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.     

Planarian fibroblast growth factor receptor homologs expressed in stem cells and cephalic ganglions

The strong regenerative capacity of planarians is considered to reside in the totipotent somatic stem cell called the 'neoblast'. However, the signal systems regulating the differentiation/growth/migration of stem cells remain unclear. The fibroblast growth factor (FGF)/FGF receptor (FGFR) system is thought to mediate various developmental events in both vertebrates and invertebrates. We examined the molecular structures and expression of DjFGFR1 and DjFGFR2, two planarian genes closely related to other animal FGFR genes. DjFGFR1 and DjFGFR2 proteins contain three and two immunoglobulin-like domains, respectively, in the extracellular region and a split tyrosine kinase domain in the intracellular region. Expression of DjFGFR1 and DjFGFR2 was observed in the cephalic ganglion and mesenchymal space in intact planarians. In regenerating planarians, accumulation of DjFGFR1-expressing cells was observed in the blastema and in fragments regenerating either a pharynx or a brain. In X-ray-irradiated planarians, which had lost regenerative capacity, the number of DjFGFR1-expressing cells in the mesenchymal space decreased markedly. These results suggest that the DjFGFR1 protein may be involved in the signal systems controlling such aspects of planarian regeneration as differentiation/growth/migration of stem cells.     

Photoperiodic modulation of cephalic melatonin in planarians

Endogenous melatonin was detected by high-performance liquid chromatography (HPLC) with electrochemical detection (EC) in the head of the planarian Dugesia dorotocephala. The identity of this elution peak was further confirmed by radioimmunoassay. In groups of planarians adapted to either normal or reversed photoperiods, the melatonin levels were always higher in those heads collected in the dark period than in those collected in the light period. This indicates that primitive animals such as planarians have already evolved a melatonin-metabolizing system that is photically driven in a manner suggestive of the way melatonin synthesis is influenced by light and dark cycles in vertebrates.     

Characterization of tyramine beta-hydroxylase in planarian Dugesia japonica: cloning and expression

The planarian Dugesia japonica has a relatively well-organized central nervous system (CNS) consisting of a brain and ventral nerve cords (VNCs), and can completely regenerate it CNS utilizing pluripotent stem cells present in the mesenchymal space. This remarkable capacity has begun to be exploited for research on neural regeneration. Recently, several kinds of molecular markers for labeling of neural subtypes have been reported in planarians. These molecular markers are useful for visualizing the distinct neural populations in planarians. In this study, we isolated a cDNA encoding tyramine beta-hydroxylase (TBH), an octopamine (OA) biosynthetic enzyme, by degenerate PCR in the planarian D. japonica, and named it DjTBH (D. japonica tyramine beta-hydroxylase). In order to examine whether DjTBH contributes to OA biosynthesis, we measured the OA content in DjTBH-knockdown planarians created by RNA interference. In addition, to examine the specificity of DjTBH for OA biosynthesis, we measured not only OA content but also noradrenaline (NA) content, because NA is synthesized by a pathway similar to that for OA. According to high-performance liquid chromatography analysis, the amount of OA, but not NA, was significantly decreased in DjTBH-knockdown planarians. In addition, we produced anti-DjTBH antibody to visualize the octopaminergic neural network. As shown by immunofluorescence analysis using anti-DjTBH antibody, DjTBH-immunopositive neurons were mainly distributed in the head region, and elongated their dendrites and/or axons along the VNCs. In order to visualize octopaminergic and dopaminergic nervous systems (phenolamine/catecholamine nervous system) in the planarian CNS, double-immunofluorescence analysis was carried out using both anti-DjTBH antibody and anti-DjTH (a planarian tyrosine hydroxylase) antibody. DjTBH-immunopositive neurons and DjTH-immunopositive neurons mainly formed distinct neural networks in the head region. Here, we demonstrated that DjTBH clearly contributes to OA biosynthesis, and DjTBH antibody is a useful tool for detecting octopaminergic neurons in planarians.     

Inhibition of planarian regeneration by melatonin

Melatonin, which is a substance produced by the pineal body in vertebrates, inhibited regeneration in the planarian Dugesia japonica japonica Ichikawa et Kawakatsu. When decapitated planarians were maintained in a 1 mmol dm^–3 solution of melatonin, formation of the head was retarded; formation of the eyes, however, was not disturbed. Similarly in animals from which the tail was cut, regeneration of the tail was retarded if the animals were kept in melatonin solution of 1 mmol dm^–3. The effect was reversible once the melatonin was removed. Retardation of regeneration did not occur with similar application of three melatonin derivatives, serotonin hydrochloride, N-acetylserotonin, and 6-hydroxymelatonin. Melatonin endogenous to the planarian could be demonstrated by means of radio-immunoassay and was more abundant in the head region than other regions of the body. Melatonin, thus, appears to play a role in regulating regeneration in planarians and conceivably provides positional information in that process.     

Expression pattern of the expanded noggin gene family in the planarian Schmidtea mediterranea

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and follistatin. Recently, it has been shown in planarians that the BMP pathway has a conserved function in the maintenance and re-establishment of the dorsoventral axis during homeostasis and regeneration. In an attempt to further characterize the BMP pathway in this model we have undertaken an in silico search of noggin genes in the genome of Schmidtea mediterranea. In contrast to other systems in which between one and four noggin genes have been reported, ten genes containing a noggin domain are present in S. mediterranea. These genes have been classified into two groups: noggin genes (two genes) and noggin-like genes (eight genes). Noggin-like genes are characterized by the presence of an insertion of 50–60 amino acids in the middle of the noggin domain. Here, we report the characterization of this expanded family of noggin genes in planarians as well as their expression patterns in both intact and regenerating animals. In situ hybridizations show that planarian noggin genes are expressed in a variety of cell types located in different regions of the planarian body.     

Reconstruction of dopaminergic neural network and locomotion function in planarian regenerates

Planarian, an invertebrate flatworm, has a high capacity for regeneration when compared with other worms and animals. We show here for the first time that the reconstructed dopamine (DA) neural network regulates locomotion and behavior in planarian regenerates. The gene encoding tyrosine hydroxylase in the planarian Dugesia japonica (DjTH) was identified. DjTH protein was coexpressed with aromatic amino acid decarboxylase-like A (DjAADCA) in the planarian central nervous system (CNS). In addition, DjTH-knockdown planarians lost the ability to synthesize DA, but showed no change in 5-hydroxytryptamine synthesis. When the planarian body was amputated, DjTH-positive neurons were regenerated in the brain newly rebuilt from the tail piece at Day 3, and the DjTH-positive axonal and dendritic neural network in the CNS (dopaminergic tiara) was reconstructed at Days 5-7. At that time, autonomic locomotion and methamphetamine-induced hyperkinesia were also suppressed in DjTH-knockdown planarians. Planarian locomotion and behavior seem to be regulated in both cilia- and muscle-dependent manners. In DjTH-knockdown planarians, muscle-mediated locomotion and behavior were significantly attenuated. These results suggest that DA neurons play a key role in the muscle-mediated movement in planarians.     

Regeneration and maintenance of the planarian midline is regulated by a slit orthologue

Several families of evolutionarily conserved axon guidance cues orchestrate the precise wiring of the nervous system during embryonic development. The remarkable plasticity of freshwater planarians provides the opportunity to study these molecules in the context of neural regeneration and maintenance. Here we characterize a homologue of the Slit family of guidance cues from the planarian Schmidtea mediterranea. Smed-slit is expressed along the planarian midline, in both dorsal and ventral domains. RNA interference (RNAi) targeting Smed-slit results in the collapse of many newly regenerated tissues at the midline; these include the cephalic ganglia, ventral nerve cords, photoreceptors, and the posterior digestive system. Surprisingly, Smed-slit RNAi knockdown animals also develop morphologically distinguishable, ectopic neural structures near the midline in uninjured regions of intact and regenerating planarians. These results suggest that Smed-slit acts not only as a repulsive cue required for proper midline formation during regeneration but that it may also act to regulate the behavior of neural precursors at the midline in intact planarians.     

The expression of planarian brain factor homologs,DjFoxG and DjFoxD

Recent accumulating evidence revealed that planarian central nervous system (CNS) has numerous functional domains distinguished by a large number of neural markers, suggesting that primitive animals which developed CNS already had the framework of the brain development. It is of interest to investigate genes which have been acquired at an early stage of evolution for brain pattern formation. One such candidate is FoxG1 (BF-1), specifically expressed in the telencephalon and implicated in brain development. We identified a FoxG1 (BF-1) homolog gene in planarians (DjFoxG). We also identified a FoxD class gene, DjFoxD. DjFoxG is expressed in the body and brain, with strong expression in the mesenchyme surrounding the gut. During regeneration, an intense anterior signal is detected, but this is not restricted to the head. DjFoxD is expressed in the mid-apex of the head, between the two lobes of the brain. Strong expression was detected in the mid-anterior blastema. Thus, FoxG and FoxD homologs do exist in planarians, but are regulated differently than those in vertebrates.     

A low percent ethanol method for immobilizing planarians

Planarians have recently become a popular model system for the study of adult stem cells, regeneration and polarity. The system is attractive for both undergraduate and graduate research labs, since planarian colonies are low cost and easy to maintain. Also in situ hybridization, immunofluorescence and RNA-interference (RNAi) gene knockdown techniques have been developed for planarian studies. However, imaging of live worms (particularly at high magnifications) is difficult because animals are strongly photophobic; they quickly move away from light sources and out of frame. The current methods available to inhibit movement in planarians include RNAi injection and exposure to cold temperatures. The former is labor and time intensive, while the latter precludes the use of many fluorescent reporter dyes. Here, we report a simple, inexpensive and reversible method to immobilize planarians for live imaging. Our data show that a short 1 hour treatment with 3% ethanol (EtOH) is sufficient to inhibit both the fine and gross movements of Schmidtea mediterranea planarians, of the typical size used (4-6 mm), with full recovery of movement within 3-4 hours. Importantly, EtOH treatment did not interfere with regeneration, even after repeated exposure, nor lyse epithelial cells (as assayed by H&E staining). We demonstrate that a short exposure to a low concentration of EtOH is a quick and effective method of immobilizing planarians, one that is easily adaptable to planarians of all sizes and will increase the accessibility of live imaging assays to planarian researchers.     

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.     

Planarian cytochrome b(561): conservation of a six transmembrane structure and localization along the central and peripheral nervous system

Cytochrome b(561) is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles in the central and peripheral nervous systems of higher animals. We succeeded in cloning a full-length cDNA encoding planarian cytochrome b(561). The deduced amino acid sequence shows a very similar six transmembrane topology to those of cytochromes b(561) of higher vertebrates and contains both putative ascorbate- and monodehydro ascorbate-binding sites. Among the six totally-conserved His residues of cytochrome b(561) in higher vertebrates, one is substituted with an Asn residue, indicating that His88 and His161 of bovine cytochrome b(561) play roles as heme b ligands at the extravesicular side. Northern- and Western-blot analyses confirmed the expression of the mRNA and protein with the expected sizes in planarians. The distributions of the mRNA and apoprotein were analyzed by in situ hybridization and immunohistochemical staining, respectively, showing two morphologically distinct structures, a pair of ventral nerve cords and the cephalic ganglion cluster in the head region. The present results suggest that the usage of ascorbate to supply electron equivalents to neuroendocrine-specific copper-containing monooxygenases is likely to have originated in organisms with a very simple nervous system.     

Planarian Phototactic Assay Reveals Differential Behavioral Responses Based on Wavelength

Planarians are free-living aquatic flatworms that possess a well-documented photophobic response to light. With a true central nervous system and simple cerebral eyes (ocelli), planarians are an emerging model for regenerative eye research. However, comparatively little is known about the physiology of their photoreception or how their behavior is affected by various wavelengths. Most phototactic studies have examined planarian behavior using white light. Here, we describe a novel planarian behavioral assay to test responses to small ranges of visible wavelengths (red, blue, green), as well as ultraviolet (UV) and infrared (IR) which have not previously been examined. Our data show that planarians display behavioral responses across a range of wavelengths. These responses occur in a hierarchy, with the shortest wavelengths (UV) causing the most intense photophobic responses while longer wavelengths produce no effect (red) or an apparent attraction (IR). In addition, our data reveals that planarian photophobia is comprised of both a general photophobic response (that drives planarians to escape the light source regardless of wavelength) and wavelength-specific responses that encompass specific behavioral reactions to individual wavelengths. Our results serve to improve the understanding of planarian phototaxis and suggest that behavioral studies performed with white light mask a complex behavioral interaction with the environment.     

Genome-wide analyses reveal a role for peptide hormones in planarian germline development

Bioactive peptides (i.e., neuropeptides or peptide hormones) represent the largest class of cell-cell signaling molecules in metazoans and are potent regulators of neural and physiological function. In vertebrates, peptide hormones play an integral role in endocrine signaling between the brain and the gonads that controls reproductive development, yet few of these molecules have been shown to influence reproductive development in invertebrates. Here, we define a role for peptide hormones in controlling reproductive physiology of the model flatworm, the planarian Schmidtea mediterranea. Based on our observation that defective neuropeptide processing results in defects in reproductive system development, we employed peptidomic and functional genomic approaches to characterize the planarian peptide hormone complement, identifying 51 prohormone genes and validating 142 peptides biochemically. Comprehensive in situ hybridization analyses of prohormone gene expression revealed the unanticipated complexity of the flatworm nervous system and identified a prohormone specifically expressed in the nervous system of sexually reproducing planarians. We show that this member of the neuropeptide Y superfamily is required for the maintenance of mature reproductive organs and differentiated germ cells in the testes. Additionally, comparative analyses of our biochemically validated prohormones with the genomes of the parasitic flatworms Schistosoma mansoni and Schistosoma japonicum identified new schistosome prohormones and validated half of all predicted peptide-encoding genes in these parasites. These studies describe the peptide hormone complement of a flatworm on a genome-wide scale and reveal a previously uncharacterized role for peptide hormones in flatworm reproduction. Furthermore, they suggest new opportunities for using planarians as free-living models for understanding the reproductive biology of flatworm parasites.     

Photoresponsivity and motility in the planarian Schmidtea mediterranea vary diurnally

ABSTRACT

The planarian flatworm has become one of the leading animal model systems for studying stem cell behavior and tissue regeneration. Recent studies have shown that components of the circadian clockwork have important roles in tissue homeostasis and repair. However, it remains unknown whether planarians exhibit circadian or diurnal rhythms in physiology or behavior. Here, we developed a behavioral assay to evaluate diurnal activity in planarians based upon their well-established propensity to swim away from light (negative phototaxis). We show evidence that the planarian Schmidtea mediterranea has diurnal variability in negative phototaxis as a function of daily variation in motility. We also demonstrate that variation in planarian motility over 48 h occurs with 24-h periodicity. Our data suggest that S. mediterranea may be a useful model for studying the interplay between the circadian system and tissue regeneration.     

Acetylcholine/dopamine interaction in planaria

Planaria represents the most primitive example of centralization and cephalization of nervous system. Previous reports indicate that planaria shows specific behavioral patterns, analogous to mammalian stereotypes, in response to drugs acting on acetylcholine or dopamine transmission. Here we further characterized these responses, and investigated the interactions between cholinergic and dopaminergic systems by means of behavioral methods. Exposure to cholinergic agonists physostigmine or nicotine produced hypokinesia with ‘bridge-like’ and ‘walnut’ positions, respectively. Blockade of muscarinic receptors by atropine produced ‘screw-like’ hyperkinesia. Exposure to dopamine agonists (nomifensine, apomorphine) produced marked hyperkinesia with ‘screw-like’ movements. Finally, exposure to dopamine antagonists produced immobility or ‘bridge-like’ position. Pre-exposure to physostigmine blocked the behavioral effects of nomifensine and reduced and markedly delayed the behavioral effects of apomorphine. Pre-exposure to apomorphine slightly reduced and delayed the behavioral changes by physostigmine. Finally, planaria exposed to atropine after either SCH23388 or sulpiride showed ‘C-like’ or ‘screw-like’ hyperkinesia, respectively. Thus, reduction of cholinergic transmission seems to play a pivotal role in determining hyperkinesia in planaria. Under these conditions, different patterns of hyperkinetic activities occur, according to the subpopulation of dopamine receptors stimulated by drugs. These findings suggest that interactions between cholinergic and dopaminergic systems occur very early in animal phylogeny.     

The mammalian anti-α-smooth muscle actin monoclonal antibody recognizes an α-actin-like protein in planaria (Dugesia lugubris s.l.)

The presence of an alpha-smooth muscle (alpha-sm) actin-like protein in planaria (Dugesia lugubris s.l.) is reported. The protein shows a 42 kDa molecular weight determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis and is specifically recognized by the mammalian anti alpha-sm actin monoclonal antibody. When a planarian is induced to regenerate by head amputation, the immunostaining of the alpha-sm actin-like molecule becomes important in the area of growing blastema, reaching a maximum between 70-120 hours after injury. Conventional electron microscopy at the 4-day-regeneration stage shows that blastema-forming cells are a homogeneous population whose morphological features resemble those of migrating mesenchyme-like cells; only the myoblasts show a recognizable phenotype. The immunocytochemical localization of alpha-sm actin-like molecule by immunoperoxidase (light microscopy) and immunogold stains (electron microscopy) was carried out on both intact and injured worms. The antigen was localized mainly at the basal portion of the epidermal cells and in the undifferentiated mesenchyme-like cells. Myoblasts, but not differentiated myofibers, were also labelled by this antibody. The results indicate that in the lower Eumetazoan planarians, as well as in vertebrates, the alpha-sm actin can be considered to be a marker for myoid differentiation. The suggestion that alpha-sm actin can be used as a marker for mesenchyme-like cells in vertebrates and in invertebrates is also discussed.