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.     

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.     

Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.     

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.     

Establishment and characterization of hepatic stem-like cell lines from normal adult rat liver

The liver is a unique organ with the potential to regenerate from injury. Hepatic stem cells contribute to liver regeneration when surviving hepatocytes in injured liver are unable to proliferate. To investigate the mechanism of liver regeneration in vitro, we established hepatic stem cell lines named HY1, HY2 and HY3, derived from a healthy liver of adult rat. HY cells showed an expression pattern similar to oval cells, and efficiently induced hepatic differentiation following sequential treatment with type I collagen, transforming growth factor-beta1 (TGF-beta1), and hepatocyte growth factor (HGF) or oncostatin M (OSM). These results suggested that HY cells are liver stem cells representing an excellent tool for in vitro studies on liver regeneration.     

Intercalary regeneration of symmetrical thighs in the axolotl,Ambystoma mexicanum

Intercalary regeneration of stylopodial and zeugopodial skeletal elements takes place in axolotl limbs composed of normal wrist blastemas autografted or homografted to double half-anterior or half-posterior thighs. Analysis of the morphological pattern of the skeleton and, in homografts, of pigmentation pattern, shows that the intercalated elements are derived from the host double half-thigh. Intercalary regeneration from double half-posterior thighs is expected since they normally can undergo complete proximal-distal regeneration, but is not necessarily expected from double half-anterior thighs, since they normally do not regenerate more distal segments. These results demonstrate that (1) cells of double half-anterior thighs are not inherently incapable of undergoing distal transformation, (2) cells of a distal blastema grafted to a more proximal level do not form patterns proximal to their level of origin, and (3) there is an inhibitory interaction between blastema cells derived from double half-anterior thighs that is expressed after simple amputation, but not when these cells are in contact with a more distal, normal blastema. Using these and other data, a three-dimensional boundary model of limb regeneration is proposed.     

Involvement of hedgehog,wingless, and dpp in the initiation of proximodistal axis formation during the regeneration of insect legs,a verification of the modified boundary model

To understand the mechanism of regeneration, many experiments have been carried out with hemimetabolous insects, since their nymphs possess the ability to regenerate amputated legs. We first succeeded in observing expression patterns of hedgehog, wingless (wg), and decapentaplegic (dpp) during leg regeneration of the cricket Gryllus bimaculatus. The observed expression patterns were essentially consistent with the predictions derived from the boundary model modified by Campbell and Tomlinson (CTBM). Thus, we concluded that the formation of the proximodistal axis of a regenerating leg is triggered at a site where ventral wg-expressing cells abut dorsal dpp-expressing cells in the anteroposterior (A/P) boundary, as postulated in the CTBM.     

Deer antler – A novel model for studying organ regeneration in mammals

Deer antler is the only mammalian organ that can fully grow back once lost from its pedicle – the base from which it grows. Therefore, antlers probably offer the most pertinent model for studying organ regeneration in mammals. This paper reviews our current understanding of the mechanisms underlying regeneration of antlers, and provides insights into the possible use for human regenerative medicine. Based on the definition, antler renewal belongs to a special type of regeneration termed epimorphic. However, histological examination failed to detect dedifferentiation of any cell type on the pedicle stump and the formation of a blastema, which are hallmark features of classic epimorphic regeneration. Instead, antler regeneration is achieved through the recruitment, proliferation and differentiation of the single cell type in the pedicle periosteum (PP). The PP cells are the direct derivatives of cells resident in the antlerogenic periosteum (AP), a tissue that exists in prepubertal deer calves and can induce ectopic antler formation when transplanted elsewhere on the deer body. Both the AP and PP cells express key embryonic stem cell markers and can be induced to differentiate into multiple cell lineages in vitro and, therefore, they are termed antler stem cells, and antler regeneration is a stem cell-based epimorphic regeneration. Comparisons between the healing process on the stumps from an amputated mouse limb and early regeneration of antlers suggest that the stump of a mouse limb cannot regenerate because of the limited potential of periosteal cells in long bones to proliferate. If we can impart a greater potential of these periosteal cells to proliferate, we might at least be able to partially regenerate limbs lost from humans. Taken together, a greater understanding of the mechanisms that regulate the regeneration of antlers may provide a valuable insight to aid the field of regenerative medicine.This article is part of a Directed Issue entitled: Regenerative Medicine: the challenge of translation.     

Notch signaling inhibits axon regeneration

Many neurons have limited capacity to regenerate their axons after injury. Neurons in the mammalian central nervous system do not regenerate, and even neurons in the peripheral nervous system often fail to regenerate to their former targets. This failure is likely due in part to pathways that actively restrict regeneration; however, only a few factors that limit regeneration are known. Here, using single-neuron analysis of regeneration in?vivo, we show that Notch/lin-12 signaling inhibits the regeneration of mature C.?elegans neurons. Notch signaling suppresses regeneration by acting autonomously in the injured cell to prevent growth cone formation. The metalloprotease and gamma-secretase cleavage events that lead to Notch activation during development are also required for its activity in regeneration. Furthermore, blocking Notch activation immediately after injury improves regeneration. Our results define a postdevelopmental role for the Notch pathway as a repressor of axon regeneration in?vivo.     

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.     

Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.     

Proximodistal patterning during limb regeneration

Regeneration is an ability that has been observed extensively throughout metazoan phylogeny. Amongst vertebrates, the urodele amphibians stand out for their exceptional capacity to regenerate body parts such as the limb. During this process, only the missing portion of the limb is precisely replaced--amputation in the upper arm results in regeneration of the entire limb, while amputation at the wrist produces a hand. Limb regeneration occurs through the formation of a local proliferative zone called the blastema. Here, we examine how proximodistal identity is established in the blastema. Using cell marking and transplantation experiments, we show that distal identities have already been established in the earliest stages of blastemas examined. Transplantation of cells into new environments is not sufficient to respecify cell identity. However, overexpression of the CD59, a cell surface molecule previously implicated in proximodistal identity during limb regeneration, causes distal blastema cells to translocate to a more proximal location and causes defects in the patterning of the distal elements of the regenerate. We suggest a model for the limb regeneration blastema where by 4 days post-amputation the blastema is already divided into distinct growth zones; the cells of each zone are already specified to give rise to upper arm, lower arm, and hand.     

Ganglion cell regeneration following whole-retina destruction in zebrafish

The retinas of adult teleost fish can regenerate neurons following injury. The current study provides the first documentation of functional whole retina regeneration in the zebrafish, Danio rerio, following intraocular injection of the cytotoxin, ouabain. Loss and replacement of laminated retinal tissue was monitored by analysis of cell death and cell proliferation, and by analysis of retina-specific gene expression patterns. The spatiotemporal process of retinal ganglion cell (RGC) regeneration was followed through the use of selective markers, and was found to largely recapitulate the spatiotemporal process of embryonic ganglion cell neurogenesis, over a more protracted time frame. However, the re-expression of some ganglion cell markers was not observed. The growth and pathfinding of ganglion cell axons was evaluated by measurement of the optic nerve head (ONH), and the restoration of normal ONH size was found to correspond to the time of recovery of two visually-mediated behaviors. However, some abnormalities were noted, including overproduction of RGCs, and progressive and excessive growth of the ONH at longer recovery times. This model system for whole-retina regeneration has provided an informative view of the regenerative process.     

Dynamic gene expression profiles during arm regeneration in the brittle star Amphiura filiformis

Echinoderms and in particular brittle stars display a remarkable ability to regenerate lost or damaged tissues. They offer an excellent model in which to study regeneration displaying extensive regenerative ability and close relationship to vertebrates providing the opportunity for comparative studies. Previous studies of gene expression during arm regeneration in brittle stars have focused on single genes commonly associated with the regenerative process. In this study we present the first microarray investigation of gene expression during arm regeneration in the brittle star Amphiura filiformis. We show the large-scale gene expression changes associated with the complex process of regeneration with over 50% of the clones measured showing a significant change at some point during the process when compared to non-regenerating arms. Particular attention is paid to genes associated with Hox gene expression regulation, neuronal development and the bone morphogenic protein BMP-1. Our data give an insight into the molecular control required during the various stages of regeneration from the stem cell rich blastema stage through to the highly differentiated regenerate. This work also forms an important basis for future gene expression investigations in this emerging model of limb regeneration.     

Vascular Regeneration in a Basal Chordate Is Due to the Presence of Immobile,Bi-Functional Cells

The source of tissue turnover during homeostasis or following injury is usually due to proliferation of a small number of resident, lineage-restricted stem cells that have the ability to amplify and differentiate into mature cell types. We are studying vascular regeneration in a chordate model organism, Botryllus schlosseri, and have previously found that following surgical ablation of the extracorporeal vasculature, new tissue will regenerate in a VEGF-dependent process within 48 hrs. Here we use a novel vascular cell lineage tracing methodology to assess regeneration in parabiosed individuals and demonstrate that the source of regenerated vasculature is due to the proliferation of pre-existing vascular resident cells and not a mobile progenitor. We also show that these cells are bi-potential, and can reversibly adopt two fates, that of the newly forming vessels or the differentiated vascular tissue at the terminus of the vasculature, known as ampullae. In addition, we show that pre-existing vascular resident cells differentially express progenitor and differentiated cell markers including the Botryllus homologs of CD133, VEGFR-2, and Cadherin during the regenerative process.     

Evolutionary loss of animal regeneration: pattern and process

The ability to regenerate lost or damaged body parts is widespread among animals and provides obvious potential benefits. It is therefore perplexing that this ability has become greatly restricted or completely lost in many lineages. Despite growing interest in the cellular and molecular basis of regeneration, our understanding of how and why regenerative abilities are lost remains rudimentary. In an effort to develop a framework for studying losses of regeneration, here I outline an approach for rigorously identifying such losses, review broad patterns of regenerative ability across animals, describe some of the clearest examples of regeneration loss, discuss some possible scenarios by which regeneration may be lost, and review recent work in annelids that is providing new insights into loss of regenerative ability.     

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.     

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.     

'Open minded' cells: how cells can change fate

It has long intrigued researchers why some but not all organisms can regenerate missing body parts. Plants are remarkable in that they can regenerate the entire organism from a small piece of tissue, or even a single cell. Epigenetic mechanisms that control chromatin organization are now known to regulate the cellular plasticity and reprogramming necessary for regeneration. Interestingly, although animals and plants have evolved different strategies and mechanisms to control developmental processes, they have maintained many similarities in the way they regulate chromatin organization. Given that plants can rapidly switch fate, we propose that an understanding of the mechanisms regulating this process in plant cells could provide a new perspective on cellular dedifferentiation in animals.