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.     

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.     

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.     

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.     

Identification and characterization of a fibroblast growth factor gene in the planarian Dugesia japonica

Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.     

TINP1 homolog is required for planarian regeneration

The planarian flatworm is an ideal system for the study of regeneration in vivo. In this study, we focus on TINP1, which is one of the most conserved proteins in eukaryotic organisms. We found that TINP1 was expressed in parenchymal region through whole body as well as central nervous system (CNS) during the course of regeneration. RNA interference targeting DjTINP1 caused lysis defects in regenerating tissues and a decreased in cell division and expression levels of DjpiwiA and Djpcna. Furthermore, the expression levels of DjTINP1 were decreased when we inhibited the TGF-β signal by knockdown of smad4, which is the sole co-smad and has been proved to control the blastema patterning and central nervous system (CNS) regeneration in planarians. These findings suggest that DjTINP1 participate in the maintenance of neoblasts and be required for proper cell proliferation in planarians as a downstream gene of the TGF-β signal pathway.     

Heterogeneity of chromatoid bodies in adult pluripotent stem cells of planarian Dugesia japonica

The robust regenerative ability of planarians is known to be dependent on adult pluripotent stem cells called neoblasts. One of the morphological features of neoblasts is cytoplasmic ribonucleoprotein granules (chromatoid bodies: CBs), which resemble germ granules present in germline cells in other animals. Previously, we showed by immuno‐electron microscopic analysis that DjCBC‐1, a planarian Me31B/Dhh1/DDX6 homologue, which is a component of ribonucleoprotein granules, was localized in CBs in the planarian Dugesia japonica. Also, recently it was reported using another planarian species that Y12 antibody recognizing symmetrical dimethylarginine (sDMA) specifically binds to CBs in which histone mRNA is co‐localized. Here, we showed by double immunostaining and RNA interference (RNAi) that DjCBC‐1‐containing CBs and Y12‐immunoreactive CBs are distinct structures, suggesting that CBs are composed of heterogeneous populations. We also found that the Y12‐immunoreactive CBs specifically contained a cytoplasmic type of planarian PIWI protein (DjPiwiC). We revealed by RNAi experiments that Y12‐immunoreactive CBs may have anti‐transposable element activity involving the DjPiwiC protein in the neoblasts.     

Effect of timing of cutting on patterning and proportion regulation during regeneration of the planarian Dugesia tigrina

The time interval between cuts that are made to obtain a tissue fragment from a planarian was found to be important to the process of its regeneration. Short fragments made by two transverse cuts across the body were more likely to regenerate abnormally when the interval between the two cuts was 5 or 12 min than when it was 1.5 min. The longer intervals specifically altered the regression line in the correlation between the length:width ratio of fragments and frequency of abnormal regenerates. This effect occured regardless of which region of the body the fragment was taken from. The time interval also affected body proportioning in regenerates and to the greatest degree in fragments derived from the region located immediately behind the head. These results indicate that events occuring shortly after a cut is made in a planarian significantly affect structure patterning and proportioning of the regenerate.     

Requirement for anti-dorsalizing morphogenetic protein in organizer patterning

The amphibian Spemann organizer is subdivided in trunk and head organizer and it is unclear how this division is regulated. The Xenopus trunk organizer expresses anti-dorsalizing morphogenetic protein (ADMP), a potent organizer antagonist. We show that ADMP represses head formation during gastrulation and that its expression is activated by BMP antagonists. A specifically acting dominant-negative ADMP anteriorizes embryos and its coexpression with BMP antagonists induces secondary embryonic axes with heads as well as expression of head inducers. Unlike other BMPs, ADMP is not inhibited by a dominant-negative BMP type I receptor, Noggin, Cerberus and Chordin but by Follistatin, suggesting that it utilizes a distinct TGF-β receptor pathway and displays differential sensitivity to BMP antagonists. The results indicate that ADMP functions in the trunk organizer to antagonize head formation, thereby regulating organizer patterning.     

Bone morphogenetic protein signaling in limb outgrowth and patterning

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor beta (TGFbeta) multigene family. Current evidence indicates that they may play different and even antagonistic roles at different stages of limb development. Refined studies of their function in these processes have been impeded in the mouse due to the early lethality of null mutants for several BMP ligands and their receptors. Recently, however, these questions have benefited from the very powerful Cre-loxP technology. In this review, I intend to summarize what has been learned from this conditional mutagenesis approach in the mouse limb, focusing on Bmp2, Bmp4 and Bmp7 while restricting my analysis to the initial phases of limb formation and patterning. Two major aspects are discussed, the role of BMPs in dorsal-ventral polarization of the limb bud, together with their relation to apical ectodermal ridge (AER) induction, and their role in controlling digit number and identity. Particular attention is paid to the methodology, its power and its limits.     

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.     

Weak extremely-low-frequency magnetic field-induced regeneration anomalies in the planarian Dugesia tigrina

We recently reported that cephalic regeneration in the planarian Dugesia tigrina was significantly delayed in populations exposed continuously to combined parallel DC and AC magnetic fields. This effect was consistent with hypotheses suggesting an underlying resonance phenomenon. We report here, in a parallel series of investigations on the same model system, that the incidence of regeneration anomalies presenting as tumor-like protuberances also increases significantly (P < .001) in association with exposure to weak 60 Hz magnetic fields, with peak intensities ranging between 1.0 and 80.0 μT. These anomalies often culminate in the complete disaggregation of the organism. Similar to regeneration rate effects, the incidence of regeneration anomalies is specifically dependent upon the planaria possessing a fixed orientation with respect to the applied magnetic field vectors. However, unlike the regeneration rate effects, the AC magnetic field alone, in the absence of any measurable DC field, is capable of producing these anomalies. Moreover, the incidence of regeneration anomalies follows a clear dose-response relationship as a function of AC magnetic field intensity, with the threshold for induced electric field intensity estimated at 5 μV/m. The addition of either 51.1 or 78.4 μT DC magnetic fields, applied in parallel combination with the AC field, enhances the appearance of anomalies relative to the 60 Hz AC field alone, but only at certain AC field intensities. Thus, whereas our previous study of regeneration rate effects appeared to involve exclusively resonance interactions, the regeneration anomalies reported here appear to result primarily from Faraday induction coupling. These results together with those reported previously point to two distinct physiological effects produced in regenerating planaria by exposure to weak extremely-low-frequency (ELF) magnetic fields. They further suggest that the planarian, which has recently been identified elsewhere as an excellent system for use in teratogenic investigations involving chemical teratogens, might be used similarly in teratogenic investigations involving ELF magnetic fields. © 1996 Wiley-Liss, Inc.     

Proportion regulation in the planarian,Dugesia tigrina following regeneration of structures

Planarian regenerates with abnormal body proportioning were followed after structure formation in order to determine if body proportion will ‘normalize’ over time. Results show proportioning will occur following structure formation since the pharynx in regenerates with proportionally larger heads and prepharyngeal area moved anteriorly over time. This occurred regardless of whether regenerates were provided with a normal feeding regime which allowed for an increase in body area or if they were on a maintenance diet which did not permit growth. An examination of mitotic indices did not demonstrate significant differences in the level of mitotic activity between pre- and postpharyngeal regions. It is concluded that the ‘normalizing’ of proportion in these abnormal regenerates does occur, but the process by which it occurs is not solely explained through normal growth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Weak extremely-low-frequency magnetic fields and regeneration in the planarian Dugesia tigrina

Extremely-low-frequency (ELF), low-intensity magnetic fields have been shown to influence cell signaling processes in a variety of systems, both in vivo and in vitro. Similar effects have been demonstrated for nervous system development and neurite outgrowth. We report that regeneration in planaria, which incorporates many of these processes, is also affected by ELF magnetic fields. The rate of cephalic regeneration, reflected by the mean regeneration time (MRT), for planaria populations regenerating under continuous exposure to combined DC (78.4 μT) and AC (60.0 Hz at 10.0 μT _peak) magnetic fields applied in parallel was found to be significantly delayed (P ? 0.001) by 48 ± 1 h relative to two different types of control populations (MRT ? 140 ± 12 h). One control population was exposed to only the AC component of this field combination, while the other experienced only the ambient geomagnetic field. All measurements were conducted in a low-gradient, low-noise magnetics laboratory under well-maintained temperature conditions. This delay in regeneration was shown to be dependent on the planaria having a fixed orientation with respect to the magnetic field vectors. Results also indicate that this orientation-dependent transduction process does not result from Faraday induction but is consistent with a Ca²⁺ cyclotron resonance mechanism. Data interpretation also permits the tentative conclusion that the effect results from an inhibition of events at an early stage in the regeneration process before the onset of proliferation and differentiation. © 1995 Wiley-Liss, Inc.     

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.     

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.     

The Dr-nanos gene is essential for germ cell specification in the planarian Dugesia ryukyuensis

Homologs of nanos are required for the formation and maintenance of germline stem cell (GSC) systems and for gametogenesis in many metazoans. Planarians can change their reproductive mode seasonally, alternating between asexual and sexual reproduction; they develop and maintain their somatic stem cells (SSCs) and GCSs from pluripotent stem cells known as neoblasts. We isolated a nanos homolog, Dr-nanos, from the expressed sequence tags (ESTs) of the sexualized form of Dugesia ryukyuensis. We examined the expression of Dr-nanos in asexual and sexualized planarians by in situ hybridization and analyzed its function using RNA interference (RNAi) together with a planarian sexualization assay. A nanos homolog, Dr-nanos, was identified in the planarian D. ryukyuensis. Dr-nanos expression was observed in the ovarian primordial cells of the asexual worms. This expression increased in proportion to sexualization and was localized in the early germline cells of the ovaries and testes. In X-ray-irradiated worms, the expression of Dr-nanos decreased to a large extent, indicating that Dr-nanos is expressed in some subpopulations of stem cells, especially in GSCs. During the sexualization process, worms in which Dr-nanos was knocked down by RNAi exhibited decreased numbers of oogonia in the ovaries and failed to develop testes, whereas the somatic sexual organs were not affected. We conclude that Dr-nanos is essential for the development of germ cells in the ovaries and testes and may have a function in the early stages of germ cell specification, but not in the development of somatic sexual organs.