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.     

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.     

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.     

Retinoic-acid-induced pattern completion in regenerating double anterior limbs of urodeles

The effects of retinoic acid on the regeneration of double anterior lower arms in the adult newt, Notophthalmus viridescens, were investigated. Normally, double anterior lower arms regenerate a hypomorphic, symmetrical pattern of structures, which are distally complete; and double anterior upper arms regenerate a hypomorphic, symmetrical but distally incomplete pattern of structures. In limbs with a normal anteroposterior axis, the major effect of retinoic acid is to alter the proximodistal (PD) positional value of cells local at the amputation level to a much more proximal value, thereby creating duplications in the regenerate of structures proximal to the amputation plane (Thoms and Stocum, '84). Therefore, we predicted that double anterior lower arms treated with retinoic acid would regenerate like double anterior upper arms. However, in a substantial number of cases, each half of these double anterior lower arms regenerated a limb that was complete in the anteroposterior (AP) axis, with asymmetry corresponding to the half of origin. In addition, these regenerates were serially duplicated in the PD axis. These results indicate that retinoic acid can posteriorize the positional value of midline cells, leading to restoration of normal AP pattern, when the set of posterior-half positional values is removed from the cross section of the limb.     

Long-range neural and gap junction protein-mediated cues control polarity during planarian regeneration

Having the ability to coordinate the behavior of stem cells to induce regeneration of specific large-scale structures would have far-reaching consequences in the treatment of degenerative diseases, acute injury, and aging. Thus, identifying and learning to manipulate the sequential steps that determine the fate of new tissue within the overall morphogenetic program of the organism is fundamental. We identified novel early signals, mediated by the central nervous system and 3 innexin proteins, which determine the fate and axial polarity of regenerated tissue in planarians. Modulation of gap junction-dependent and neural signals specifically induces ectopic anterior regeneration blastemas in posterior and lateral wounds. These ectopic anterior blastemas differentiate new brains that establish permanent primary axes re-established during subsequent rounds of unperturbed regeneration. These data reveal powerful novel controls of pattern formation and suggest a constructive model linking nervous inputs and polarity determination in early stages of 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.     

Early planarian brain regeneration is independent of blastema polarity mediated by the Wnt/β-catenin pathway

Analysis of anteroposterior (AP) axis specification in regenerating planarian flatworms has shown that Wnt/β-catenin signaling is required for posterior specification and that the FGF-like receptor molecule nou-darake (ndk) may be involved in restricting brain regeneration to anterior regions. The relationship between re-establishment of AP identity and correct morphogenesis of the brain is, however, still poorly understood. Here we report the characterization of two axin paralogs in the planarian Schmidtea mediterranea. Although Axins are well known negative regulators of Wnt/β-catenin signaling, no role in AP specification has previously been reported for axin genes in planarians. We show that silencing of Smed-axin genes by RNA interference (RNAi) results in two-tailed planarians, a phenotype previously reported after silencing of Smed-APC-1, another β-catenin inhibitor. More strikingly, we show for the first time that while early brain formation at anterior wounds remains unaffected, subsequent development of the brain is blocked in the two-tailed planarians generated after silencing of Smed-axin genes and Smed-APC-1. These findings suggest that the mechanisms underlying early brain formation can be uncoupled from the specification of AP identity by the Wnt/β-catenin pathway. Finally, the posterior expansion of the brain observed following Smed-ndk RNAi is enhanced by silencing Smed-APC-1, revealing an indirect relationship between the FGFR/Ndk and Wnt/β-catenin signaling systems in establishing the posterior limits of brain differentiation.     

Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. We have previously identified a zebrafish frizzled8a (fz8a) gene expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. We have investigated the role of Fz8a-mediated Wnt8b signalling in anterior brain patterning in zebrafish. We show that in zebrafish embryos: (1) Wnt signalling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation; (2) Fz8a plays an important role in mediating anterior brain patterning; (3) Wnt8b and Fz8a can functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos; and (4) Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signalling. In conclusion, we suggest that a gradient of Fz8a-mediated Wnt8b signalling may play crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula.     

Wnt signaling is required for antero-posterior patterning of the planarian brain

Wnt signaling functions in axis formation and morphogenesis in various animals and organs. Here we report that Wnt signaling is required for proper brain patterning during planarian brain regeneration. We showed here that one of the Wnt homologues in the planarian Dugesia japonica, DjwntA, was expressed in the posterior region of the brain. When DjwntA-knockdown planarians were produced by RNAi, they could regenerate their heads at the anterior ends of the fragments, but formed ectopic eyes with irregular posterior lateral branches and brain expansion. This suggests that the Wnt signal may be involved in antero-posterior (A-P) patterning of the planarian brain, as in vertebrates. We also investigated the relationship between the DjwntA and nou-darake/FGFR signal systems, as knockdown planarians of these genes showed similar phenotypes. Double-knockdown planarians of these genes did not show any synergistic effects, suggesting that the two signal systems function independently in the process of brain regeneration, which accords with the fact that nou-darake was expressed earlier than DjwntA during brain regeneration. These observations suggest that the nou-darake/FGFR signal may be involved in brain rudiment formation during the early stage of head regeneration, and subsequently the DjwntA signal may function in A-P patterning of the brain rudiment.     

Smad5 determines murine amnion fate through the control of bone morphogenetic protein expression and signalling levels

Smad5 is an intracellular mediator of bone morphogenetic protein (Bmp) signalling. It is essential for primordial germ cell (PGC) development, for the development of the allantois and for amnion closure, as demonstrated by loss of Bmp signalling. By contrast, the appearance of ectopic PGC-like cells and regionalized ectopic vasculogenesis and haematopoiesis in thickened Smad5(m1/m1) amnion are amnion defects that have not been associated with loss of Bmp signalling components. We show that defects in amnion and allantois can already be detected at embryonic day (E) 7.5 in Smad5 mutant mice. However, ectopic Oct4-positive (Oct4(+)) and alkaline phosphatase-positive (AP(+)) cells appear suddenly in thickened amnion at E8.5, and at a remote distance from the allantois and posterior primitive streak, suggesting a change of fate in situ. These ectopic Oct4(+), AP(+) cells appear to be Stella negative and hence cannot be called bona fide PGCs. We demonstrate a robust upregulation of Bmp2 and Bmp4 expression, as well as of Erk and Smad activity, in the Smad5 mutant amnion. The ectopic expression of several Bmp target genes in different domains and the regionalized presence of cells of several Bmp-sensitive lineages in the mutant amnion suggest that different levels of Bmp signalling may determine cell fate. Injection of rBMP4 in the exocoelom of wild-type embryos can induce thickening of amnion, mimicking the early amnion phenotype in Smad5 mutants. These results support a model in which loss of Smad5 results paradoxically in gain of Bmp function defects in the amnion.     

Inhibitory Smads and bone morphogenetic protein (BMP) modulate anterior photoreceptor cell number during planarian eye regeneration

Planarians represent an excellent model to study the processes of body axis and organ re-specification during regeneration. Previous studies have revealed a conserved role for the bone morphogenetic protein (BMP) pathway and its intracellular mediators Smad1/5/8 and Smad4 in planarian dorsoventral (DV) axis re-establishment. In an attempt to gain further insight into the role of this signalling pathway in planarians, we have isolated and functionally characte-rized the inhibitory Smads (I-Smads) in Schmidtea mediterranea. Two I-Smad homologues have been identified: Smed-smad6/7-1 and Smed-smad6/7-2. Expression of smad6/7-1 was detected in the parenchyma, while smad6/7-2 was found to be ex-pressed in the central nervous system and the eyes. Neither single smad6/7-1 and smad6/7-2 nor double smad6/7-1,-2 silencing gave rise to any apparent disruption of the DV axis. However, both regenerating and intact smad6/7-2 (RNAi) planarians showed defects in eye morphogenesis and displayed small, rounded eyes that lacked the anterior subpopulation of photoreceptor cells. The number of pigment cells was also reduced in these animals at later stages of regeneration. In contrast, after low doses of Smed-bmp(RNAi), planarians regenerated larger eyes in which the anterior subpopulation of photoreceptor cells was expanded. Our results suggest that Smed-smad6/7-2 and Smed-bmp control the re-specification and maintenance of anterior photoreceptor cell number in S. mediterranea.     

Regionalised signalling within the extraembryonic ectoderm regulates anterior visceral endoderm positioning in the mouse embryo

The development of the anterior-posterior (AP) axis in the mammalian embryo is controlled by interactions between embryonic and extraembryonic tissues. It is well established that one of these extraembryonic tissues, the anterior visceral endoderm (AVE), can repress posterior cell fate and that signalling from the other, the extraembryonic ectoderm (ExE), is required for posterior patterning. Here, we show that signals from the prospective posterior ExE repress AVE gene expression and affect the distribution of the AVE cells. Surgical ablation of the prospective posterior, but not the anterior, extraembryonic region at 5.5 days of development (E5.5) perturbs the characteristic distal-to-anterior distribution of AVE cells and leads to a dramatic expansion of the AVE domain. Time-lapse imaging studies show that this increase is due to the ectopic expression of an AVE marker, which results in a symmetrical positioning of the AVE. Surgical ablation of this same ExE region after the distal-to-anterior migration has already commenced, at E5.75, does not affect the localisation of the AVE, indicating that this effect takes place within a short time window. Conversely, transplanting the prospective posterior, but not the anterior, extraembryonic region onto isolated E5.5 embryonic explants drastically reduces the AVE domain. Further, transplantation experiments demonstrate that the signalling regulating AVE gene expression originates from the posterior ExE, rather than its surrounding VE. Together, our results show that signals emanating from the future posterior ExE within a temporal window both restrict the AVE domain and promote its specific positioning. This indicates for the first time that the ExE is already regionalised a day before the onset of gastrulation in order to correctly set the orientation of the AP axis of the mouse embryo. We propose a reciprocal function of the posterior ExE and the AVE in establishing a balance between the antagonistic activities of these two tissues, essential for AP patterning.     

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

The dorsal ectoderm of the vertebrate gastrula was proposed by Nieuwkoop to be specified towards an anterior neural fate by an activation signal, with its subsequent regionalization along the anteroposterior (AP) axis regulated by a graded transforming activity, leading to a properly patterned forebrain, midbrain, hindbrain and spinal cord. The activation phase involves inhibition of BMP signals by dorsal antagonists, but the later caudalization process is much more poorly characterized. Explant and overexpression studies in chick, Xenopus, mouse and zebrafish implicate lateral/paraxial mesoderm in supplying the transforming influence, which is largely speculated to be a Wnt family member. We have analyzed the requirement for the specific ventrolaterally expressed Wnt8 ligand in the posteriorization of neural tissue in zebrafish wild-type and Nodal-deficient embryos (Antivin overexpressing or cyclops;squint double mutants), which show extensive AP brain patterning in the absence of dorsal mesoderm. In different genetic situations that vary the extent of mesodermal precursor formation, the presence of lateral wnt8-expressing cells correlates with the establishment of AP brain pattern. Cell tracing experiments show that the neuroectoderm of Nodal-deficient embryos undergoes a rapid anterior-to-posterior transformation in vivo during a short period at the end of the gastrula stage. Moreover, in both wild-type and Nodal-deficient embryos, inactivation of Wnt8 function by morpholino (MO(wnt8)) translational interference dose-dependently abrogates formation of spinal cord and posterior brain fates, without blocking ventrolateral mesoderm formation. MO(wnt8) also suppresses the forebrain deficiency in bozozok mutants, in which inactivation of a homeobox gene causes ectopic wnt8 expression. In addition, the bozozok forebrain reduction is suppressed in bozozok;squint;cyclops triple mutants, and is associated with reduced wnt8 expression, as seen in cyclops;squint mutants. Hence, whereas boz and Nodal signaling largely cooperate in gastrula organizer formation, they have opposing roles in regulating wnt8 expression and forebrain specification. Our findings provide strong support for a model of neural transformation in which a planar gastrula-stage Wnt8 signal, promoted by Nodal signaling and dorsally limited by Bozozok, acts on anterior neuroectoderm from the lateral mesoderm to produce the AP regional patterning of the CNS.     

Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo.

Fragments of the germ layer tissues isolated from the early-primitive-streak (early-streak) stage mouse embryos were tested for axis induction activity by transplantation to late-gastrula (late-streak to early-bud) stage host embryos. The posterior epiblast fragment that contains the early gastrula organizer was able to recruit the host tissues to form an ectopic axis. However, the most anterior neural gene that was expressed in the ectopic axis was Krox20 that marks parts of the hindbrain, but markers of the mid- and forebrain (Otx2 and En1) were not expressed. Anterior visceral endoderm or the anterior epiblast alone did not induce any ectopic neural tissue. However, when these two anterior germ layer tissues were transplanted together, they can induce the formation of ectopic host-derived neural tissues but these tissues rarely expressed anterior neural genes and did not show any organization of an ectopic axis. Therefore, although the anterior endoderm and epiblast together may display some inductive activity, they do not act like a classical organizer. Induction of the anterior neural genes in the ectopic axis was achieved only when a combination of the posterior epiblast fragment, anterior visceral endoderm and the anterior epiblast was transplanted to the host embryo. The formation of anterior neural structures therefore requires the synergistic interaction of the early gastrula organizer and anterior germ layer tissues.     

Regeneration of oral siphon pigment organs in the ascidian Ciona intestinalis

Ascidians have powerful capacities for regeneration but the underlying mechanisms are poorly understood. Here we examine oral siphon regeneration in the solitary ascidian Ciona intestinalis. Following amputation, the oral siphon rapidly reforms oral pigment organs (OPO) at its distal margin prior to slower regeneration of proximal siphon parts. The early stages of oral siphon reformation include cell proliferation and re-growth of the siphon nerves, although the neural complex (adult brain and associated organs) is not required for regeneration. Young animals reform OPO more rapidly after amputation than old animals indicating that regeneration is age dependent. UV irradiation, microcautery, and cultured siphon explant experiments indicate that OPOs are replaced as independent units based on local differentiation of progenitor cells within the siphon, rather than by cell migration from a distant source in the body. The typical pattern of eight OPOs and siphon lobes is restored with fidelity after distal amputation of the oral siphon, but as many as 16 OPOs and lobes can be reformed following proximal amputation near the siphon base. Thus, the pattern of OPO regeneration is determined by cues positioned along the proximal distal axis of the oral siphon. A model is presented in which columns of siphon tissue along the proximal-distal axis below pre-existing OPO are responsible for reproducing the normal OPO pattern during regeneration. This study reveals previously unknown principles of oral siphon and OPO regeneration that will be important for developing Ciona as a regeneration model in urochordates, which may be the closest living relatives of vertebrates.     

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.     

Optic chiasm formation in planarian I: Cooperative netrin- and robo-mediated signals are required for the early stage of optic chiasm formation

Freshwater planarians can regenerate a brain, including eyes, from the anterior blastema, and coordinately form an optic chiasm during eye and brain regeneration. To investigate the role of the netrin- and slit-signaling systems during optic chiasm formation, we cloned three receptor genes (Djunc5A, Djdcc and DjroboA) expressed in visual neurons and their ligand genes (DjnetB and Djslit) and analyzed their functions by RNA interference (RNAi). Although each of DjroboA(RNAi), Djunc5A(RNAi) and DjnetB(RNAi) showed a weak phenotype and Djslit(RNAi) showed a severe defect of eye formation, we did not observe any defect of crossing of visual axons over the midline among single knockdown planarians. However, among double knockdown planarians, some of DjnetB(RNAi);DjroboA(RNAi) and Djunc5A(RNAi);DjroboA(RNAi) showed complete disconnection between the visual axons from the two sides, suggesting that some combination of netrin- and robo-mediated signals may be required for crossing over the midline. Finally, we carefully investigated the distribution patterns of cells expressing DjNetB protein, DjnetB, and Djslit at the early stage of regeneration, and found that visual axons projected along a path sandwiched between DjNetB protein and Djslit-positive cells. These results suggest that two different collaborative or combinatory signals may be required for midline crossing at the early stage of chiasm formation during eye and brain regeneration.     

What role do annelid neoblasts play? A comparison of the regeneration patterns in a neoblast-bearing and a neoblast-lacking enchytraeid oligochaete

The term 'neoblast' was originally coined for a particular type of cell that had been observed during annelid regeneration, but is now used to describe the pluripotent/totipotent stem cells that are indispensable for planarian regeneration. Despite having the same name, however, planarian and annelid neoblasts are morphologically and functionally distinct, and many annelid species that lack neoblasts can nonetheless substantially regenerate. To further elucidate the functions of the annelid neoblasts, a comparison was made between the regeneration patterns of two enchytraeid oligochaetes, Enchytraeus japonensis and Enchytraeus buchholzi, which possess and lack neoblasts, respectively. In E. japonensis, which can reproduce asexually by fragmentation and subsequent regeneration, neoblasts are present in all segments except for the eight anterior-most segments including the seven head-specific segments, and all body fragments containing neoblasts can regenerate a complete head and a complete tail, irrespective of the region of the body from which they were originally derived. In E. japonensis, therefore, no antero-posterior gradient of regeneration ability exists in the trunk region. However, when amputation was carried out within the head region, where neoblasts are absent, the number of regenerated segments was found to be dependent on the level of amputation along the body axis. In E. buchholzi, which reproduces only sexually and lacks neoblasts in all segments, complete heads were never regenerated and incomplete (hypomeric) heads could be regenerated only from the anterior region of the body. Such an antero-posterior gradient of regeneration ability was observed for both the anterior and posterior regeneration in the whole body of E. buchholzi. These results indicate that the presence of neoblasts correlates with the absence of an antero-posterior gradient of regeneration ability along the body axis, and suggest that the annelid neoblasts are more essential for efficient asexual reproduction than for the regeneration of missing body parts.     

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.