The mechanism of anterior-posterior polarity control in planarians

The substance which inhibits brain formation in the regenerating planarian Dugesia etrusca was found to be a large molecule, at least in part protein, which electrophoreses as an electronegative moiety in pH 6.8 buffer. A model is presented, based on this finding and previous studies, which proposes an electrochemical mechanism for the control of polarity and possibly for the maintenance of tissue organization in planarians. It is proposed that a bioelectric field exists and moves the electronegative brain-inhibiting substance in a posterior direction, establishing polarity. This model explains the polarity reversal experiments using external fields and many of the previously unexplained classical planarian experiments. Data are presented demonstrating the existence, magnitude, and polarity of this bioelectric field, which is not greatly altered upon decapitation, all in accord with predictions of the model.     

An insight into planarian regeneration

BackgroundPlanarian has attracted increasing attentions in the regeneration field for its usefulness as an important biological model organism attributing to its strong regeneration ability. Both the complexity of multiple regulatory networks and their coordinate functions contribute to the maintenance of normal cellular homeostasis and the process of regeneration in planarian. The polarity, size, location and number of regeneration tissues are regulated by diverse mechanisms. In this review we summarize the recent advances about the importance genetic and molecular mechanisms for regeneration control on various tissues in planarian.MethodsA comprehensive literature search of original articles published in recent years was performed in regards to the molecular mechanism of each cell types during the planarian regeneration, including neoblast, nerve system, eye spot, excretory system and epidermal.ResultsAvailable molecular mechanisms gave us an overview of regeneration process in every tissue. The sense of injuries and initiation of regeneration is regulated by diverse genes like follistatin and ERK signaling. The Neoblasts differentiate into tissue progenitors under the regulation of genes such as egfr‐3. The regeneration polarity is controlled by Wnt pathway, BMP pathway and bioelectric signals. The neoblast within the blastema differentiate into desired cell types and regenerate the missing tissues. Those tissue specific genes regulate the tissue progenitor cells to differentiate into desired cell types to complete the regeneration process.ConclusionAll tissue types in planarian participate in the regeneration process regulated by distinct molecular factors and cellular signaling pathways. The neoblasts play vital roles in tissue regeneration and morphology maintenance. These studies provide new insights into the molecular mechanisms for regulating planarian regeneration.

Genetic and molecular mechanisms for regeneration control on various tissues in planarian.     

Differentiation During Regeneration Caused by Migration of Repressors in Bioelectric Fields

Experiments are reviewed which have led to the conclusion thatdifferentiation occurs during regeneration in Tubularia onlyif a bioelectric field of sufficient strength is maintained.The electrical polarity seems to determine the polarity of morphogenesisby controlling the movement of charged repressors. The repressorswork in this totipotent system by preventing recipient regionsfrom becoming what the donor regions are becoming. It appearsthat any level can become the most anterior structure not forminganterior to it. Thus by a series of repressions in totipotentmaterial all levels of a hydranth are established.     

The bioelectric field of the catfish Ictalurus nebulosus.

The variability of the bioelectric field of the electrosensitive catfish, Ictalurus nebulosus, was investigated by recording the potential variation occurring when the fish passed a stationary electrode, and by recording the field of a stationary fish by a 15-electrode array. A good first order approximation of the recorded field of a 20 cm long fish is a dipole dc source with the source and sink about 7 cm apart, carrying a current of about 1 microA in water with a specific resistivity of 3.3 kohm cm. At 5 cm distance from the dipole axis such a source generates an electric potential swing in the order of 50 microV in free space, head negative, tail positive. Superimposed on the basic component are respiration related fluctuations, and fluctuations related to the activity of the alimentary canal, gills, and skin. Novel stimuli, or stressors like investigators approaching the aquarium, evoke sudden increases in field strength. which last about 15 min. Demineralization of the aquarium water causes changes in field strength and reversal of field polarity. The administration of food causes field variations in the vicinity of the anal opening. The bioelectric field shows diurnal fluctuations of 100 microV. The peak is at about 04:00, the dip at 14:00. The fluctuations of the bioelectric field are sufficiently strong and specilic to serve as electrical stimuli to other electrosensitive catfish. It is suggested that the field changes allow a simple form of electrocommunication. i.e. inform conspecifics about some physiological properties of the field source. The cellular mechanisms underlying the fluctuations of the bioelectric field are homeostatic processes mediated by ion pumps and ion channels.     

Differentiation of epidermal cells in the regenerating planarian Dugesia japonica

Distribution of the cytoplasmic components in planarian epidermal cells is highly polarized, just as in vertebrate epithelia. Differentiating epidermal cells of the planarian Dugesia japonica Ichikawa et Kawakatsu were found to have relatively conspicuous accumulations of microtubules in their apical cytoplasm. When colchicine, a microtubule-disrupting drug, was applied to regenerating worms, it reversibly disorganized the polarity of differentiating epidermal cells. Cytochalasin B, which depolymerizes actin filaments, had no significant effect on the polarization, however. Tubulin could be localized by immunocytochemistry in the cytoplasm of differentiating epidermal cells; this reaction was inhibited by treatment with colchicine for 20 h. These observations indicate that microtubules play a role in establishing polarity during cell differentiation.     

A polar transport model of planarian regeneration

Abstract. A mathematical model based on the hypothesis of polar transport is presented for pattern formation and regeneration in planarian worms. The planarian body pattern is determined by two perpendicularly oriented morphogen distributions, one of which is bilaterally symmetrical. The mathematically simulated regeneration responses of this model are in good agreement with experimental observations. Further experimental work is needed to test the polar transport hypothesis.     

Autophagy meets planarians

This review aims to demonstrate the importance of freshwater planarians as model organisms, particularly emphasizing those characteristics of the animal that make them a good model to study autophagy. The aim of this review is to provide a better understanding of autophagy in this model for the non-planarian reader, and elucidate the relevance of autophagy research in this peculiar model organism. Furthermore, I will try to synthesize the evidence showing the importance of autophagy in planarian body remodelling, and I will discuss some ideas about the role of autophagy in stem cell biology. In light of these new developments, it is likely that the planarian field will make an important contribution to the study of the molecular mechanisms involved in autophagy in the future.     

Oblique dipole layer potentials applied to electrocardiology

We study the properties of the potential field generated by an oblique dipole layer. This field arises, for instance, in describing the potential elicited by a depolarization wavefront spreading in the myocardium when a dependence of the potential on the cardiac fiber orientation is introduced. The representation of cardiac bioelectric sources by means of an oblique dipole layer leads to a mathematical structure which generalizes the classical solid angle theory used in electrocardiology, which has been challenged by recent experimental evidence, and links models previously proposed with a view to adequately reproduce the potential observed in experiments. We investigate also the relationship between our model and an intracellular current model and we derive potential jump formulae for some models which account for the anisotropic structure of the myocardium. The potential generated by an oblique dipole layer is considered both for unbounded and bounded domains. In the latter case an integral boundary equation is derived and we study its solvability. A numerical procedure for solving this integral equation by means of the finite element method with collocation is outlined.     

Planarian embryology in the era of comparative developmental biology

During the last decade, the field of evolutionary developmental biology (evo-devo) has emerged as a major research discipline in modern biology and an essential approach to understanding evolutionary relationships in the animal kingdom. At the same time, planarians have become a useful and important model with which to address basic questions regarding the molecular and cellular basis of regeneration, tissue repair and stem cells in adult organisms. Nevertheless, little attention has been paid to their embryonic development, even though this provides a unique opportunity for studying how molecular developmental mechanisms are re-deployed during adult regeneration or the independent losses of spiral cleavage that took place in different lophotrochozoan lineages. In this paper, we review the most relevant works on planarian embryos from a historical point of view. In doing so, we highlight the questions that have recurrently intrigued researchers, most of which remain unanswered. Finally, we present a comprehensive scenario for planarian embryogenesis in an attempt to provide a testable hypothesis that will help to bridge the gap between this divergent mode of development, the ancestral canonical spiral cleavage, and adult planarian regeneration.     

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities¹. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a ''trunk'' fragment is cut from an intact worm, a new ''head'' will regenerate anteriorly and a ''tail'' will regenerate posteriorly restoring the original ''head-to-tail'' polarity of body structures prior to amputation (Figure 1A).Regeneration is driven by planarian stem cells, known as ''neoblasts'' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity^2-9.The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters¹⁰, represent an important system for studying evolution of the central nervous system^{11, 12} and have behavioral screening potential^{13, 14.} Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs^{8, 9, 15}. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals^16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.     

Synergistic interactions between temporal coupling of complex light and magnetic pulses upon melanoma cell proliferation and planarian regeneration

Synergisms between a physiologically patterned magnetic field that is known to enhance planarian growth and suppress proliferation of malignant cells in culture and three light emitting diode (LED) generated visible wavelengths (blue, green, red) upon planarian regeneration and melanoma cell numbers were discerned. Five days of hourly exposures to either a physiologically patterned (2.5–5.0 μT) magnetic field, one of three wavelengths (3 kLux) or both treatments simultaneously indicated that red light (680 nm), blue light (470 nm) or the magnetic field significantly facilitated regeneration of planarian compared to sham field exposed planarian. Presentation of both light and magnetic field conditions enhanced the effect. Whereas the blue and red light diminished the growth of malignant (melanoma) cells, the effect was not as large as that produced by the magnetic field. Only the paired presentation of the blue light and magnetic field enhanced the suppression. On the other hand, the changes following green light (540 nm) exposure did not differ from the control condition and green light presented with the magnetic field eliminated its effects for both the planarian and melanoma cells. These results indicate specific colors affect positive adaptation that is similar to weak, physiologically patterned frequency modulated (8–24 Hz) magnetic fields and that the two forms of energy can synergistically summate or cancel.     

A reference curve for axial bioelectric potentials in rabbit tibia

A high coefficient of variation is characteristic of the bioelectric potentials recorded from living long bones. As a consequence, the data collected from animal and human experiments are difficult to use in practical ways. A distribution curve for bone bioelectric potentials has been calculated using polynomial regression analysis to process the voltage values recorded on the whole length of rabbit tibiae, with reference electrodes positioned in three different points of the bone. The distribution curve so obtained is presented to fulfill the need for a reference curve for the bioelectric potentials recorded from rabbit tibial surfaces.     

Oblique double layer potentials for the direct and inverse problems of electrocardiology

A mathematical analysis of a model of the cardiac bioelectric sources generating the potential field in a bounded conducting volume is carried out. A boundary integral representation of the potential is given, which shows that, if the anisotropy of the cardiac muscle is taken into account, the bioelectric sources are characterized by an “oblique” double layer on the excitation wavefront. Boundedness conditions for the potential and jump relationships across the front are investigated. Uniqueness results for the inverse problem (i.e. the determination of the wavefront surface, given the potential on the volume boundary) are established.     

Signification of the sexualizing substance produced by the sexualized planarians

Asexual worms of an exclusively fissiparous strain (the OH strain) of the planarian Dugesia ryukyuensis keep developing hermaphroditic reproductive organs and eventually undergo sexual reproduction instead of asexual reproduction, namely fission, if they are fed with sexually mature worms of an exclusively oviparous planarian, Bdellocephala brunnea, suggesting that the sexually mature worms has a sexualizing substance(s). The fully sexualized worms no longer need the feeding on sexual worms to maintain the sexuality. Here, we demonstrate that the sexualized worms produce enough of their own sexualizing substance similar to that contained in B. brunnea. In case of surgical ablation of the sexualized worms, the fragments with sexual organs regenerate to become sexual, while those without sexual organs, namely head fragments, regenerate to return to the asexual state. The asexual regenerants from the sexualized worms are also fully sexualized by being fed with B. brunnea. Additionally, it was reported that head region in sexually mature worms lacks the putative sexualizing substance necessary for complete sexualization (Sakurai, 1981). These results suggest that the fragments without sexual organ lack enough of an amount of the putative sexualizing substance and the sexuality is maintained by the sexualizing substance contained in the sexualized worms.     

The past and present of planarians--an interview with Vittorio Gremigni. Interview by Alessandra Salvetti and Leonardo Rossi

Vittorio Gremigni is a scientific leader in the field of planarian biology with a very long historical perspective. By using electron microscopy, he contributed to the reconstruction of the phylogenesis of free living "Turbellaria", and he pioneered the study of the origin of blastema cells by using chromosomal markers. In this interview, Professor Gremigni describes the steps that moved his career towards the planarian field, the main scientific achievements he obtained and the changes that are taking place in the field. He also discusses recent progress and unanswered questions on planarian neoblasts and regeneration.     

The Electroosmotic Effects Arising from the Interaction of the Selectively Anion and Selectively Cation Permeable Parts of Mosaic Membranes

It has been previously shown, theoretically and in model system experiments, that mosaic membranes composed of anion-selective (electropositive) and cation-selective (electronegative) parts interposed between electrolytic solutions of different concentrations give rise to local electrical circuits. In this work with model systems it is shown that these currents produce electroosmosis. In systems with permselective electronegative membranes and KCl solutions, the electroosmotic water transport was 16 moles/faraday. With the permselective electronegative membrane replaced by more porous electronegative membranes, the electroosmotic effects were about twice as high. With Li salts, the water transport was considerably larger. A system with a permselective electropositive membrane of 50 cm² effective area and an electronegative membrane of 120 cm² gave internally generated currents up to 20 ma. In extrapolating from the results with macromodels to effects with true mosaics, i.e. microsystems, it is stressed that current depends on the linear distance over which membranes interact. In true mosaic membranes, the current pathways will be of the same order as the dimensions of individual membrane microelements; the sum of all local microcurrents will be correspondingly larger than the current in the macromodel, and the electroosmotic effects will be proportionately greater. Electroosmotic effects with true charge-mosaic membranes may be of the same order or larger than the liquid transport by normal and anomalous osmosis which might occur across the individual parts of the charge-mosaic.     

The body margin of the planarian Dugesia japonica: characterization by the expression of an intermediate filament gene

We have cloned and sequenced a cDNA encoding an intermediate filament protein (IF) from the planarian Dugesia japonica named DjIFb. The deduced amino acid sequence of DjIFb has similarity to those of protostomic IFs and lamins, supporting a previous hypothesis that the protostomic IFs, including DjIFb, are evolutionarily closer to lamins than to vertebrate cytoplasmic IFs. In addition, analysis of the exon/intron organization revealed that 8 out of 10 introns of DjIFb were coincident in their position, even in the codon phase, with those of the non-neuronal IF of the snail Helix aspersa. This suggests that the Platyhelminthes are not the most primitive Bilateria but instead are evolutionarily close to the Mollusca. The DjIFb gene was expressed in particular cells, probably a kind of adhesive gland cell, which were present in the marginal region encircling the planarian body. The localization of DjIFb protein suggests that it plays an important role in the secretion of an adhesive substance. The specific expression pattern of the DjIFb gene enabled us to monitor how the body margin forms during planarian regeneration.     

涡虫生殖生物学研究进展

涡虫由于具有极强的再生能力而成为发育生物学及再生生物学研究的模式生物。此外,其在有性生殖方面所表现出来的独特性也备受人们关注。目前,涡虫生殖生物学研究领域主要围绕两个热点问题开展工作：1.无性生殖向有性生殖转化的诱因及机制的探讨;2.生殖相关基因的克隆、表达及功能分析。有关生殖转化机制方面的研究主要集中在涡虫的性化相关事件以及性化物质的本质探索;截至目前已克隆并对其表达和功能进行探讨的涡虫生殖相关基因主要有DjPTK1、vasa-like 基因、DeY1、Dryg、nanos相关基因以及Drpiwi-1等。此外,本文也对有关涡虫生殖生物学方面存在的问题及未来该领域的发展趋势进行了总结和展望。