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.     

Simple blood‐feeding method for live imaging of gut tube remodeling in regenerating planarians

Live cell imaging is a powerful technique to study cellular dynamics in vivo during animal development and regeneration. However, few live imaging methods have been reported for studying planarian regeneration. Here, we developed a simple method for steady visualization of gut tube remodeling during regeneration of a living freshwater planarian, Dugesia japonica. When planarians were fed blood several times, gut branches were well‐visualized in living intact animals under normal bright‐field illumination. Interestingly, tail fragments derived from these colored planarians enabled successive observation of the processes of the formation of a single anterior gut branch in the prepharyngeal region from the preexisting two posterior gut branches in the same living animals during head regeneration. Furthermore, we combined this method and RNA interference (RNAi) and thereby showed that a D. japonica raf‐related gene (DjrafA) and mek‐related gene (DjmekA) we identified both play a major role in the activation of extracellular signal‐regulated kinase (ERK) signaling during planarian regeneration, as indicated by their RNAi‐induced defects on gut tube remodeling in a time‐saving initial screening using blood‐feeding without immunohistochemical detection of the gut. Thus, this blood‐feeding method is useful for live imaging of gut tube remodeling, and provides an advance for the field of regeneration study in planarians.     

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

Heat shock proteins (HSPs) play a crucial role in the protection of cells. In the present study, we have identified an hsp90-related gene (Djhsp90) encoding a cytosolic form of HSP90 that is primarily expressed in gastrodermis of the planarian Dugesia japonica. Djhsp90 becomes significantly induced after traumatic amputation or other stress stimuli, such as exposure to X-ray or ultraviolet radiations, heat shock, or prolonged starvation. When Djhsp90 is silenced by ribonucleic acid interference (RNAi), planarians dramatically decrease in size, becoming unable to eat, and die in a few weeks. Our results indicate that this gene plays an essential cytoprotective role in the gastrodermis of planarians and suggest that this chaperone can be involved in autophagic processes that are activated by this tissue.     

Spontaneous Behaviors and Wall-Curvature Lead to Apparent Wall Preference in Planarian

The planarian Dugesia japonica tends to stay near the walls of its breeding containers and experimental dishes in the laboratory, a phenomenon called “wall preference”. This behavior is thought to be important for environmental adaptation, such as hiding by planarians in nature. However, the mechanisms regulating wall-preference behavior are not well understood, since this behavior occurs in the absence of any particular stimulation. Here we show the mechanisms of wall-preference behavior. Surprisingly, planarian wall-preference behavior was also shown even by the head alone and by headless planarians. These results indicate that planarian “wall-preference” behavior only appears to be a “preference” behavior, and is actually an outcome of spontaneous behaviors, rather than of brain function. We found that in the absence of environmental cues planarians moved basically straight ahead until they reached a wall, and that after reaching a wall, they changed their direction of movement to one tangential to the wall, suggesting that this spontaneous behavior may play a critical role in the wall preference. When we tested another spontaneous behavior, the wigwag movement of the planarian head, using computer simulation with various wigwag angles and wigwag intervals, large wigwag angle and short wigwag interval reduced wall-preference behavior. This indicated that wigwag movement may determine the probability of staying near the wall or leaving the wall. Furthermore, in accord with this simulation, when we tested planarian wall-preference behavior using several assay fields with different curvature of the wall, we found that concavity and sharp curvature of walls negatively impacted wall preference by affecting the permissible angle of the wigwag movement. Together, these results indicate that planarian wall preference may be involuntarily caused by the combination of two spontaneous planarian behaviors: moving straight ahead until reaching a wall and then moving along it in the absence of environmental cues, and wigwag movements of the head.     

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.     

Early planarian brain regeneration is independent of blastema polarity mediated by the Wnt/β-catenin pathway

Analysis of anteroposterior (AP) axis specification in regenerating planarian flatworms has shown that Wnt/β-catenin signaling is required for posterior specification and that the FGF-like receptor molecule nou-darake (ndk) may be involved in restricting brain regeneration to anterior regions. The relationship between re-establishment of AP identity and correct morphogenesis of the brain is, however, still poorly understood. Here we report the characterization of two axin paralogs in the planarian Schmidtea mediterranea. Although Axins are well known negative regulators of Wnt/β-catenin signaling, no role in AP specification has previously been reported for axin genes in planarians. We show that silencing of Smed-axin genes by RNA interference (RNAi) results in two-tailed planarians, a phenotype previously reported after silencing of Smed-APC-1, another β-catenin inhibitor. More strikingly, we show for the first time that while early brain formation at anterior wounds remains unaffected, subsequent development of the brain is blocked in the two-tailed planarians generated after silencing of Smed-axin genes and Smed-APC-1. These findings suggest that the mechanisms underlying early brain formation can be uncoupled from the specification of AP identity by the Wnt/β-catenin pathway. Finally, the posterior expansion of the brain observed following Smed-ndk RNAi is enhanced by silencing Smed-APC-1, revealing an indirect relationship between the FGFR/Ndk and Wnt/β-catenin signaling systems in establishing the posterior limits of brain differentiation.     

The BMP pathway is essential for re-specification and maintenance of the dorsoventral axis in regenerating and intact planarians

The bone morphogenetic protein (BMP) pathway has been shown to play an important role in the establishment of the dorsoventral axis during development in both vertebrate and invertebrate species. In an attempt to unravel the role of BMPs in pattern formation during planarian regeneration, we studied this signaling pathway in Schmidtea mediterranea. Here, we functionally characterize planarian homologues of two key elements of the pathway: Smed-BMP and Smed-Smad1. Whole-mount in situ hybridization showed that Smed-BMP is expressed at the planarian dorsal midline, suggesting a role in dorsoventral patterning, while Smed-Smad1 is widely expressed throughout the mesenchyme and in the central nervous system. RNA interference (RNAi) knockdowns of Smed-BMP or Smed-Smad1 led to the disappearance of dorsal markers along with the ectopic expression of ventral markers on the dorsal side of the treated animals. In almost all cases, a duplicated central nervous system differentiated dorsally after Smed-BMP or Smed-Smad1 RNAi. These defects were observed not only during regeneration but also in intact non-regenerating animals. Our results suggest that the BMP signaling pathway is conserved in planarians and that it plays a key role in the regeneration and maintenance of the dorsoventral axis.     

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.     

Characterization of a flatworm inositol (1,4,5) trisphosphate receptor (IP3R) reveals a role in reproductive physiology

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels that elevate cytoplasmic Ca²⁺ in response to the second messenger IP₃. Here, we describe the identification and in vivo functional characterization of the planarian IP₃R, the first intracellular Ca²⁺ channel to be defined in flatworms. A single IP₃R gene in Dugesia japonica encoded a 2666 amino acid protein (Dj.IP₃R) that shared well conserved structural features with vertebrate IP₃R counterparts. Expression of an NH₂-terminal Dj.IP₃R region (amino acid residues 223–585) recovered high affinity ³H-IP₃ binding (0.9 ± 0.1 nM) which was abolished by a single point mutation of an arginine residue (R495L) important for IP₃ coordination. In situ hybridization revealed that Dj.IP₃R mRNA was most strongly expressed in the pharynx and optical nerve system as well as the reproductive system in sexualized planarians. Consistent with this observed tissue distribution, in vivo RNAi of Dj.IP₃R resulted in a decreased egg-laying behavior suggesting Dj.IP₃R plays an upstream role in planarian reproductive physiology.     

Regeneration of photoreceptor organs in freshwater planarians at different levels of accumulation of natural methylmercury compounds

The effects of natural methylmercury compounds on regeneration of photoreceptor organs were studied in three freshwater planarians: Polycelis tenuis, Dugesia lugubris, and D. tigrina. Accumulation of methyl mercury in the planarian body suppressed regeneration of P. tenuis with numerous photoreceptor organs to a greater extent than in two other planarians that have only two eyes. High methyl mercury concentrations inhibited the restoration of photoreceptor organs in asexual and sexual D. tigrina races     

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, ?3 and ?4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo.     

Spoltud-1 is a chromatoid body component required for planarian long-term stem cell self-renewal

Freshwater planarians exhibit a striking power of regeneration, based on a population of undifferentiated totipotent stem cells, called neoblasts. These somatic stem cells have several characteristics resembling those of germ line stem cells in other animals, such as the presence of perinuclear RNA granules (chromatoid bodies). We have isolated a Tudor domain-containing gene in the planarian species Schmidtea polychroa, Spoltud-1, and show that it is expressed in neoblast cells, germ line cells and central nervous system, and during embryonic development. Within the neoblasts, Spoltud-1 protein is enriched in chromatoid bodies. Spoltud-1 RNAi eliminates protein expression after 3 weeks, and abolishes the power of regeneration of planarians after 7 weeks. Neoblast cells are eliminated by the RNAi treatment, disappearing at the end rather than gradually during the process. Neoblasts with no detectable Spoltud-1 protein are able to proliferate and differentiate. These results suggest that Spoltud-1 is required for long term stem cell self renewal.     

Clathrin-mediated endocytic signals are required for the regeneration of, as well as homeostasis in, the planarian CNS

Planarians have a well-organized central nervous system (CNS), including a brain, and can regenerate the CNS from almost any portion of the body using pluripotent stem cells. In this study, to identify genes required for CNS regeneration, genes expressed in the regenerating CNS were systematically cloned and subjected to functional analysis. RNA interference (RNAi) of the planarian clathrin heavy chain (DjCHC) gene prevented CNS regeneration in the intermediate stage of regeneration prior to neural circuit formation. To analyze DjCHC gene function at the cellular level, we developed a functional analysis method using primary cultures of planarian neurons purified by fluorescence-activated cell sorting (FACS) after RNAi treatment. Using this method, we showed that the DjCHC gene was not essential for neural differentiation, but was required for neurite extension and maintenance, and that DjCHC-RNAi-treated neurons entered a TUNEL-positive apoptotic state. DjCHC-RNAi-treated uncut planarians showed brain atrophy, and the DjCHC-RNAi planarian phenotype was mimicked by RNAi-treated planarians of the mu-2 (micro2) gene, which is involved in endocytosis, but not the mu-1 (micro1) gene, which is involved in exocytosis. Thus, clathrin-mediated endocytic signals may be required for not only maintenance of neurons after synaptic formation, but also axonal extension at the early stage of neural differentiation.