of several stages of differentiation were grafted in normal orientation to stump levels proximal or distal to their level of origin, normal limbs regenerated. Histological and autoradiographic studies of the development of these regulated limbs showed that the grafted blastemas formed only structures normally distal to their level of origin. In the case of a blastema transplanted proximally, regulation occurred by intercalary regeneration from the stump, whereas, when blastemas were transplanted distally, regulation appeared to take place within the blastema itself by a distal shift in its pattern of organization. The results suggest that the proximal limit of the limb regenerate is determined by level-specific properties of the limb cells but that these properties allow for interactions leading to regulation when different levels of stump and blastema are brought together.     

The planarian <Emphasis Type="Italic">nanos</Emphasis>-like gene Smednos is expressed in germline and eye precursor cells during development and regeneration

Planarians are highly regenerative organisms with the ability to remake all their cell types, including the germ cells. The germ cells have been suggested to arise from totipotent neoblasts through epigenetic mechanisms. Nanos is a zinc-finger protein with a widely conserved role in the maintenance of germ cell identity. In this work, we describe the expression of a planarian nanos-like gene Smednos in two kinds of precursor cells namely, primordial germ cells and eye precursor cells, during both development and regeneration of the planarian Schmidtea mediterranea. In sexual planarians, Smednos is expressed in presumptive male primordial germ cells of embryos from stage 8 of embryogenesis and throughout development of the male gonads and in the female primordial germ cells of the ovary. Thus, upon hatching, juvenile planarians do possess primordial germ cells. In the asexual strain, Smednos is expressed in presumptive male and female primordial germ cells. During regeneration, Smednos expression is maintained in the primordial germ cells, and new clusters of Smednos-positive cells appear in the regenerated tissue. Remarkably, during the final stages of development (stage 8 of embryogenesis) and during regeneration of the planarian eye, Smednos is expressed in cells surrounding the differentiating eye cells, possibly corresponding to eye precursor cells. Our results suggest that similar genetic mechanisms might be used to control the differentiation of precursor cells during development and regeneration in planarians. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

TCAV-1, a monoclonal antibody specific to epithelial pharyngeal cells in the planarian Dugesia (Girardia) tigrina. Application to pattern formation of the pharynx during regeneration

During regeneration in planarians, anterior (head and prepharyngeal) and posterior (postpharyngeal and tail) fragments rebuild one of the most peculiar structures of planarians: the pharynx and the pharynx cavity. Previous studies (see Brønsted, 1969, for a general review, and Asai, 1990, 1991, for anterior regeneration) have shown that within postpharyngeal pieces both structures appear in the old stump from clusters of undifferentiated cells. However, the lineage and differentiation of their elements (inner and outer epithelial cells, muscle layers, gland cells, nerve rings) and the overall pattern of growth and differentiation is not clear.     

Brain regeneration from pluripotent stem cells in planarian

How can planarians regenerate their brain? Recently we have identified many genes critical for this process. Brain regeneration can be divided into five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) pattern formation, (4) neural network formation, and (5) functional recovery. Here we will describe the structure and process of regeneration of the planarian brain in the first part, and then introduce genes involved in brain regeneration in the second part. Especially, we will speculate about molecular events during the early steps of brain regeneration in this review. The finding providing the greatest insight thus far is the discovery of the nou-darake (ndk; ‘brains everywhere’ in Japanese) gene, since brain neurons are formed throughout the entire body as a result of loss of function of the ndk gene. This finding provides a clue for elucidating the molecular and cellular mechanisms underlying brain regeneration. Here we describe the molecular action of the nou-darake gene and propose a new model to explain brain regeneration and restriction in the head region of the planarians.     

Neoblast-enriched fraction rescues eye formation in eye-defective planarian 'menashi' Dugesia ryukyuensis

Planarians are well known for their remarkable regenerative capacity. This capacity to regenerate is thought to be due to the presence of totipotent somatic stem cells known as ‘neoblasts’, which have particular morphological characteristics. The totipotency of neoblasts was supported by Baguñà's experiment, which involved the introduction of donor cells into irradiated hosts. However, since Baguñà's experiment did not include the use of a phenotypic marker, the donor cells could not be traced. In the current study, a genetic mutant planarian, menashi, an eye‐defective mutant that lacks the pigmented area in the eyes, was established. This planarian is excellent for tracing the fate of cells after their introduction into irradiated hosts. To investigate the differentiation potency more directly, a neoblast‐rich fraction obtained from normal worms was transplanted into an X‐ray‐irradiated menashi strain. Planarians that survive X‐ray irradiation were developed, and we observed the pigment of the area in the eyes of the regenerating planarians. This result suggests that the neoblast‐rich fraction contains cells that can proliferate and differentiate. These cells can replace the cells and structures lost by X‐ray irradiation and ablation, and they can also differentiate into eye pigment cells.