Yoshihiko Umesono

STRUCTURE OF THE PLANARIAN CENTRAL NERVOUS SYSTEM (CNS) REVEALED BY NEURONAL CELL MARKERS

Abstract Planarians are considered to be among the most primitive animals which developed the central nervous system (CNS). To understand the origin and evolution of the CNS, we have isolated a neural marker gene from a planarian, Dugesia japonica, and analyzed the structure of the planarian CNS by in situ hybridization. The planarian CNS is located on the ventral side of the body, and composed of a mass of cephalic ganglions in the head region and a pair of ventral nerve cords (VNC). Cephalic ganglions cluster independently from VNC, are more dorsal than VNC, and form an inverted U-shaped brain-like structure with nine branches on each outer side. Two eyes are located on the dorsal side of the 3rd branch and visual axons form optic chiasma on the dorsal-inside region of the inverted U-shaped brain. The 6th–9th branches cluster more closely and form auricles on the surface which may function as the sensory organ of taste. We found that the gross structure of the planarian CNS along the anterior-posterior (A–P) axis is strikingly similar to the distribution pattern of the “primary” neurons of vertebrate embryos which differentiate at the neural plate stage to provide a fundamental nervous system, although the vertebrate CNS is located on the dorsal side. These data suggest that the basic plan for the CNS development along the A–P axis might have been acquired at an early stage of evolution before conversion of the location of the CNS from the ventral to the dorsal side.

FGFR-related gene nou-darake restricts brain tissues to the head region of planarians

The study of planarian regeneration may help us to understand how we can rebuild organs and tissues after injury, disease or ageing. The robust regenerative abilities of planarians are based upon a population of totipotent stem cells (neoblasts), and among the organs regenerated by these animals is a well-organized central nervous system. In recent years, methodologies such as whole-mount in situ hybridizations and double-stranded RNA have been extended to planarians with the aim of unravelling the molecular basis of their regenerative capacities. Here we report the identification and characterization of nou-darake (ndk), a gene encoding a fibroblast growth factor receptor (FGFR)-like molecule specifically expressed in the head region of the planarian Dugesia japonica. Loss of function of ndk by RNA interference results in the induction of ectopic brain tissues throughout the body. This ectopic brain formation was suppressed by inhibition of two planarian FGFR homologues (FGFR1 and FGFR2). Additionally, ndk inhibits FGF signalling in Xenopus embryos. The data suggest that ndk may modulate FGF signalling in stem cells to restrict brain tissues to the head region of planarians.

Planarian Hedgehog/Patched establishes anterior–posterior polarity by regulating Wnt signaling

Despite long-standing interest, the molecular mechanisms underlying the establishment of anterior–posterior (AP) polarity remain among the unsolved mysteries in metazoans. In the planarians (a family of flatworms), canonical Wnt/β-catenin signaling is required for posterior specification, as it is in many animals. However, the molecular mechanisms regulating the posterior-specific induction of Wnt genes according to the AP polarity have remained unclear. Here, we demonstrate that Hedgehog (Hh) signaling is responsible for the establishment of AP polarity via its regulation of the transcription of Wnt family genes during planarian regeneration. We found that RNAi gene knockdown of Dugesia japonica patched (Djptc) caused ectopic tail formation in the anterior blastema of body fragments, resulting in bipolar-tails regeneration. In contrast, RNAi of hedgehog (Djhh) and gli (Djgli) caused bipolar-heads regeneration. We show that Patched-mediated Hh signaling was crucial for posterior specification, which is established by regulating the transcription of Wnt genes via downstream Gli activity. Moreover, differentiated cells were responsible for the posterior specification of undifferentiated stem cells through Wnt/β-catenin signaling. Surprisingly, Djhh was expressed in neural cells all along the ventral nerve cords (along the AP axis), but not in the posterior blastema of body fragments, where the expression of Wnt genes was induced for posteriorization. We therefore propose that Hh signals direct head or tail regeneration according to the AP polarity, which is established by Hh signaling activity along the bodys preexisting nervous system.

Morphological and Functional Recovery of the Planarian Photosensing System during Head Regeneration

Abstract 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.

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.

The molecular logic for planarian regeneration along the anterior-posterior axis

The planarian Dugesia japonica can regenerate a complete individual from a head, trunk or tail fragment via activation of somatic pluripotent stem cells. About a century ago, Thomas Hunt Morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative “head stuff” and “tail stuff” along the anterior–posterior axis. However, Morgan’s hypothesis remains open to debate. Here we show that extracellular signal-related kinase (ERK) and Wnt/β-catenin signalling pathways establish a solid framework for planarian regeneration. Our data suggest that ERK signalling forms a spatial gradient in the anterior region during regeneration. The fibroblast growth factor receptor-like gene nou-darake (which serves as an output of ERK signalling in the differentiating head) and posteriorly biased β-catenin activity negatively regulate ERK signalling along the anterior–posterior axis in distinct manners, and thereby posteriorize regenerating tissues outside the head region to reconstruct a complete head-to-tail axis. On the basis of this knowledge about D. japonica, we proposed that β-catenin signalling is responsible for the lack of head-regenerative ability of tail fragments in the planarian Phagocata kawakatsui, and our confirmation thereof supports the notion that posterior β-catenin signalling negatively modulates the ERK signalling involved in anteriorization across planarian species. These findings suggest that ERK signalling has a pivotal role in triggering globally dynamic differentiation of stem cells in a head-to-tail sequence through a default program that promotes head tissue specification in the absence of posteriorizing signals. Thus, we have confirmed the broad outline of Morgan’s hypothesis, and refined it on the basis of our proposed default property of planarian stem cells.

Evolution and regeneration of the planarian central nervous system

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.

Identification and distribution of tryptophan hydroxylase (TPH)-positive neurons in the planarian Dugesia japonica.

We identified a full-length tryptophan hydroxylase (TPH) gene of planarian Dugesia japonica from a head EST database, and named it DjTPH. Based on whole-mount in situ hybridization and immunofluorescence analyses, DjTPH mRNA and protein were mainly expressed in the nervous system, especially ventral nerve cords and eye pigment cells. Furthermore, DjTPH immunoreactivity was clearly detected at commissure axonal connections in the ventral nerve cords. 5-HT was significantly decreased in DjTPH-knockdown planarians compared with control animals. These results suggest that DjTPH is required for 5-HT biosynthesis, and DjTPH antibody is a useful marker for serotonergic neurons in planarians.

ERK signaling controls blastema cell differentiation during planarian regeneration

The robust regenerative ability of planarians depends on a population of somatic stem cells called neoblasts, which are the only mitotic cells in adults and are responsible for blastema formation after amputation. The molecular mechanism underlying neoblast differentiation associated with blastema formation remains unknown. Here, using the planarian Dugesia japonica we found that DjmkpA, a planarian mitogen-activated protein kinase (MAPK) phosphatase-related gene, was specifically expressed in blastema cells in response to increased extracellular signal-related kinase (ERK) activity. Pharmacological and genetic [RNA interference (RNAi)] approaches provided evidence that ERK activity was required for blastema cells to exit the proliferative state and undergo differentiation. By contrast, DjmkpA RNAi induced an increased level of ERK activity and rescued the differentiation defect of blastema cells caused by pharmacological reduction of ERK activity. These observations suggest that ERK signaling plays an instructive role in the cell fate decisions of blastema cells regarding whether to differentiate or not, by inducing DjmkpA as a negative regulator of ERK signaling during planarian regeneration.

Identification of glutamic acid decarboxylase gene and distribution of GABAergic nervous system in the planarian Dugesia japonica

The planarian Dugesia japonica has a relatively well-organized CNS that includes the brain and the ventral nerve cords, and also has high regenerative capacity derived from pluripotent stem cells present in the mesenchymal space throughout the body. Glutamic acid decarboxylase (GAD) is the enzyme that converts glutamic acid into GABA, a major inhibitory neurotransmitter. In this study, we first identified a full-length GAD gene (DjGAD, D. japonica glutamic acid decarboxylase) in the planarian D. japonica. Whole-mount in situ hybridization revealed that a few cells expressed DjGAD mRNA, and these cells were located in both the head and pharynx regions. In order to examine the distribution pattern of DjGAD protein, we generated a mouse monoclonal anti-DjGAD antibody. The distribution pattern of DjGAD protein was very similar to that of DjGAD mRNA. A neural network of DjGAD-immunopositive cells was also clearly observed. In addition, we examined the immunofluorescence during the process of regeneration of the head from the tail piece. At day 3 of regeneration, we could detect newly formed DjGAD-immunopositive neurons in the anterior region. During day 5-7 of regeneration, reconstruction of the neural network of DjGAD-immunopositive cells occurred. DjGAD-immunoreactivity was lost in DjGAD-knockdown planarians obtained by RNA interference. The amount of GABA was significantly decreased in DjGAD-knockdown planarians, which lost negative phototaxis but not locomotion activity. These results suggest that DjGAD is clearly required for GABA biosynthesis and photosensitivity in planarians, and expression of DjGAD as detected by anti-DjGAD antibody is a useful marker for GABAergic neurons.