Morphological and functional recovery of the planarian photosensing system during head regeneration

When exposed to light, planarians display a distinctive light avoidance behavior known as negative phototaxis. Such behavior is temporarily suppressed when animals are decapitated, and it is restored once the animals regenerate their heads. Head regeneration and the simple but reproducible phototactic response of planarians provides an opportunity to study the association between neuronal differentiation and the establishment of behavior in a simple, experimentally tractable metazoan. We have devised a phototaxis assay system to analyze light response recovery during head regeneration and determined that light evasion is markedly re-established 5 days after amputation. Immunohistological and in situ hybridization studies indicate that the photoreceptors and optic nerve connections to the brain begin by the fourth day of cephalic regeneration. To experimentally manipulate the light response recovery, we performed gene knockdown analysis using RNA interference (RNAi) on two genes (1020HH and eye53) previously reported to be expressed at 5 days after amputation and in the dorso-medial region of the brain (where the optic nerves project). Although RNAi failed to produce morphological defects in either the brain or the visual neurons, the recovery of the phototactic response normally observed in 5-day regenerates was significantly suppressed. The data suggest that 1020HH and eye53 may be involved in the functional recovery and maintenance of the visual system, and that the phototaxis assay presented here can be used to reliably quantify the negative phototactic behavior of planarians.     

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.     

Formation of the function of the photosensing system in early development

The function of simple eyes in two planarian species, two-eyed Girardia tigrina and multi-eyed Polycelis tenuis, has been studied. When exposed to light, planarians display a light avoidance reaction known as negative phototaxis. This reaction has been investigated in intact animals and in head and tail fragments in the course of eye regeneration after their section. Specific features of the phototaxis reaction have been described in all groups of animals. The differences in light response recovery were shown between two planarian species and two regenerating fragments. No correlation has been found between phototactic reactions and restoration of eye structure, the number of eyes, maturation of the ganglion, growth of regenerative blastema, and motor system. The phototactic response occurred two days after the recovery of the morphology of eyes and their connection with the brain. The participation of conserved and novel genes in early development of the eye function is discussed.     

Formation of the photosensing system function in early development

The function of simple prototypic eyes in two planarian species, the two ocular Girardia tigrina and the multiocular Polycelis tenuis, has been studied. When exposed to light, planarians display the light avoidance reaction known as negative phototaxis. This reaction has been investigated in intact animals and in head and tail fragments after their section in the course of eye regeneration. Specific features of the phototaxis reaction have been described in all groups of animals. The differences in light response recovery were shown between two planarian species and two regenerating fragments. No correlation between phototaxic reactions and the restoration of the eye structure, the number of eyes, the maturation of ganglion, the growth of regenerative blastema, and motor system has been found. The phototaxic response occurred two days after the recovery of the morphology of eyes and their connection with the brain. The participation of conserved and novel genes in early development of the eye function is discussed.     

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.     

Bone morphogenetic protein is required for dorso-ventral patterning in the planarian Dugesia japonica

In order to clarify the function of the Djbmp (Dugesia japonica bone morphogenetic protein) gene in planarian body patterning, we carried out knockdown of this gene by RNA interference. When the planarians were treated with double-stranded RNA of Djbmp, a bulge formed on the dorsal side, with a dent in the middle of the bulge, and the body surface inside the dent was smoothened and less pigmented. In situ hybridization of the DjIFb gene, which is expressed in the body margin, revealed that the additional body margin was formed ectopically at the region surrounding the dent. The Djbmp-knockdown planarians often had a pair of incomplete nerve cords in the dorsal side, in addition to the original pair of ventral nerve cords. Taken together, we concluded that the Djbmp-knockdown induced formation of an ectopic ventral side, suggesting that Djbmp is required for the dorso-ventral body patterning in the planarian.     

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.     

Brain regeneration from pluripotent stem cells in planarian

How can planarians regenerate their brain? Recently we have identified many genes critical for this process. Brain regeneration can be divided into five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) pattern formation, (4) neural network formation, and (5) functional recovery. Here we will describe the structure and process of regeneration of the planarian brain in the first part, and then introduce genes involved in brain regeneration in the second part. Especially, we will speculate about molecular events during the early steps of brain regeneration in this review. The finding providing the greatest insight thus far is the discovery of the nou-darake (ndk; ‘brains everywhere’ in Japanese) gene, since brain neurons are formed throughout the entire body as a result of loss of function of the ndk gene. This finding provides a clue for elucidating the molecular and cellular mechanisms underlying brain regeneration. Here we describe the molecular action of the nou-darake gene and propose a new model to explain brain regeneration and restriction in the head region of the planarians.     

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.     

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.     

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.     

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.     

Specific features of eye regeneration in multiocular planarians Polycelis tenuis

Regeneration and negative phototaxis were studied in planarians Polycelis tenuis, in which the anterior body end is fringed with many eyes. Comparative data for the same indices are given for binocular planarians Girardia tigrina. Multiple eyes regenerated gradually with a decrease in the rate of regeneration and independently from the rate of restoration of the anterior body end, where they are located. Negative phototaxis was restored independently from the total amount of regenerated eyes. It was unstable in both planarian species.     

The role of the segmentation gene <Emphasis Type="Italic">hairy</Emphasis> in <Emphasis Type="Italic">Tribolium</Emphasis>

Hairy stripes in Tribolium are generated during blastoderm and germ band extension, but a direct role for Tc-h in trunk segmentation was not found. We have studied here several aspects of hairy function and expression in Tribolium, to further elucidate its role. First, we show that there is no functional redundancy with other hairy paralogues in Tribolium. Second, we cloned the hairy orthologue from Tribolium confusum and show that its expression mimics that of Tribolium castaneum, implying that stripe expression should be functional in some way. Third, we show that the dynamics of stripe formation in the growth zone is not compatible with an oscillatory mechanism comparable to the one driving the expression of hairy homologues in vertebrates. Fourth, we use parental RNAi experiments to study Tc-h function and we find that mandible and labium are particularly sensitive to loss of Tc-h, reminiscent of a pair-rule function in the head region. In addition, lack of Tc-h leads to cell death in the gnathal region at later embryonic stages, resulting in a detachment of the head. Cell death patterns are also altered in the midline. Finally, we have analysed the effect of Tc-h knockdown on two of the target genes of hairy in Drosophila, namely fushi tarazu and paired. We find that the trunk expression of Tc-h is required to regulate Tc-ftz, although Tc-ftz is itself also not required for trunk segmentation in Tribolium. Our results imply that there is considerable divergence in hairy function between Tribolium and Drosophila.     

The sll0886 gene, controlling light-activated heterotrophic growth, is involved in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6893

The sll0886 gene, controlling light-activated heterotrophic growth (LAHG), was tested for the role in regulating phototaxis in cyanobacterium Synechocystis sp. PCC 6803. Insertional inactivation of the gene in the genome of a wildtype strain did not affect positive (toward light) or negative (away from high light) phototaxis. However, cells lost motility when sll0886 inactivation was combined with the prqRL17Q mutation, which determined negative phototaxis at low light. Immotile cells with the prqRL17Q mutation and the inactivated sll0886 gene did not display any defect in the formation of type IV pili, essential for phototaxis. Hence, the function, rather than biogenesis, of pili was affected. It was concluded that the sll0886 gene, coding for a TPR family protein, is involved in controlling negative phototaxis of cyanobacteria at the level of photoreception and signal transduction and that its role is mediated by the unidentified redundant gene whose function is suppressed by the prqRL17Q mutation.     

Optic chiasm formation in planarian I: Cooperative netrin- and robo-mediated signals are required for the early stage of optic chiasm formation

Freshwater planarians can regenerate a brain, including eyes, from the anterior blastema, and coordinately form an optic chiasm during eye and brain regeneration. To investigate the role of the netrin- and slit-signaling systems during optic chiasm formation, we cloned three receptor genes (Djunc5A, Djdcc and DjroboA) expressed in visual neurons and their ligand genes (DjnetB and Djslit) and analyzed their functions by RNA interference (RNAi). Although each of DjroboA(RNAi), Djunc5A(RNAi) and DjnetB(RNAi) showed a weak phenotype and Djslit(RNAi) showed a severe defect of eye formation, we did not observe any defect of crossing of visual axons over the midline among single knockdown planarians. However, among double knockdown planarians, some of DjnetB(RNAi);DjroboA(RNAi) and Djunc5A(RNAi);DjroboA(RNAi) showed complete disconnection between the visual axons from the two sides, suggesting that some combination of netrin- and robo-mediated signals may be required for crossing over the midline. Finally, we carefully investigated the distribution patterns of cells expressing DjNetB protein, DjnetB, and Djslit at the early stage of regeneration, and found that visual axons projected along a path sandwiched between DjNetB protein and Djslit-positive cells. These results suggest that two different collaborative or combinatory signals may be required for midline crossing at the early stage of chiasm formation during eye and brain regeneration.     

Specific features of eye regeneration in multiocular planarians <Emphasis Type="Italic">Polycelis tenuis</Emphasis>

Regeneration and negative phototaxis were studied in planarians Polycelis tenuis, in which the anterior body end is fringed with many eyes. Comparative data for the same indices are given for binocular planarians Girardia tigrina. Multiple eyes regenerated gradually with a decrease in the rate of regeneration and independently from the rate of restoration of the anterior body end, where they are located. Negative phototaxis was restored independently from the total amount of regenerated eyes. It was unstable in both planarian species.     

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.