Monoclonal antibodies as markers of specific cell types and regional antigens in the freshwater planarian Dugesia (G.) tigrina

We have produced monoclonal antibodies (mAb's) against antigens of the fresh-water planarian Dugesia (G.) tigrina (Girard) using standard protocols. Labeling these mAb's with PAP (peroxidase-antiperoxidase) and indirect-immunofluorescence methods, we then determined the distribution of their antigens in the planarian. Out of 112 mAb's that showed some specificity for restricted parts of the planarian, 71 were found to be cell- or tissue-specific — among them 36 for parenchymal cells, 7 for muscle cells, 11 for epidermal cells, 8 for gastrodermis, and 7 to basement membrane. Another 41 showed different, but overlapping, regional specificities, namely to pharynx and parenchyma. So far, we have been unable to isolate specific mAb's against undifferentiated cells (neoblasts). These mAb's should be important tools in study of tissue and cell morphology, regeneration, and growth and degrowth.     

TCEN-49, a monoclonal antibody that identifies a central body antigen in the planarian Dugesia (Girardia) tigrina. Implications for pattern formation and positional signalling mechanisms

We have produced monoclonal antibodies (mAbs) against antigens of the freshwater planarian Dugesia (G.) tigrina (Girard) using standard protocols. One of these mAbs, TCEN-49, detects an antigen (TCEN-49Ag) present in most cells of the central area of the body, including the pharynx. Labelled cells seem more related by position than by lineage, suggesting that TCEN-49Ag is involved somehow in the expression of central body positional identity. The spatial and temporal changes in TCEN-49Ag expression during growth/degrowth and regeneration have been monitored and the implications of these results are discussed.     

Morphological stages of regeneration in the planarian Dugesia tigrina: A light and electron microscopic study

A precise sequence of four morphological stages of head regeneration in the planarian Dugesia tigrina has been determined by light and electron microscopy. Each stage is identified by a particular morphogenetic process: I, wound healing; II, blastema development; III, growth; IV, differentiation. A wound epidermis consisting of a thin, sheet-like layer of cells, rapidly forms from undamaged epidermal cells at the wound margin. The early blastema is comprised of neoblasts which mature into regeneration cells. The maturational changes include the appearance of a nucleolus, nuclear pores, and perinuclear dense aggregates of granulofibrillar material in these cells. These elements are not evident in the neoblasts of the younger blastema. No mitotic cells are encountered in the blastema or wound epidermis. Cytoplasmic expansion of the regeneration cells is correlated with the formation of numerous microtubules radiating from a juxtanuclear centrosphere. During differentiation of muscle cells, distended, granule-studded cisternae, having moderately fibrillar contents, are regularly disposed adjacent to small patches of myofilaments. Presumptive epidermal cells are recognized by prominent “islands” of finely fibrillar cytoplasm. These cytoplasmic zones persist for a time during definitive differentiation when Golgi bodies, vacuoles, mucous droplets, and rhabdites become evident. The newly formed epidermal cells become inserted among the cells of the wound epidermis. Thus, cells of both the blastema and of the wound epidermis contribute to the reconstituted epidermis.     

Ultrastructural observations on gastrodermal regeneration in the planarian Dugesia japonica (Turbellaria)

The earliest detectable change during regeneration of the gastrodermis in Dugesia japonica was an aggregation of regenerative cells underneath the gastrodermis remaining at the wound margin. The gastrodermal cells in experimental regenerates retained some of their original characters and presented no indication of cell dedifferentiation. The regenerative cells came into contact with the basal surface of gastrodermal cells, forming stratified cell layers. Differentiation of these cells into gastrodermal cells was initiated by the development of synthetic organelles within their cytoplasm. These differentiating cells gave rise to two different types of gastrodermal cells, namely phagocytic cells and sphere cells. In later stages, there was an apparent movement of differentiated gastrodermal cells towards the parenchyma.     

Cell-, tissue-, and position-specific monoclonal antibodies against the planarian Dugesia (Girardia) tigrina

 To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the freshwater planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians. Accepted: 16 September 1996     

Quantitative Estimation of HRP-Labeled Sensory and Motor Neurons during Nerve-Dependent and Nerve-Independent Periods of Urodele Limb Regeneration

The relationship between urodele regeneration and the possibility of regeneration in mammals is unclear, but the idea of possible regeneration of neural elements in man is being studied because of its potential clinical importance. One of the great challenges is to gain sufficient knowledge about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is known that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibers connected to their cell bodies. The nerve fibers severed at the level of limb amputation regrow and penetrate the blastema, providing blastema cells with indispensable factors. These factors are produced in the perikarya of neurons and transported via their axons to the blastema. Numerous studies have been performed to elucidate the quantitative relationships between nerve fibers and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of this investigation was to analyze the quantitative participation and qualitative distinctions of different nerve cells innervating the regenerating parts of the urodele limb and their possible roles in the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral part of the spinal cord gray matter (motor neurons). The direct involvement of these neurons in different regeneration periods was visualized by means of horseradish peroxidase (HRP) labeling. A total of 34 animals (21 experimental and 13 control) were used to study fluctuations in the numbers of labeled nerve cells. The results are summarized as follows: (a) the first nerve cells incorporating HRP within 5 days after amputation are found in the dorsal ganglia, whereas motor neurons in the gray matter are labeled within 7 days; (b) the number of labeled perikarya increases during the nerve-dependent regeneration period (0–21 days after amputation), with the percentage of implicated sensory neurons exceeding that found in the control series; and (c) during the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration on the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.     

Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.     

Morphogenesis in Planarians Dugesia tigrina

We carried out computer morphometry in regenerates of planarians Dugesia tigrina. The blastema growth was analyzed in fragments of planarians after their fission and after transverse transection at different body levels. The blastema was growing at a higher rate on tail fragments than on the head fragments and the growth rate was the higher, the closer the transection was to the head end. After fission, the blastema was growing at a slower rate than after transection in the fission zone. The growth of adjacent blastemas formed on both sides after fission or transection proceeded at different rates as a function of new body polarity.     

A fine structural study of regeneration after fission in the planarian Dugesia japonica

We examined morphologically the process of regeneration before and after fission in a sexual strain of the freshwater planarian Dugesia japonica. Usually fission takes place in the post-pharyngeal region. Decapitation significantly accelerates the rate of fissioning. When decapitated worms were treated with substance P and neuropeptide K separately, the rate of fission markedly decreased in both cases. Before the onset of fission, a presumptive region of fission was recognized in the post-pharyngeal portion where undifferentiated cells, regenerative cells and newly differentiated cells were localized. Moreover a functional network of fixed parenchyma cells was noted in this region. After fission, cell distribution in the blastema became quite different from that of artificially amputated worms. This difference seems to be due to the process that occurs in the presumptive region of fission. This revised version was published online in July 2006 with corrections to the Cover Date.     

Adenylate cyclase in regenerating tissues of the planarian Dugesia lugubris (O. Schmidt)

Adenylate cyclase (AC) was localized ultracytochemically in certain tissues of the regenerating planarian Dugesia lugubris. Studies were carried out from one hour after injury up to the 5th day of regeneration. It was found that the greatest amount of active AC appears during the initial hours of regeneration in the membranes of the muscle cells near the wound, in the epithelial cells surrounding the wound, and in rhabdite-forming cells and neoblasts.     

p53同系物Djp53在涡虫胚基早期发育阶段的组织特异性表达及其在再生中的作用

为了探索东亚三角涡虫Djp53基因在涡虫组织中的表达和功能,利用整体原位杂交、RT-PCR技术,检测了涡虫Djp53基因在组织中的表达分布特点,结果表明,Djp53基因在再生1、3、5天的胚基中具有较强的阳性信号,且3天的表达量最高;而在再生7、10天和成虫的实质组织中表达较弱．RNA干扰后的RT-PCR检测显示,Djp53表达量显著下降,涡虫不能正常再生或出现眼点缺陷．由此推断,东亚三角涡虫Djp53基因在早期胚基发育阶段,通过调节多功能干细胞的迁移和增殖分化影响早期胚基的形成,是涡虫早期胚基发育必不可少的一个基因,并且在涡虫成体和再生后期对多功能干细胞的维持具有重要的作用.     

Inhibition of regeneration in the planarian Dugesia polychroa (Schmidt) by treatment with magnesium chloride: a morphological study of wound closure

At a concentration of 0.2% (21 m M) in culture water, magnesium chloride impaired muscle contraction and completely inhibited head regeneration in specimens of Dugesia polychroa cut prepharyngeally. The wound stayed open for nine days, with neoblasts accumulating beneath the wound without any signs of differentiation. Extremely delayed wound closure occurred by spreading epithelial cells, and was completed after 30 days in the magnesium chloride solution. Histological examination confirmed the absence of any regenerated head structures. Interestingly, the inhibitory effect was removed when such headless fragments were cut once more and kept in normal culture water: complete head regeneration then occurred at a normal rate. Among several possible explanations for the failure to regenerate, the following hypothesis is an attractive alternative: direct contact between parenchyma and epithelial cells during the period following injury seems to be an essential stimulus for the start of cell differentiation within the blastema, and the lack of such contact as a result of the drug action prevents normal regeneration. When the wound has eventually closed, a continuous basement membrane separates epithelium from parenchyma. Thus a direct contact between these tissues is never established. This revised version was published online in July 2006 with corrections to the Cover Date.     

Epidermal cell migration during wound healing in Dugesia lugubris

The epidermal cells that migrate over the surface during the wound closure stage of head regeneration in Dugesia lugubris s.l. were examined by scanning electron microscopy. The effect of cytochalasin B on epidermal cell migration was also examined. During the first few hours after decapitation epidermal cells at the edges of the wound showed significant changes of shape related to the process of migration that was accomplished approximately 10 h after wounding. Flattening of the marginal cells was associated with active epidermal spreading throughout the healing period. Suitable support for migrating cells appeared to be a rhabditic network attached to the wound tissue. Epidermal cell migration was inhibited by cytochalasin B. These results demonstrate that the basis for cell movement in planarians is similar to that of many other systems.     

Positional information and gonadal differentiation in the planarian Dugesia lugubris (Turbellaria)

The planarian Dugesia lugubris is a balanced hermaphrodite, meaning that male genetic factors are in equilibrium with female factors. Differentiation of the gonads is controlled by the region in which they develop. According to the classical theory of germ cell formation, these cells stem from neoblasts that are induced to differentiate by factors specific to the gonadal regions, factors presumably due to gradients formed by neurosecretory activity of the cephalic ganglia and longitudinal nerve cords. A more recently proposed theory holds that germ cells in regenerates originate not from neoblasts but from dedifferentiated cells and that characteristics of the gonadal regions are determined by direct interactions of cells here. Results of our experiments with homo- and autoplastic grafst support the classical theory. Prepharyngeal portions grafted onto posterior body portions retained their ability to maintain or induce development of ovaries. Postpharyngeal portions grafted onto anterior portions produced only testes even though the brain developed normally in these regenerates. Under these experimental conditions, gonad regeneration took longer than it does in normal regeneration (i.e., that in which body regions are not displaced).Translated, from the French, by Marianne Klauser and Seth Tyler.     

A resource of single-cell gene expression profiles in a planarian Dugesia japonica

The freshwater planarian Dugesia japonica maintains an abundant heterogeneous cell population called neoblasts, which include adult pluripotent stem cells. Thus, it is an excellent model organism for stem cell and regeneration research. Recently, many single-cell RNA sequencing (scRNA-seq) databases of several model organisms, including other planarian species, have become publicly available; these are powerful and useful resources to search for gene expression in various tissues and cells. However, the only scRNA-seq dataset for D. japonica has been limited by the number of genes detected. Herein, we collected D. japonica cells, and conducted an scRNA-seq analysis. A novel, automatic, iterative cell clustering strategy produced a dataset of 3,404 cells, which could be classified into 63 cell types based on gene expression profiles. We introduced two examples for utilizing the scRNA-seq dataset in this study using D. japonica. First, the dataset provided results consistent with previous studies as well as novel functionally relevant insights, that is, the expression of DjMTA and DjP2X-A genes in neoblasts that give rise to differentiated cells. Second, we conducted an integrative analysis of the scRNA-seq dataset and time-course bulk RNA-seq of irradiated animals, demonstrating that the dataset can help interpret differentially expressed genes captured via bulk RNA-seq. Using the R package “Seurat” and GSE223927, researchers can easily access and utilize this dataset.     

Distribution of S-phase cells during the regeneration of Drosophila imaginal wing discs

We investigated the distribution of S-phase cells during regeneration of the imaginal wing disc of Drosophila melanogaster following excision of 30 degrees, 90 degrees, and 150 degrees sectors of tissue. The fragments were cultured in adult abdomens for 1-5 days, labeled in vitro with tritiated thymidine, serially sectioned, and subjected to autoradiography. There was negligible thymidine incorporation in unoperated controls and in the undamaged parts of the operated discs, indicating that DNA synthesis in undamaged tissue is terminated during the first day of the culture period. Almost all of the fragments from which tissue had been removed, as well as controls which were simply cut without the removal of any tissue, showed a cluster of labeled cells (blastema) even after only 1 day of culture. The blastemas in control discs were short-lived, with over 50% of these discs showing no blastema by the third day in culture. Blastemas in discs from which sectors were removed were more persistent; the time at which 50% of the fragments no longer showed a blastema was 4 days for the -30 degrees fragments, 5 days for the -90 degrees fragments, and greater than 5 days for the -150 degrees fragments. The average blastema size, measured as number of labeled cells, was directly related to the amount of tissue removed, and in most cases did not change significantly during the culture period. Both wound edges incorporated tritiated thymidine initially and the S-phase cells remained tightly clustered throughout regeneration; maximum blastema width varied from about 8 to 25 cell diameters. The results are consistent with the idea that regenerative cell proliferation is stimulated and maintained by positional information discontinuities, and terminated when these discontinuities are resolved by the addition of an appropriate number of new cells.     

Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration

A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9420-9) contains supplementary material, which is available to authorized users.     

Morphogenesis in planarians Dugesia tigrina

We carried out computer morphometry in regenerates of planarians Dugesia tigrina. The blastema growth was analyzed in fragments of planarians after their fission and after transverse transection at different body levels. The blastema was growing at a higher rate on tail fragments than on the head fragments and the growth rate was the higher, the closer the transection was to the head end. After fission, the blastema was growing at a slower rate than after transection in the fission zone. The growth of adjacent blastemas formed on both sides after fission or transection proceeded at different rates as a function of new body polarity.