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A unique aspect of planarians is that they can regenerate a brain from somatic pluripotent stem cells called neoblasts, which have the ability to produce themselves (self-renew) and to give rise to all missing cell types during regeneration. Recent molecular studies have revealed that the planarian brain is composed of many distinct neuronal populations, which are evolutionarily and functionally conserved ones, and acts as an information-processing center to elicit distinct behavioral traits depending on a variety of signals arising from the external environment. How can planarians regenerate such a brain? On the basis of our recent findings, here we review the cellular and molecular mechanisms that regulate the stem cell dynamics involved in the brain regeneration of the planarian Dugesia japonica. Our findings suggest the possible value of in vivo planarian studies for guiding regenerative medicine to treat neurodegenerative diseases via interlinking stem cell biology and regeneration biology., (© 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

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.

Recently, planarians have received much attention because of their contributions to research on the basic science of stem cell systems, neural regeneration, and regenerative medicine. Planarians can regenerate complete organs, including a well-organized central nervous system (CNS), within about 7 days. This high regenerative capacity is supported by pluripotent stem cells present in the mesenchymal space throughout the body. Interestingly, planarians can regenerate their brain via a molecular mechanism similar to that of mammalian brain development. The regeneration process of the planarian brain can be divided into five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) brain pattern formation, (4) neural network formation, and (5) functional recovery, with several kinds of genes and molecular cascades acting at each step. Recently, we have identified a planarian tyrosine hydroxylase (TH) gene, a rate-limiting enzyme for dopamine (DA) biosynthesis, and produced TH-knockdown planarians by the RNA interference technique. Studies of TH-knockdown planarians showed that DA has an important role of the modification in behavioral movement in planarians. Using monoclonal anti-planarian TH antibody, we also found that dopaminergic neurons are mainly localized in the planarian brain. When the planarian body was amputated, newly generated TH-immunopositive neurons were detected in the anterior region at day 3 of regeneration (i.e., the period of neural network formation), and the TH-immunopositive axonal and dendritic neural network in the CNS was reconstructed during day 5-7 of regeneration. In this article, recent advances in elucidating the molecular mechanism of planarian brain regeneration and dopaminergic neurons are reviewed, and its future prospects for contribution of this system to basic science and medical science research are described.

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.

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.

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., (Copyright 2003 Elsevier Science B.V.)

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.

Research on individual behaviors can help to reveal the processes and mechanisms that mediate an animal's habits and interactions with the environment. Importantly, individual behaviors arise as outcomes of genetic programs, morphogenesis, physiological processes, and neural functions; thus, behavioral analyses can be used to detect disorders in these processes. Planarians belong to an early branching bilateral group of organisms that possess a simple central nervous system. Furthermore, planarians display various behavioral responses to the environment via their nervous system. Planarians also have remarkable regenerative abilities, including whole‐brain regeneration. Therefore, the combination of planarians' phylogenetic position, behavioral properties, regenerative ability, and genetic accessibility provides a unique opportunity to understand the basic mechanisms underlying the anatomical properties of neural morphogenesis and the dynamic physiological processes and neural function. Here, we describe a step‐by‐step protocol for conducting simple behavioral analyses in planarians with the aim of helping to introduce researchers to the utility of performing behavioral analyses in planarians. Since the conditions of planarians impact experimental results and reproducibility, this protocol begins with a method for maintaining planarians. Next, we introduce the behavioral tests as well as the methods for quantifying them using minimal and cost‐effective equipment and materials. Finally, we present a unique RNAi technique that enables conditional silencing of neural activity in the brain of planarians. [ABSTRACT FROM AUTHOR]

More than 100 years ago, early workers realized that planarians offer an excellent system for regeneration studies. Another unique aspect of planarians is that they occupy an interesting phylogenetic position with respect to the nervous system in that they possess an evolutionarily primitive brain structure and can regenerate a functional brain from almost any tiny body fragment. Recent molecular studies have revisited planarian regeneration and revealed key information about the cellular and molecular mechanisms underlying brain regeneration in planarians. One of our great advances was identification of a gene, nou-darake, which directs the formation of a proper extrinsic environment for pluripotent stem cells to differentiate into brain cells in the planarian Dugesia japonica. Our recent findings have provided mechanistic insights into stem cell biology and also evolutionary biology. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In previous studies, we have shown that dorsoventral (DV) interaction evokes not only blastema formation, but also morphogenetic events similar to those that occur in regeneration. However, it is still unclear what kinds of signal molecules are involved in the DV interaction. To investigate the signal systems involved in the DV interaction, we focused on a noggin-like gene (Djnlg) identified by the planarian EST project. Djnlg is the first noggin homologue isolated from an invertebrate. In DjNLG, the positions of nine cysteine residues which may be essential for dimer formation were well conserved, but overall, the amino acid sequence of DjNLG did not show high similarity to the sequences of vertebrate Noggins. Expression of Djnlg was observed only in the proximal region of the branch structures in the brain of intact planarians, suggesting that Djnlg may have a role in pattern formation in the brain. Interestingly, transient strong expression of Djnlg was observed in the amputated region of regenerating planarians. Djnlg-expressing cells were detected beneath the muscle 9 h after amputation and were then detected in the ventral subepidermal region of the blastema. The induction of Djnlg expression by amputation was not affected by X-ray irradiation, even though the stem cells were completely eliminated, implying the existence of signal-producing cells which may provide a positional cue to the stem cells. In DV reversed grafting, expression of Djnlg was strongly induced in the DV boundary between the host and donor. These results suggest that ectopic DV interaction may induce expression of Djnlg in the positional cue-producing cells, and that it might be involved in stimulation of blastema formation as well as DV patterning of the body. [Copyright &y& Elsevier]

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level. [Copyright &y& Elsevier]