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.     

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-dependent conditional gene knockdown (Readyknock) in planarian: demonstration of requirement for Djsnap-25 expression in the brain for negative phototactic behavior

Freshwater planarians have a simple and evolutionarily primitive brain structure. Here, we identified the Djsnap-25 gene encoding a homolog of the evolutionarily conserved synaptic protein SNAP-25 from the planarian Dugesia japonica and assessed its role in brain function. Djsnap-25 was expressed widely in the nervous system. To investigate the specific role of Djsnap-25 in the brain, we developed a unique technique of RNA interference (RNAi), regeneration-dependent conditional gene knockdown (Readyknock), exploiting the high regenerative capacity of planarians, and succeeded in selectively eliminating the DjSNAP-25 activity in the head region while leaving the DjSNAP-25 activity in the trunk region intact. These knockdown animals showed no effect on brain morphology or on undirected movement of the trunk itself. Light-avoidance behavior or negative phototaxis was used to quantitatively analyze brain function in the knockdown animals. The results suggested that the DjSNAP-25 activity within the head region is required for two independent sensory-processing pathways that regulate locomotive activity and directional movement downstream of distinct primary sensory outputs coming from the head margin and the eyes, respectively, during negative phototaxis. Our approach demonstrates that planarians are a powerful model organism to study the molecular basis of the brain as an information-processing center.     

Visual Exploration during Locomotion Limited by Fear of Heights

Background

Visual exploration of the surroundings during locomotion at heights has not yet been investigated in subjects suffering from fear of heights.

Methods

Eye and head movements were recorded separately in 16 subjects susceptible to fear of heights and in 16 non-susceptible controls while walking on an emergency escape balcony 20 meters above ground level. Participants wore mobile infrared eye-tracking goggles with a head-fixed scene camera and integrated 6-degrees-of-freedom inertial sensors for recording head movements. Video recordings of the subjects were simultaneously made to correlate gaze and gait behavior.

Results

Susceptibles exhibited a limited visual exploration of the surroundings, particularly the depth. Head movements were significantly reduced in all three planes (yaw, pitch, and roll) with less vertical head oscillations, whereas total eye movements (saccade amplitudes, frequencies, fixation durations) did not differ from those of controls. However, there was an anisotropy, with a preference for the vertical as opposed to the horizontal direction of saccades. Comparison of eye and head movement histograms and the resulting gaze-in-space revealed a smaller total area of visual exploration, which was mainly directed straight ahead and covered vertically an area from the horizon to the ground in front of the feet. This gaze behavior was associated with a slow, cautious gait.

Conclusions

The visual exploration of the surroundings by susceptibles to fear of heights differs during locomotion at heights from the earlier investigated behavior of standing still and looking from a balcony. During locomotion, anisotropy of gaze-in-space shows a preference for the vertical as opposed to the horizontal direction during stance. Avoiding looking into the abyss may reduce anxiety in both conditions; exploration of the “vertical strip” in the heading direction is beneficial for visual control of balance and avoidance of obstacles during locomotion.     

Action Recognition Depends on Observer’s Level of Action Control and Social Personality Traits

Humans recognize both the movement (physical) goals and action (conceptual) goals of individuals with whom they are interacting. Here, we assessed whether spontaneous recognition of others’ goals depends on whether the observers control their own behavior at the movement or action level. We also examined the relationship between individual differences in empathy and ASD-like traits, and the processing of other individual’s movement and action goals that are known to be encoded in the “mirroring” and “mentalizing” brain networks. In order to address these questions, we used a computer-based card paradigm that made it possible to independently manipulate movement and action congruency of observed and executed actions. In separate blocks, participants were instructed to select either the right or left card (movement-control condition) or the higher or lower card (action-control condition), while we manipulated action- and movement-congruency of both actors’ goals. An action-congruency effect was present in all conditions and the size of this effect was significantly correlated with self-reported empathy and ASD-like traits. In contrast, movement-congruency effects were only present in the movement-control block and were strongly dependent on action-congruency. These results illustrate that spontaneous recognition of others’ behavior depends on the control scheme that is currently adopted by the observer. The findings suggest that deficits in action recognition are related to abnormal synthesis of perceived movements and prior conceptual knowledge that are associated with activations in the “mirroring” and “mentalizing” cortical networks.     

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.     

In-Kennel Behavior Predicts Length of Stay in Shelter Dogs

Previous empirical evaluations of training programs aimed at improving dog adoption rates assume that dogs exhibiting certain behaviors are more adoptable. However, no systematic data are available to indicate that the spontaneous behavior of shelter dogs has an effect on adopter preference. The aim of the present study was to determine whether any behaviors that dogs exhibit spontaneously in the presence of potential adopters were associated with the dogs'' length of stay in the shelter. A sample of 289 dogs was videotaped for 1 min daily throughout their stay at a county shelter. To account for differences in adopter behavior, experimenters varied from solitary passive observers to pairs of interactive observers. Dogs behaved more attentively to active observers. To account for adopter preference for morphology, dogs were divided into “morphologically preferred” and “non-preferred” groups. Morphologically preferred dogs were small, long coated, ratters, herders, and lap dogs. No theoretically significant differences in behavior were observed between the two different dog morphologies. When accounting for morphological preference, three behaviors were found to have a significant effect on length of stay in all dogs: leaning or rubbing on the enclosure wall (increased median length of stay by 30 days), facing away from the front of the enclosure (increased by 15 days), and standing (increased by 7 days). When combinations of behaviors were assessed, back and forth motion was found to predict a longer stay (increased by 24 days). No consistent behavioral changes were observed due to time spent at the shelter. These findings will allow shelters to focus behavioral modification efforts only on behaviors likely to influence adopters'' choices.     

Pharmacological assessment of methamphetamine-induced behavioral hyperactivity mediated by dopaminergic transmission in planarian Dugesia japonica

The freshwater planarian Dugesia japonica has a simple central nervous system (CNS) and can regenerate complete organs, even a functional brain. Recent studies demonstrated that there is a great variety of neuronal-related genes, specifically expressed in several domains of the planarian brain. We identified a planarian dat gene, named it D. japonica dopamine transporter (Djdat), and analyzed its expression and function. Both in situ hybridization and immunofluorescence revealed that localization of Djdat mRNA and protein was the same as that of D. japonica tyrosine hydroxylase (DjTH). Although, dopamine (DA) content in Djdat(RNAi) planarians was not altered, Djdat(RNAi) planarians showed increased spontaneous locomotion. The hyperactivity in the Djdat(RNAi) planarians was significantly suppressed by SCH23390 or sulpiride pretreatment, which are D₁ or D₂ receptor antagonists, respectively. These results suggest that planarians have a Djdat ortholog and the ability to regulate dopaminergic neurotransmission and association with spontaneous locomotion.     

Inferring Adaptive Codon Preference to Understand Sources of Selection Shaping Codon Usage Bias

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify “adaptive codon preference,” a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated “preference” largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.     

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.     

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.     

Search for the evolutionary origin of a brain: planarian brain characterized by microarray

The origin of the brain remains a challenging problem in evolutionary studies. To understand when and how the structural brain emerged, we analyzed the central nervous system (CNS) of a lower invertebrate, planarian. We conducted a large-scale screening of the head part-specific genes in the planarian by constructing a cDNA microarray. Competitive hybridization of cDNAs between a head portion and the other body portion of planarians revealed 205 genes with head part-specific spikes, including essential genes in the vertebrate nervous system. The expression patterns of the top 30 genes showing the strongest spikes implied that the planarian brain has undergone functional regionalization. We demonstrate the complex cytoarchitecture of the planarian brain, despite its simple superficiality of the morphology.     

The preferential accumulation of cadmium in the head portion of the freshwater planarian, Dugesia japonica (Platyhelminthes: Turbellaria)

Free-living freshwater planarians are considered to have the potential for development as an experimental model for toxicological studies on xenobiotics, including metals. However, little was known about the distribution patterns of metals in the body of treated planarians. This study was conducted to determine the tissue distribution patterns of cadmium (Cd) in different body portions of the treated planarian, Dugesia japonica. Results showed that Cd accumulated in the head of planarians at a significantly higher concentration than in the tail. After examining the level of metallothionein (MT), we suggested that the tissue distribution pattern of Cd might be related to MT induction patterns. In contrast, in planarians treated with copper (Cu), neither the tissue accumulation of Cu nor the multiples of induction of MTs significantly differed between different portions. Furthermore, a higher Cd accumulation rate in the head of planarians caused more-severe oxidative stress to appear in this portion and also a higher susceptibility to a lethal concentration of Cd. Finally, both in vitro and in vivo acetylcholinesterase activities in both body portions of planarians were inhibited by Cd. The present study provides the first report that different metals are distributed in various body portions with different patterns in the planarian.     

Nucleosome Remodeling and Epigenetics

Eukaryotic chromatin is kept flexible and dynamic to respond to environmental, metabolic, and developmental cues through the action of a family of so-called “nucleosome remodeling” ATPases. Consistent with their helicase ancestry, these enzymes experience conformation changes as they bind and hydrolyze ATP. At the same time they interact with DNA and histones, which alters histone–DNA interactions in target nucleosomes. Their action may lead to complete or partial disassembly of nucleosomes, the exchange of histones for variants, the assembly of nucleosomes, or the movement of histone octamers on DNA. “Remodeling” may render DNA sequences accessible to interacting proteins or, conversely, promote packing into tightly folded structures. Remodeling processes participate in every aspect of genome function. Remodeling activities are commonly integrated with other mechanisms such as histone modifications or RNA metabolism to assemble stable, epigenetic states.     

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.     

A biophysical model for plant cell plate maturation based on the contribution of a spreading force

Plant cytokinesis, a fundamental process of plant life, involves de novo formation of a “cell plate” partitioning the cytoplasm of dividing cells. Cell plate formation is directed by orchestrated delivery, fusion of cytokinetic vesicles, and membrane maturation to form a nascent cell wall by timely deposition of polysaccharides. During cell plate maturation, the fragile membrane network transitions to a fenestrated sheet and finally a young cell wall. Here, we approximated cell plate sub-structures with testable shapes and adopted the Helfrich-free energy model for membranes, including a stabilizing and spreading force, to understand the transition from a vesicular network to a fenestrated sheet and mature cell plate. Regular cell plate development in the model was possible, with suitable bending modulus, for a two-dimensional late stage spreading force of 2–6 pN/nm, an osmotic pressure difference of 2–10 kPa, and spontaneous curvature between 0 and 0.04 nm⁻¹. With these conditions, stable membrane conformation sizes and morphologies emerged in concordance with stages of cell plate development. To reach a mature cell plate, our model required the late-stage onset of a spreading/stabilizing force coupled with a concurrent loss of spontaneous curvature. Absence of a spreading/stabilizing force predicts failure of maturation. The proposed model provides a framework to interrogate different players in late cytokinesis and potentially other membrane networks that undergo such transitions. Callose, is a polysaccharide that accumulates transiently during cell plate maturation. Callose-related observations were consistent with the proposed model’s concept, suggesting that it is one of the factors involved in establishing the spreading force.

The late-stage onset of an “areal” spreading and stabilizing force is essential for regular plant cell plate development and maturation.     

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.     

Neuropharmacology and behavior in planarians: translations to mammals

Planarians are the simplest animals to exhibit a body plan common to all vertebrates and many invertebrates, characterized by bilateral rather than radial symmetry, dorsal and ventral surfaces, and a rostrocaudal axis with a head and a tail, including specialized sense organs and an aggregate of nerve cells in the head. Neurons in planarian more closely resemble those of vertebrates than those of advanced invertebrates, exhibiting typical vertebrate features of multipolar shape, dendritic spines with synaptic boutons, a single axon, expression of vertebrate-like neural proteins, and relatively low spontaneously generated electrical activity. Here we report the most relevant contribution to the knowledge of the neuropharmacology of planarians, with particular reference to the behavioral consequences of the exposure to drugs acting on neural transmission. Neurochemical and histochemical data indicate the presence of several neurotransmitter-receptor systems in planarians. Moreover, a variety of experimental studies characterized specific behavioral patterns of these animals following the exposure to drugs acting on neural transmission. There is also evidence of the interactions between discrete neurotransmitter-receptor systems in modulating behavior in planarians. Finally, the model has proved efficacy for investigating the neurotoxicology of the dopamine neurons, and for the initial screening of the neuroprotective potential of drugs. In conclusion, these findings indicate that interactions between discrete neurotransmitter-receptor systems occur very early along phylogeny, although they may have evolved from very fundamental behaviors, such as motor activity in planarian, to more complex and integrated functions in vertebrates.     

Two populations of pluripotent stem cells in planarians <Emphasis Type="Italic">Girardia tigrina</Emphasis>

The positional differences in the regenerative capability of individual body parts of the planarian Girardia (Dugesia) tigrina were analyzed. The paper shows the significance of the size and positional differences of individual fragments of planarians for their regenerative capabilities, as well as the fundamental difference in the mechanisms of the head and tail blastema formation. A scheme of regeneration that includes two populations of pluripotent stem cells called neoblasts is suggested. The two populations of neoblasts differ in their role and distribution along the planarian body. Specifically, the population of neoblasts involved in the formation of any blastema migrates to the nearest blastema, and the population participating only in the creation of the head blastema migrates along the planarian body, following the gradient of biomass of the damaged axons arising after the amputation of the head end. The maximal size of the head blastema was found in the fragment obtained after cutting off the head fragment at the eye level, and the maximal portion of all pluripotent stem cells migrating into two blastemas was found in the fragment obtained by cutting the planarian above the mouth, followed by cutting off the head fragment at the eye level.