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Identification of glutamic acid decarboxylase gene and distribution of GABAergic nervous system in the planarian Dugesia japonica
Neuroscience 153 (2008) 1103–1114
IDENTIFICATION OF GLUTAMIC ACID DECARBOXYLASE GENE AND DISTRIBUTION OF GABAergic NERVOUS SYSTEM IN THE PLANARIAN DUGESIA JAPONICA K. NISHIMURA,a Y. KITAMURA,a1* Y. UMESONO,b K. TAKEUCHI,c K. TAKATA,a T. TANIGUCHIa AND K. AGATAb1*
is a useful marker for GABAergic neurons. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: planarian, GABA, neural network, regeneration, RNA interference.
a
Department of Neurobiology, and 21st Century Center of Excellence Program, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
b
Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Recent studies of the freshwater planarian Dugesia japonica have provided many unique insights into the mechanism of regeneration of the nervous system. Planarians have a relatively well-organized and functional brain, and can regenerate the brain after injury from nearly any portion of their bodies by utilizing somatic pluripotent stem cells (Agata and Watanabe, 1999; Agata, 2003). The planarian CNS consists of the brain in the anterior region and a pair of ventral nerve cords (VNCs). Its brain forms an inverted U-shaped structure with lateral branches (Agata et al., 1998; Tazaki et al., 1999; Okamoto et al., 2005). Previous studies clearly indicated that the planarian brain is reconstructed during regeneration by similar molecular mechanisms to those operating in mammalian brain development. Fibroblast growth factor receptor–mediated signaling is indispensable for brain regeneration (Ogawa et al., 2002a; Cebrià et al., 2002a). A noggin-like gene is expressed in the future brain-forming region (Ogawa et al., 2002b). Brain patterning along the anterior–posterior axis and the medial–lateral axis is regulated by Wnt-family genes and Otx/otd-related genes, respectively (Umesono et al., 1997, 1999; Kobayashi et al., 2007). Moreover, several crucial genes involved in functional brain regeneration have been identified from planarians using the RNA interference (RNAi) technique (Inoue et al., 2004, 2007; Takano et al., 2007). Detailed neural networks and distinct domains in the planarian brain have been clearly visualized by using fluorescence dye-tracing and molecular marker staining (Sakai et al., 2000; Mineta et al., 2003; Nakazawa et al., 2003; Okamoto et al., 2005; Fusaoka et al., 2006). In a previous study, we identified a gene encoding planarian tyrosine hydroxylase (DjTH), the rate-limiting enzyme for dopamine (DA) biosynthesis, and produced DjTH-knockdown planarians by the RNAi method, and using them demonstrated that DA has an important role in muscle-mediated movement in planarians (Nishimura et al., 2007a). We also found by immunostaining with a monoclonal anti-DjTH antibody that dopaminergic neurons are distributed in a regular manner in the main lobes of the planarian brain. In a similar way, we also isolated a gene for tryptophan hydroxylase, the rate-limiting enzyme of 5-HT biosynthesis, and showed that the protein it encodes is mainly distributed at VNCs (Nishimura et al., 2007b).
c
Department of Anatomy and Developmental Biology, Kyoto Prefectual University of Medicine, Kawaramachi, Kamigyo-ku, Kyoto 6028566, Japan
Abstract—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 1
These contributors both served as senior authors. *Corresponding authors: Tel: ⫹81-75-595-4706; fax: ⫹81-75-5954796 (Y. Kitamura); Tel: ⫹81-75-753-4200; fax: ⫹81-75-753-4203 (K. Agata). E-mail address: [email protected] (Y. Kitamura), agata@ mdb.biophys.kyoto-u.ac.jp (K. Agata). Abbreviations: ANOVA, analysis of variance; cDNA, complementary DNA; DA, dopamine; DIG, digoxigenin; DjGAD, D. japonica glutamic acid decarboxylase; DjPC2, D. japonica pro-hormone convertase 2 homologue; dsRNA, double-stranded RNA; ECD, electrochemical detection; EDTA, ethylenediaminetetraacetic acid; GAD, glutamic acid decarboxylase; GSP, gene-specific primer; PCR, polymerase chain reaction; RNAi, RNA interference; VNC, ventral nerve cord; 5=RACE, 5=rapid amplification of cDNA ends.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.03.026
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Glutamic acid decarboxylase (GAD; EC4.1.1.15) is the rate-limiting enzyme for GABA biosynthesis, and converts glutamic acid into GABA (Fig. 1A). Two independent GAD
genes (i.e. GAD65 and GAD67) have been identified in vertebrates (Erlander et al., 1991; Bu et al., 1992). These two genes show strong conservation of the sequence in
Fig. 1. DjGAD encodes a glutamic acid decarboxylase. Pathway of GABA biosynthesis (A). Nucleotide and predicted amino acid sequence of DjGAD cDNA (B). The arrows labeled “GSP” show the primers for 5=RACE. The arrows labeled “GAD” show the degenerate PCR primers and specific primers used for the stepwise dilution cloning method. Asterisks indicate stop codons.
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their C-terminal region (Fenalti et al., 2007). Only a single GAD gene was identified in invertebrates such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster (Jackson et al., 1990; McIntire et al., 1993). The amino acid GABA is a major inhibitory neurotransmitter which exists widely in both vertebrates and invertebrates (Jackson et al., 1990; McIntire et al., 1993; Tillakaratne et al., 1995; Martyniuk et al., 2007). Several reports have already demonstrated that several kinds of neurotransmitters are detected in planarians (Ness et al., 1996; Itoh and Igarashi, 2000; Rawls et al., 2006). However, the GAD gene has not hitherto been identified in planarians. In addition, the detailed distribution of GABAsynthetic neurons (GABAergic neurons) and the process of regeneration of the GABAergic neural network are still not known in planarians. Here we focused on GABAergic neurons, and identified the planarian GAD gene (DjGAD, D. japonica glutamic acid decarboxylase), showed its distribution patterns in both intact and regenerating planarians using an anti-DjGAD antibody, and demonstrated that DjGAD is clearly required for GABA biosynthesis in planarians.
EXPERIMENTAL PROCEDURES Animals Planarians (Dugesia japonica) of SSP strain were cultured in autoclaved tap water at 24 °C in subdued light and fed chicken liver paste once a week. Planarians 5–7 mm in length were used in the present study. Animals were starved for at least 7 days before experiments. All procedures conformed to the guidelines of local and the National Institutes of Health for Laboratory Animal Science. Every effort was made to minimize the number of animals used and their suffering.
RNA extraction and complementary DNA (cDNA) preparation Total RNA was extracted from 300 head pieces of planarians using Isogene-LS (Nippon Gene, Toyama, Japan). mRNA was purified from total RNA using an Oligotex-dT30 ⬍super⬎mRNA purification kit (Takara, Kyoto, Japan). cDNA was synthesized from mRNA using a first-strand cDNA synthesis kit (Amersham Biosciences, Arlington Heights, IL, USA).
Cloning of DjGAD cDNA Degenerate oligonucleotides were designed for regions conserved in GAD among various animals, namely, Caenorhabditis elegans UNC25, Drosophila melanogaster GAD, Mus musculus GAD-65/67 and Homo sapiens GAD-65/67. These primers were as follows: GAD F1 5=-GTNAARYTNGTNAARTGYAAYGAR-3= GAD F2 5=-AARTGYAAYGARMGNGGNAARATG-3= GAD R1 5=-CATNGCYTTRTGNGGRTTCC-3= GAD R2 5=-NARNGTNACNGCCATNGCYTTATG-3= (N⫽A/T/G/C, Y⫽C/T, R⫽A/G, W⫽T/A, S⫽C/G, M⫽A/C) The polymerase chain reaction (PCR) was carried out using primers GAD F1 and GAD R1 sequentially with cycling consisting of an initial denaturation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. Subsequently, nested PCR was carried out using primers GAD F2 and GAD R2. Finally, a 100-bp cDNA fragment was isolated. We then carried out the screening of a cDNA library constructed from poly(A)⫹ RNA of planarian head pieces in ZAPII vector (Stratagene, La Jolla, CA,
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USA) by the stepwise dilution method (Watanabe et al., 1997) using a specific primer set designed from the 100-bp cDNA fragment. These primers were as follows: GAD F3 5=-AAGAGGAAAAATGGATACTGAAAACC-3= GAD R3 5=-ATTTTCAATTCCATTCAGTAAATAGCG-3=. The cycling consisted of an initial denaturation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. To determine the 5=-end sequence of DjGAD, 5=rapid amplification of cDNA ends (5=RACE) was carried out using a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA, USA). The genespecific primer (GSP) sequences were as follow: GSP R1 5=-GGGCCATTGCTTGTTTAAGTTGGTGAGGATGG-3= GSP R2 5=-GGCCTTGTTTGTCAAGTGAATAGC-3=. The cycling consisted of an initial denaturation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. Subsequently, nested PCR was carried out using GSP R2. Finally, a 1984-bp cDNA fragment was isolated.
RNA probe synthesis and whole-mount in situ hybridization The plasmid pBluescript SK (⫺) containing DjGAD cDNA was linearized with BamHI. Digoxigenin (DIG) -antisense RNA probe was synthesized using T7 RNA polymerase. Planarians were treated with 2% HCl in 5/8 Holtfreter’s solution for 5 min at 4 °C and were fixed in 4% paraformaldehyde containing 5% methanol for 3 h at 4 °C. Hybridization was carried out using DIG-labeled complementary RNA probe as previously described (Umesono et al., 1997; Agata et al., 1998).
Generation of a mouse monoclonal anti-DjGAD antibody and whole-mount immunofluorescence A mouse monoclonal anti-DjGAD antibody was generated against a fusion protein produced in E. coli XL10-GOLD strain (Stratagene). A fragment encoding amino acid residues 31–125 of DjGAD was inserted into pQE30 (Qiagen, Chatsworth, CA, USA). A fusion protein with a histidine tag was induced by isopropyl-D-thiogalactopyranoside and purified using Ni-NTA agarose (Qiagen). For further purification, the gel slice containing the target protein was cut out after SDS-PAGE, and the purified protein was isolated by electroelution. Antibody generation was performed by Kohjin-Bio (Saitama, Japan). Planarians were treated with 2% HCl in 5/8 Holtfreter’s solution for 5 min at 4 °C and fixed in Carnoy’s solution (ethanol: chloroform: acetic acid anhydrate in proportions 6:3:1, respectively) for 2 h at 4 °C. Immunofluorescence analysis was performed as previously described (Nishimura et al., 2007a). Primary antibodies used in this study were: mouse monoclonal antibodies against DjGAD and DjSYT (synaptotagmin: Tazaki et al., 1999) to visualize GABAergic neurons and pan-neural cells, respectively, and rabbit polyclonal antibody against DjTH (Nishimura et al., 2007a) and Djarrestin (Sakai et al., 2000) to visualize dopaminergic neurons and photoreceptor neurons, respectively.
Double-stranded RNA (dsRNA) synthesis and microinjection The plasmid pBluescript SK (⫺) containing the longest DjGAD cDNA was linearized with KpnI and BamHI. Sense or antisense RNAs were synthesized with T3 or T7 RNA polymerase, respectively. After incubation with RQI DNase for 1 h at 37 °C, the samples were denatured for 20 min at 65 °C and annealed for 40 min at 37 °C (Sánchez Alvarado and Newmark 1999). Intact planarians were injected three times (32 nL/injection) with dsRNA (once per day for 3 days) using a Drummond Scientific Nanoject injector (Broomall, PA, USA). Control
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animals were injected with water instead of dsRNA. At 1 day after the last injection, planarians were amputated immediately posterior to the pharynx and 7-day-regenerated animals from the tail piece were used in these experiments.
analysis of variance (ANOVA). Further statistical analysis for post hoc comparisons was performed using the Bonferroni/Dunn test (StatView, Abacus Concepts, Berkeley, CA, USA).
Phototaxis assay HPLC analysis of GABA Quantification of GABA was performed using HPLC with precolumn o-phthaldialdehyde-mercaptoethanol derivatization (OPAME) and electrochemical detection (ECD). Samples were homogenized by sonication in 100 L of 0.1 M perchloric acid containing 100 M EDTA. After centrifugation at 15,000 r.p.m. for 15 min at 4 °C, the supernatant was filtered through a 0.22-m pore membrane. Ten microliters of the filtrate was added to 30 L of condensation buffer containing: 0.1 M K2CO3, 2% ethanol, 4 mM OPA and 0.04% ME (pH⫽9.5). After incubation of the mixture for 150 s, samples were injected onto an SC-5ODS column (3.0⫻150 mm; Eicom Corporation, Kyoto, Japan), and detected using an electrochemical detector (HTEC-500; Eicom Corporation). The working electrode potential was maintained at ⫹600 mV against Ag/AgCl. A CA-ODS column (3.0⫻4 mm; Eicom Corporation) was placed between the injector and the analytical column. The temperature was constant at 30 °C. The mobile phase (pH⫽2.8) was composed of 50 mM phosphate buffer, 50% methanol and 5 mg/L EDTA, and was delivered at a 0.5 mL/min flow rate. The retention time of GABA was approximately 14.3 min.
Statistical evaluation The amount of GABA is given as mean⫾standard error of the mean (S.E.M.). The significance of differences was determined by
The negative phototaxis assay was carried out as previously described (Inoue et al., 2004). Ten individual planarians were used in this assay. The time spent in the target quadrant and the total distance of movement were given as mean⫾S.E.M. Statistical analysis was performed using the Student’s t-test (StatView).
RESULTS Molecular cloning of DjGAD cDNA A partial planarian GAD cDNA was obtained by a reversetranscription PCR strategy with degenerate primers designed based on the amino acid sequence highly conserved in several animals. Subsequently, a full-length sequence was obtained by head cDNA library screening and 5=RACE (see Experimental Procedures). This cDNA sequence had a 1749-bp coding region, and was predicted to encode a protein of 583 amino acid residues (Fig. 1B). This predicted amino acid sequence was similar to the sequence of GAD of other species. This sequence also contained the pyridoxal-phosphate binding domain that is a functional domain of GAD (Fig. 2A). Therefore, this gene was named DjGAD. The full-length cDNA sequence of
Fig. 2. Multiple alignment of DjGAD sequences. Comparison of amino acid sequence of DjGAD with GAD of C. elegans, D. melanogaster and H. sapiens. The box encloses the core region of the pyridoxal-phosphate binding domain, a functional domain of GAD. Conserved amino acids are indicated by dots. Gaps are indicated by hyphens. The accession numbers of the aligned sequence are as follow: C. elegans UNC-25 (AF109378), D. melanogaster-GAD (NM_079190), H. sapiens-GAD65 (NM_000818) and H. sapiens-GAD67 (NM_000817).
K. Nishimura et al. / Neuroscience 153 (2008) 1103–1114
DjGAD has been deposited in DDBJ/EMBL/GenBank under accession number AB332029. Expression pattern of DjGAD mRNA and protein Expression analysis of DjGAD was carried out in intact animals by whole mount in situ hybridization with a DIGlabeled antisense RNA probe (Fig. 3B–E). DjGAD expression was detected in very few cells compared with the number of pan-neural cells that were stained with DjPC2 (D. japonica pro-hormone convertase 2 homologue) -probe (Fig. 3A) (Agata et al., 1998). In the head region, DjGAD-expressing cells were distributed in a dotted pattern in the brain; some of them were aligned in an inverted U-shape, and the others were distributed in the medial part of the brain (Fig. 3C). In the transverse view of the head region, DjGAD-expressing cells were detected in both the dorsal and ventral regions of the brain (Fig. 3D). DjGAD-expressing cells were detected around the pharynx and at its tip (Fig. 3E). In order to visualize the distribution of neural cell bodies and the axonal projections of DjGAD-positive cells, we generated a mouse monoclonal anti-DjGAD antibody. An axonal network and neural morphology were clearly detected by immunofluorescence analysis using anti-DjGAD antibody. The distribution pattern of DjGAD-immunopositive cells was very similar to that of DjGAD-mRNA stained by in situ hybridization (Fig. 3B, F). DjGAD-immunopositive neurons could be classified into three types according to cell morphology and distribution. Two types of DjGADimmunopositive neurons were located in the dorsal and ventral sides of the brain, respectively (Fig. 3G, H). DjGAD-immunopositive neuronal cell bodies and axons were connected with each other in the ventral part of the brain (Fig. 3G, H). The third type of DjGAD-immunopositive cells was detected around the pharynx and at its tip (Fig. 3I). We then measured the amount of GABA by HPLC-ECD in the whole body and in separate body pieces (i.e. the head, trunk, and tail) that were amputated anterior and posterior to the pharynx (Fig. 3J). The amount of GABA in the whole body was 8.0⫾1.0 pmol/mg wet weight of planarian body. The amounts of GABA in the head, trunk, and tail region were 4.4⫾0.4, 11.3⫾1.0, and 1.8⫾0.5 pmol/mg wet weight, respectively. Thus, the amount of GABA in the trunk region was higher than the amounts in both the head and tail regions. In addition, the amount of GABA in the head region was higher than the amount of GABA in the tail region. Process of regeneration of DjGAD-immunopositive cells Planarians can regenerate complete organs after the amputation of various body pieces. The planarian brain can be completely regenerated from even a body piece with no brain tissue within 7 days (Cebrià et al., 2002b). We examined the process of regeneration of head DjGAD-immunopositive neurons from the tail piece that was amputated posterior to the pharynx. Immunoreactivity of DjGAD was undetectable on the first day after amputation (Fig. 4A). On the third day, several new DjGAD-immunopositive neurons
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became detectable in the anterior region (Fig. 4B). On the fifth day, the number of new DjGAD-immunopositive neurons was increased compared with that on the third day of regeneration, and these neurons could be seen to possess axons (Fig. 4C). On the seventh day, the number of new DjGAD-immunopositive neurons was increased and the neural network was reconstructed and expanded, in comparison with those on the fifth day of regeneration (Fig. 4D). In addition, DjGAD-immunoreactivity was also observed at the tip of the pharynx. We further examined the process of regeneration of DjGAD-immunopositive cells at the tip of the pharynx in animals regenerated from the tail piece. Immunoreactivity of DjGAD was undetectable on the first and third day after amputation (Fig. 4E). Interestingly, the pharynx was newly regenerated from the residual tail piece on the second day (Kobayashi et al., 1999). Several DjGAD-immunopositive cells became newly detectable in the tip of the pharynx (Fig. 4F) on the fifth day, and then DjGAD immunoreactive intensity was increased on the seventh day (Fig. 4G). Comparison of DjGAD-immunopositive neurons and DjTH-immunopositive neurons in the planarian CNS Previously, we characterized the DjTH gene, which is required for DA biosynthesis, and determined the distribution of DjTH by immunofluorescence analysis (Nishimura et al., 2007a). In order to compare the distribution patterns of GABAergic neurons and dopaminergic neurons, we performed double-immunofluorescence analysis using antiDjGAD antibody and anti-DjTH antibody. DjGAD-immunopositive neurons and DjTH-immunopositive neurons were present in distinct locations in the dorsal region of the head (Fig. 5A, B). In addition, DjGAD-immunopositive neurons were located in the middle region between lateral arrays of ventrally localized DjTH-immunopositive neurons (Fig. 5C, D). DjGAD-knockdown planarians GAD is the rate-limiting enzyme for GABA biosynthesis in both vertebrates and invertebrates. To examine whether DjGAD is required for GABA biosynthesis, the amount of GABA was measured in DjGAD-knockdown planarians created by RNAi. We used an RNAi method in which the mRNA level is knocked down in the newly regenerating region. This regeneration-dependent conditional gene knockdown is called Readyknock (meaning ready-toknock down) (Takano et al., 2007). DjGAD immunoreactivity analysis revealed that this DjGAD-knockdown caused a reduction of DjGAD at least from day 7 to day 14 after the injection of DjGAD-dsRNA. As DjGAD-knockdown planarians, we used the animals regenerated from the tail piece at 7 days after amputation. DjGAD-immunoreactivity was clearly detected in the newly regenerated head region of control (vehicleinjected, amputated) planarians (Fig. 6A), while DjGADimmunoreactivity was almost lost in DjGAD-knockdown planarians (Fig. 6B). HPLC analysis revealed that the amount of GABA was significantly decreased in the newly regenerated head region in DjGAD-knockdown planarians
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Fig. 3. Distribution pattern of DjGAD-positive cells in planarians. Expression of DjPC2 mRNA, a pan-neural marker in planarians (A). Expression patterns of DjGAD mRNA (B–E) and protein (F–I). Ventral view of whole body (A, B, F) and head region (C, G, H), and transverse view of head region (D) and pharynx region (E, I). Different focal planes of the same field of view (G, H). DjGAD-expressing cells in the dorsal region are indicated by arrows (D). DjGAD-expressing cells in the ventral region are indicated by arrowheads (D). Scale bars⫽500 m (A, B, F), 100 m (C–E, G–I). The amount of GABA in each body piece analyzed by HPLC (J). Whole bodies (three planarians/HPLC sample) and body parts (i.e. the head, trunk and tail) (each part, nine pieces/HPLC sample). Each value is the mean⫾S.E.M. of three independent samples. Significance (Bonferroni/Dunn test post hoc comparisons after ANOVA): * P⬍0.05; *** P⬍0.001.
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Fig. 4. Process of regeneration of head and pharynx DjGAD-immunopositive cells. Planarians were amputated immediately posterior to the pharynx. The distribution of head DjGAD-immunopositive neurons was visualized at 1, 3, 5 and 7 days after amputation (A–D). The distribution of DjGAD-immunopositive cells in the pharynx was visualized at 5 and 7 days after amputation (E–G). Scale bars⫽100 m. The lower panel shows a schematic drawing of the regeneration process. Blue represents the brain. Magenta represents DjGAD-positive cells. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
compared with that in vehicle-control animals (Fig. 6C). These results clearly suggest that DjGAD is required for GABA biosynthesis in planarians. On the other hand, either the process, speed, or size of head regeneration was not markedly different between vehicle and DjGAD-knockdown planarians (Fig. 6A, B). In addition, a simple feeding assay revealed that DjGAD-knockdown planarians fed normally on chicken liver paste (data not shown). We further examined DjGAD-knockdown planarians using a phototaxis assay (Inoue et al., 2004). Negative phototactic behavior was clearly observed in control animals (Fig. 7B). However, DjGAD-knockdown planarians did not avoid the direction of the light source, and could not move to the dark area (Fig. 7C, D). The total distance of movement did not change in DjGAD-knockdown animals compared with that in vehicle controls (Fig. 7E).
By immunohistochemical analysis using antibodies against Djarrestin (a marker of photoreceptor neurons) and DjSYT (a pan-neural marker), no marked morphological difference of the neural circuit of photoreceptor cells (Fig. 7F, G) or the pan-neural structure (Fig. 7H, I) was detected between DjGAD-knockdown and vehicle-treated planarian brains.
DISCUSSION Distribution of GABAergic neurons in planarian brain Here, we have identified an orthologue of the GAD gene and determined the distribution pattern of its mRNA and protein in a lower invertebrate, the planarian Dugesia japonica. Immunofluorescence analysis revealed that DjGAD-immunopositive neurons were mainly distributed in the
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Fig. 5. Double-immunofluorescence of DjGAD (green) and DjTH (magenta). Dorsal (A, B) and ventral (C, D) regions of the head. (B, D) More highly magnified views of (A) and (C), respectively. Scale bars⫽100 m (A, C), 50 m (B, D). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
head region and the pharynx (Fig. 3F–I). In DjGAD-knockdown planarians, DjGAD-immunopositive neurons and the amount of GABA were significantly reduced (Fig. 6), sug-
gesting that DjGAD is required for GABA biosynthesis in planarians. Thus, DjGAD-immunopositive neurons in the planarian CNS are GABAergic neurons.
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Fig. 6. Immunofluorescence and the amount of GABA in DjGAD-knockdown planarians. Immunoreactivity of DjGAD-immunopositive neurons in heads regenerated from tail pieces that were injected with water (vehicle, A), or DjGAD-dsRNA (B). Scale bar⫽100 m. The amount of GABA in newly regenerated heads in DjGAD-knockdown planarians (C). Five head pieces/sample were analyzed by HPLC. Each value is the mean⫾S.E.M. of three independent samples. Significance (Student’s t-test): ** P⬍0.005 vs. vehicle.
DjGAD-immunopositive neurons consisted of only a small number of cells in both the dorsal and ventral sides of the brain (Fig. 3G, H). The dorsally localized DjTHimmunopositive neurons were also located in this region. However, we never detected doubly DjGAD- and DjTHimmunopositive neurons (Fig. 5). DjTH-immunopositive neural cell bodies and axons existed close to DjGAD-immunopositive neurons (Fig. 5), suggesting that GABAergic neurons may be functionally associated with dopaminergic neurons in the planarian CNS. GABAergic neural regeneration and function After amputation of planarians posterior to the pharynx, DjGAD-immunoreactivity could not be detected in the anterior region of the tail piece until the third day of regeneration (Fig. 4B). Then, newly generated DjGAD-immunopositive neurons were detected in the anterior region on the third day of regeneration (Fig. 4B). This is about the same time at which DjTH-immunopositive neurons are first detected during regeneration (Nishimura et al., 2007a). At this stage, netrin and netrin receptor homologues begin to be expressed in the head region (Cebrià et al., 2002b; Cebrià and Newmark, 2005). Cell adhesion molecules (DjCAMs) are also activated at this time in the head region (Fusaoka et al., 2006). The formation of the neural network of the brain at this stage and the initiation of the expression of neurotransmitter-related genes (DjGAD and DjTH) are likely to be functionally related. During the fifth to seventh days of regeneration, the formation of DjGAD-immunopositive axonal projections and the neural network pro-
gressed (Fig. 4C). On the seventh day of regeneration, the DjGAD-immunopositive neural network was almost completely reconstructed (Fig. 4D). Our finding that the head and pharynx were normally regenerated even in DjGADknockdown planarians implies that DjGAD may not influence planarian regeneration. In recent studies, several factors, including Pitx2 and Lhx7, that contribute to GABAergic neuronal differentiation have been identified in vertebrates (Westmoreland et al., 2001; Bachy and Rétaux, 2006). However, it is unknown whether these factors contribute to GABAergic neural differentiation and regeneration in planarians. Usually, planarians display a distinctive light avoidance behavior known as negative phototaxis. Planarian locomotion is regulated by both the photosensing system and motor system (Inoue et al., 2004; Nishimura et al., 2007a). Regarding the time-course of regeneration of phototactic behavior, although most 4-day regenerants could not recognize light and moved in random directions, most 5-day regenerants moved to the dark side, indicating that the functional connection between the photosensing system and motor system may be reestablished during the fifth day after amputation (Inoue et al., 2004). In our planarian gene-knockdown study, we amputated the body of planarians into which dsRNA had been microinjected 1 day earlier, and then examined the regenerants. Basically, the head and brain seemed to be normally regenerated and developed even in DjGAD-knockdown planarians. Therefore, we further examined the regenerants using the phototaxis assay. DjGAD-knockdown planarians showed ab-
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normal negative phototactic behavior (Fig. 7C). In brief, they could not avoid the light area. However, the total distance of movement did not change in DjGAD-knockdown planarians (Fig. 7E). The neural circuit of photoreceptor cells (photosensing system) was observed to be located extremely close to the GABAergic neural network in the dorsal side of the brain (Fig. 7F). In the DjGADknockdown planarian brain, although almost all DjGADimmunoreactivity was lost, the photosensing system and the brain structure were not morphologically changed (Fig. 7G, I). Recently, we have found that: i) the knockdown of DjDSCAM (a planarian homolog of Down syndrome cell adhesion molecule) causes disorganization of the brain neural network and severe defects in locomotion activity, but normal recognition of light stimulation (Fusaoka et al., 2006); ii) planarians with knockdown of DjSNAP-25 (a planarian homolog of synaptosome associated protein 25kDa) lose both locomotion activity and negative phototaxis, but have normal brain structure (Takano et al., 2007); and iii) DjTH-knockdown planarians show normal brain structure, negative phototaxis and normal locomotion activity (cilia-mediated movement), but show a lack of musclemediated movement (Nishimura et al., 2007a). In the present study, DjGAD-immunopositive neurons were seen to be located extremely close to the neural circuit of photoreceptor cells (photosensing system) and dopaminergic tiara (DA neural network) in the normal planarian brain (Figs. 5, 7F). Based on these observations, we speculate that planarian GABAergic neurons may play key role(s) in light information-processing, recognition, and/or the choice of the direction for negative phototaxis, after the reception of light signals by the photosensing system. Subsequently, GABAergic stimuli may output to or modulate the motor system coordinating the cilia-mediated movement and dopaminergic muscle-mediated movement. We also speculate that DjDSCAM and DjSNAP-25 may promote the formation and maintenance of functional synapses between the photosensing system and their target neurons, including GABAergic and dopaminergic neurons. Thus, GABAergic neurons may mediate between the photosensing system and the motor system, but the detailed regulatory mechanisms remain to be determined in future studies. GABAergic cells in planarian pharynx
Fig. 7. Phototaxis assay and immunofluorescence in DjGAD-knockdown planarians. (A) A schematic drawing of the container compartment in the phototaxis assay. The circle indicates the start area. Distribution of 90-s trajectories representing the movement of vehicle (B) and DjGAD-knockdown (C) animals. (D) The time spent in the target quadrant is given as mean⫾S.E.M. of 10 independent samples. Significance (Student’s t-test): *** P⬍0.001 vs. vehicle. (E) The total distance of movement. Each value is the mean⫾S.E.M. of 10 independent samples. Significance (Student’s t-test): Not significantly different. Immunofluorescence of vehicle (F) and DjGAD-knockdown (G) animals using antibodies against DjGAD (green) and Djarrestin (magenta). Immunofluorescence of vehicle (H) and DjGAD-knockdown (I) animals using anti-DjSYT (synaptotagmin) antibody. No morphological
In previous studies, we did not detect any immunoreactivity for DjTH or DjTPH (dopaminergic and serotonergic markers, respectively) on the pharynx tip (Nishimura et al., 2007a,b). In the present study, DjGAD-immunoreactivity was detected around the pharynx and its tip and the level of GABA was high in the trunk piece (Fig. 3). Although pharynx tip cells that expressed both DjGAD mRNA and protein did not have morphologically typical neural shapes, DjPC2 mRNA (Fig. 3A) and DjSYT immunoreactivity (data
changes were detected in either the brain or photoreceptor neurons in DjGAD-knockdown planarians compared with vehicle-treated planarians. Scale bars⫽100 m (G), 200 m (I). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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not shown) were also detected around the pharynx tip. Therefore, pharynx GABAergic cells may be neuron-like cells. Similar to the regeneration of the head, brain and photosensing system, the pharynx was normally regenerated and developed in DjGAD-knockdown planarians. In addition, since DjGAD-knockdown planarians fed normally on chicken liver paste, whether GABAergic function is important in the pharynx is unknown.
CONCLUSION A full-length GAD gene (DjGAD) was newly identified in planarians. Moreover, we clearly showed that DjGAD was expressed in the planarian CNS by both whole-mount in situ hybridization and immunofluorescence. Since planarians have a relatively well-organized CNS, the GABAergic system of the planarian CNS can be considered a prototype of the GABAergic system. Research on the GABAergic nervous system of planarians should yield many insights into primitive GABAergic functions in the animal kingdom. Acknowledgments—We thank Elizabeth Nakajima for critical reading of the manuscript. We also thank Eicom Corporation (Kyoto, Japan) for support with the HPLC analysis, and Eisai Co. Ltd. (Tsukuba, Japan) and KAN Research Institute Inc. (Kobe, Japan) for support with the generation of a mouse monoclonal anti-DjGAD antibody. This study was supported in part by the 21st Century Center of Excellence (COE) Program (Y.K. and T.T.), Frontier Research Program (Y.K. and T.T.), in part by the Global COE Program (K.A.), Grants-in-Aid for Creative Research (K.A.) and Scientific Research on Priority Areas (K.A.), and Grants-inAid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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(Accepted 10 March 2008) (Available online 22 March 2008)
IDENTIFICATION OF GLUTAMIC ACID DECARBOXYLASE GENE AND DISTRIBUTION OF GABAergic NERVOUS SYSTEM IN THE PLANARIAN DUGESIA JAPONICA K. NISHIMURA,a Y. KITAMURA,a1* Y. UMESONO,b K. TAKEUCHI,c K. TAKATA,a T. TANIGUCHIa AND K. AGATAb1*
is a useful marker for GABAergic neurons. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: planarian, GABA, neural network, regeneration, RNA interference.
a
Department of Neurobiology, and 21st Century Center of Excellence Program, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan
b
Department of Biophysics, Graduate School of Science, Kyoto University, Oiwake, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
Recent studies of the freshwater planarian Dugesia japonica have provided many unique insights into the mechanism of regeneration of the nervous system. Planarians have a relatively well-organized and functional brain, and can regenerate the brain after injury from nearly any portion of their bodies by utilizing somatic pluripotent stem cells (Agata and Watanabe, 1999; Agata, 2003). The planarian CNS consists of the brain in the anterior region and a pair of ventral nerve cords (VNCs). Its brain forms an inverted U-shaped structure with lateral branches (Agata et al., 1998; Tazaki et al., 1999; Okamoto et al., 2005). Previous studies clearly indicated that the planarian brain is reconstructed during regeneration by similar molecular mechanisms to those operating in mammalian brain development. Fibroblast growth factor receptor–mediated signaling is indispensable for brain regeneration (Ogawa et al., 2002a; Cebrià et al., 2002a). A noggin-like gene is expressed in the future brain-forming region (Ogawa et al., 2002b). Brain patterning along the anterior–posterior axis and the medial–lateral axis is regulated by Wnt-family genes and Otx/otd-related genes, respectively (Umesono et al., 1997, 1999; Kobayashi et al., 2007). Moreover, several crucial genes involved in functional brain regeneration have been identified from planarians using the RNA interference (RNAi) technique (Inoue et al., 2004, 2007; Takano et al., 2007). Detailed neural networks and distinct domains in the planarian brain have been clearly visualized by using fluorescence dye-tracing and molecular marker staining (Sakai et al., 2000; Mineta et al., 2003; Nakazawa et al., 2003; Okamoto et al., 2005; Fusaoka et al., 2006). In a previous study, we identified a gene encoding planarian tyrosine hydroxylase (DjTH), the rate-limiting enzyme for dopamine (DA) biosynthesis, and produced DjTH-knockdown planarians by the RNAi method, and using them demonstrated that DA has an important role in muscle-mediated movement in planarians (Nishimura et al., 2007a). We also found by immunostaining with a monoclonal anti-DjTH antibody that dopaminergic neurons are distributed in a regular manner in the main lobes of the planarian brain. In a similar way, we also isolated a gene for tryptophan hydroxylase, the rate-limiting enzyme of 5-HT biosynthesis, and showed that the protein it encodes is mainly distributed at VNCs (Nishimura et al., 2007b).
c
Department of Anatomy and Developmental Biology, Kyoto Prefectual University of Medicine, Kawaramachi, Kamigyo-ku, Kyoto 6028566, Japan
Abstract—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 1
These contributors both served as senior authors. *Corresponding authors: Tel: ⫹81-75-595-4706; fax: ⫹81-75-5954796 (Y. Kitamura); Tel: ⫹81-75-753-4200; fax: ⫹81-75-753-4203 (K. Agata). E-mail address: [email protected] (Y. Kitamura), agata@ mdb.biophys.kyoto-u.ac.jp (K. Agata). Abbreviations: ANOVA, analysis of variance; cDNA, complementary DNA; DA, dopamine; DIG, digoxigenin; DjGAD, D. japonica glutamic acid decarboxylase; DjPC2, D. japonica pro-hormone convertase 2 homologue; dsRNA, double-stranded RNA; ECD, electrochemical detection; EDTA, ethylenediaminetetraacetic acid; GAD, glutamic acid decarboxylase; GSP, gene-specific primer; PCR, polymerase chain reaction; RNAi, RNA interference; VNC, ventral nerve cord; 5=RACE, 5=rapid amplification of cDNA ends.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.03.026
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Glutamic acid decarboxylase (GAD; EC4.1.1.15) is the rate-limiting enzyme for GABA biosynthesis, and converts glutamic acid into GABA (Fig. 1A). Two independent GAD
genes (i.e. GAD65 and GAD67) have been identified in vertebrates (Erlander et al., 1991; Bu et al., 1992). These two genes show strong conservation of the sequence in
Fig. 1. DjGAD encodes a glutamic acid decarboxylase. Pathway of GABA biosynthesis (A). Nucleotide and predicted amino acid sequence of DjGAD cDNA (B). The arrows labeled “GSP” show the primers for 5=RACE. The arrows labeled “GAD” show the degenerate PCR primers and specific primers used for the stepwise dilution cloning method. Asterisks indicate stop codons.
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their C-terminal region (Fenalti et al., 2007). Only a single GAD gene was identified in invertebrates such as the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster (Jackson et al., 1990; McIntire et al., 1993). The amino acid GABA is a major inhibitory neurotransmitter which exists widely in both vertebrates and invertebrates (Jackson et al., 1990; McIntire et al., 1993; Tillakaratne et al., 1995; Martyniuk et al., 2007). Several reports have already demonstrated that several kinds of neurotransmitters are detected in planarians (Ness et al., 1996; Itoh and Igarashi, 2000; Rawls et al., 2006). However, the GAD gene has not hitherto been identified in planarians. In addition, the detailed distribution of GABAsynthetic neurons (GABAergic neurons) and the process of regeneration of the GABAergic neural network are still not known in planarians. Here we focused on GABAergic neurons, and identified the planarian GAD gene (DjGAD, D. japonica glutamic acid decarboxylase), showed its distribution patterns in both intact and regenerating planarians using an anti-DjGAD antibody, and demonstrated that DjGAD is clearly required for GABA biosynthesis in planarians.
EXPERIMENTAL PROCEDURES Animals Planarians (Dugesia japonica) of SSP strain were cultured in autoclaved tap water at 24 °C in subdued light and fed chicken liver paste once a week. Planarians 5–7 mm in length were used in the present study. Animals were starved for at least 7 days before experiments. All procedures conformed to the guidelines of local and the National Institutes of Health for Laboratory Animal Science. Every effort was made to minimize the number of animals used and their suffering.
RNA extraction and complementary DNA (cDNA) preparation Total RNA was extracted from 300 head pieces of planarians using Isogene-LS (Nippon Gene, Toyama, Japan). mRNA was purified from total RNA using an Oligotex-dT30 ⬍super⬎mRNA purification kit (Takara, Kyoto, Japan). cDNA was synthesized from mRNA using a first-strand cDNA synthesis kit (Amersham Biosciences, Arlington Heights, IL, USA).
Cloning of DjGAD cDNA Degenerate oligonucleotides were designed for regions conserved in GAD among various animals, namely, Caenorhabditis elegans UNC25, Drosophila melanogaster GAD, Mus musculus GAD-65/67 and Homo sapiens GAD-65/67. These primers were as follows: GAD F1 5=-GTNAARYTNGTNAARTGYAAYGAR-3= GAD F2 5=-AARTGYAAYGARMGNGGNAARATG-3= GAD R1 5=-CATNGCYTTRTGNGGRTTCC-3= GAD R2 5=-NARNGTNACNGCCATNGCYTTATG-3= (N⫽A/T/G/C, Y⫽C/T, R⫽A/G, W⫽T/A, S⫽C/G, M⫽A/C) The polymerase chain reaction (PCR) was carried out using primers GAD F1 and GAD R1 sequentially with cycling consisting of an initial denaturation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 52 °C for 1 min, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. Subsequently, nested PCR was carried out using primers GAD F2 and GAD R2. Finally, a 100-bp cDNA fragment was isolated. We then carried out the screening of a cDNA library constructed from poly(A)⫹ RNA of planarian head pieces in ZAPII vector (Stratagene, La Jolla, CA,
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USA) by the stepwise dilution method (Watanabe et al., 1997) using a specific primer set designed from the 100-bp cDNA fragment. These primers were as follows: GAD F3 5=-AAGAGGAAAAATGGATACTGAAAACC-3= GAD R3 5=-ATTTTCAATTCCATTCAGTAAATAGCG-3=. The cycling consisted of an initial denaturation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. To determine the 5=-end sequence of DjGAD, 5=rapid amplification of cDNA ends (5=RACE) was carried out using a SMART RACE cDNA amplification kit (Clontech, Palo Alto, CA, USA). The genespecific primer (GSP) sequences were as follow: GSP R1 5=-GGGCCATTGCTTGTTTAAGTTGGTGAGGATGG-3= GSP R2 5=-GGCCTTGTTTGTCAAGTGAATAGC-3=. The cycling consisted of an initial denaturation at 94 °C for 10 min, followed by 40 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. Subsequently, nested PCR was carried out using GSP R2. Finally, a 1984-bp cDNA fragment was isolated.
RNA probe synthesis and whole-mount in situ hybridization The plasmid pBluescript SK (⫺) containing DjGAD cDNA was linearized with BamHI. Digoxigenin (DIG) -antisense RNA probe was synthesized using T7 RNA polymerase. Planarians were treated with 2% HCl in 5/8 Holtfreter’s solution for 5 min at 4 °C and were fixed in 4% paraformaldehyde containing 5% methanol for 3 h at 4 °C. Hybridization was carried out using DIG-labeled complementary RNA probe as previously described (Umesono et al., 1997; Agata et al., 1998).
Generation of a mouse monoclonal anti-DjGAD antibody and whole-mount immunofluorescence A mouse monoclonal anti-DjGAD antibody was generated against a fusion protein produced in E. coli XL10-GOLD strain (Stratagene). A fragment encoding amino acid residues 31–125 of DjGAD was inserted into pQE30 (Qiagen, Chatsworth, CA, USA). A fusion protein with a histidine tag was induced by isopropyl-D-thiogalactopyranoside and purified using Ni-NTA agarose (Qiagen). For further purification, the gel slice containing the target protein was cut out after SDS-PAGE, and the purified protein was isolated by electroelution. Antibody generation was performed by Kohjin-Bio (Saitama, Japan). Planarians were treated with 2% HCl in 5/8 Holtfreter’s solution for 5 min at 4 °C and fixed in Carnoy’s solution (ethanol: chloroform: acetic acid anhydrate in proportions 6:3:1, respectively) for 2 h at 4 °C. Immunofluorescence analysis was performed as previously described (Nishimura et al., 2007a). Primary antibodies used in this study were: mouse monoclonal antibodies against DjGAD and DjSYT (synaptotagmin: Tazaki et al., 1999) to visualize GABAergic neurons and pan-neural cells, respectively, and rabbit polyclonal antibody against DjTH (Nishimura et al., 2007a) and Djarrestin (Sakai et al., 2000) to visualize dopaminergic neurons and photoreceptor neurons, respectively.
Double-stranded RNA (dsRNA) synthesis and microinjection The plasmid pBluescript SK (⫺) containing the longest DjGAD cDNA was linearized with KpnI and BamHI. Sense or antisense RNAs were synthesized with T3 or T7 RNA polymerase, respectively. After incubation with RQI DNase for 1 h at 37 °C, the samples were denatured for 20 min at 65 °C and annealed for 40 min at 37 °C (Sánchez Alvarado and Newmark 1999). Intact planarians were injected three times (32 nL/injection) with dsRNA (once per day for 3 days) using a Drummond Scientific Nanoject injector (Broomall, PA, USA). Control
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animals were injected with water instead of dsRNA. At 1 day after the last injection, planarians were amputated immediately posterior to the pharynx and 7-day-regenerated animals from the tail piece were used in these experiments.
analysis of variance (ANOVA). Further statistical analysis for post hoc comparisons was performed using the Bonferroni/Dunn test (StatView, Abacus Concepts, Berkeley, CA, USA).
Phototaxis assay HPLC analysis of GABA Quantification of GABA was performed using HPLC with precolumn o-phthaldialdehyde-mercaptoethanol derivatization (OPAME) and electrochemical detection (ECD). Samples were homogenized by sonication in 100 L of 0.1 M perchloric acid containing 100 M EDTA. After centrifugation at 15,000 r.p.m. for 15 min at 4 °C, the supernatant was filtered through a 0.22-m pore membrane. Ten microliters of the filtrate was added to 30 L of condensation buffer containing: 0.1 M K2CO3, 2% ethanol, 4 mM OPA and 0.04% ME (pH⫽9.5). After incubation of the mixture for 150 s, samples were injected onto an SC-5ODS column (3.0⫻150 mm; Eicom Corporation, Kyoto, Japan), and detected using an electrochemical detector (HTEC-500; Eicom Corporation). The working electrode potential was maintained at ⫹600 mV against Ag/AgCl. A CA-ODS column (3.0⫻4 mm; Eicom Corporation) was placed between the injector and the analytical column. The temperature was constant at 30 °C. The mobile phase (pH⫽2.8) was composed of 50 mM phosphate buffer, 50% methanol and 5 mg/L EDTA, and was delivered at a 0.5 mL/min flow rate. The retention time of GABA was approximately 14.3 min.
Statistical evaluation The amount of GABA is given as mean⫾standard error of the mean (S.E.M.). The significance of differences was determined by
The negative phototaxis assay was carried out as previously described (Inoue et al., 2004). Ten individual planarians were used in this assay. The time spent in the target quadrant and the total distance of movement were given as mean⫾S.E.M. Statistical analysis was performed using the Student’s t-test (StatView).
RESULTS Molecular cloning of DjGAD cDNA A partial planarian GAD cDNA was obtained by a reversetranscription PCR strategy with degenerate primers designed based on the amino acid sequence highly conserved in several animals. Subsequently, a full-length sequence was obtained by head cDNA library screening and 5=RACE (see Experimental Procedures). This cDNA sequence had a 1749-bp coding region, and was predicted to encode a protein of 583 amino acid residues (Fig. 1B). This predicted amino acid sequence was similar to the sequence of GAD of other species. This sequence also contained the pyridoxal-phosphate binding domain that is a functional domain of GAD (Fig. 2A). Therefore, this gene was named DjGAD. The full-length cDNA sequence of
Fig. 2. Multiple alignment of DjGAD sequences. Comparison of amino acid sequence of DjGAD with GAD of C. elegans, D. melanogaster and H. sapiens. The box encloses the core region of the pyridoxal-phosphate binding domain, a functional domain of GAD. Conserved amino acids are indicated by dots. Gaps are indicated by hyphens. The accession numbers of the aligned sequence are as follow: C. elegans UNC-25 (AF109378), D. melanogaster-GAD (NM_079190), H. sapiens-GAD65 (NM_000818) and H. sapiens-GAD67 (NM_000817).
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DjGAD has been deposited in DDBJ/EMBL/GenBank under accession number AB332029. Expression pattern of DjGAD mRNA and protein Expression analysis of DjGAD was carried out in intact animals by whole mount in situ hybridization with a DIGlabeled antisense RNA probe (Fig. 3B–E). DjGAD expression was detected in very few cells compared with the number of pan-neural cells that were stained with DjPC2 (D. japonica pro-hormone convertase 2 homologue) -probe (Fig. 3A) (Agata et al., 1998). In the head region, DjGAD-expressing cells were distributed in a dotted pattern in the brain; some of them were aligned in an inverted U-shape, and the others were distributed in the medial part of the brain (Fig. 3C). In the transverse view of the head region, DjGAD-expressing cells were detected in both the dorsal and ventral regions of the brain (Fig. 3D). DjGAD-expressing cells were detected around the pharynx and at its tip (Fig. 3E). In order to visualize the distribution of neural cell bodies and the axonal projections of DjGAD-positive cells, we generated a mouse monoclonal anti-DjGAD antibody. An axonal network and neural morphology were clearly detected by immunofluorescence analysis using anti-DjGAD antibody. The distribution pattern of DjGAD-immunopositive cells was very similar to that of DjGAD-mRNA stained by in situ hybridization (Fig. 3B, F). DjGAD-immunopositive neurons could be classified into three types according to cell morphology and distribution. Two types of DjGADimmunopositive neurons were located in the dorsal and ventral sides of the brain, respectively (Fig. 3G, H). DjGAD-immunopositive neuronal cell bodies and axons were connected with each other in the ventral part of the brain (Fig. 3G, H). The third type of DjGAD-immunopositive cells was detected around the pharynx and at its tip (Fig. 3I). We then measured the amount of GABA by HPLC-ECD in the whole body and in separate body pieces (i.e. the head, trunk, and tail) that were amputated anterior and posterior to the pharynx (Fig. 3J). The amount of GABA in the whole body was 8.0⫾1.0 pmol/mg wet weight of planarian body. The amounts of GABA in the head, trunk, and tail region were 4.4⫾0.4, 11.3⫾1.0, and 1.8⫾0.5 pmol/mg wet weight, respectively. Thus, the amount of GABA in the trunk region was higher than the amounts in both the head and tail regions. In addition, the amount of GABA in the head region was higher than the amount of GABA in the tail region. Process of regeneration of DjGAD-immunopositive cells Planarians can regenerate complete organs after the amputation of various body pieces. The planarian brain can be completely regenerated from even a body piece with no brain tissue within 7 days (Cebrià et al., 2002b). We examined the process of regeneration of head DjGAD-immunopositive neurons from the tail piece that was amputated posterior to the pharynx. Immunoreactivity of DjGAD was undetectable on the first day after amputation (Fig. 4A). On the third day, several new DjGAD-immunopositive neurons
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became detectable in the anterior region (Fig. 4B). On the fifth day, the number of new DjGAD-immunopositive neurons was increased compared with that on the third day of regeneration, and these neurons could be seen to possess axons (Fig. 4C). On the seventh day, the number of new DjGAD-immunopositive neurons was increased and the neural network was reconstructed and expanded, in comparison with those on the fifth day of regeneration (Fig. 4D). In addition, DjGAD-immunoreactivity was also observed at the tip of the pharynx. We further examined the process of regeneration of DjGAD-immunopositive cells at the tip of the pharynx in animals regenerated from the tail piece. Immunoreactivity of DjGAD was undetectable on the first and third day after amputation (Fig. 4E). Interestingly, the pharynx was newly regenerated from the residual tail piece on the second day (Kobayashi et al., 1999). Several DjGAD-immunopositive cells became newly detectable in the tip of the pharynx (Fig. 4F) on the fifth day, and then DjGAD immunoreactive intensity was increased on the seventh day (Fig. 4G). Comparison of DjGAD-immunopositive neurons and DjTH-immunopositive neurons in the planarian CNS Previously, we characterized the DjTH gene, which is required for DA biosynthesis, and determined the distribution of DjTH by immunofluorescence analysis (Nishimura et al., 2007a). In order to compare the distribution patterns of GABAergic neurons and dopaminergic neurons, we performed double-immunofluorescence analysis using antiDjGAD antibody and anti-DjTH antibody. DjGAD-immunopositive neurons and DjTH-immunopositive neurons were present in distinct locations in the dorsal region of the head (Fig. 5A, B). In addition, DjGAD-immunopositive neurons were located in the middle region between lateral arrays of ventrally localized DjTH-immunopositive neurons (Fig. 5C, D). DjGAD-knockdown planarians GAD is the rate-limiting enzyme for GABA biosynthesis in both vertebrates and invertebrates. To examine whether DjGAD is required for GABA biosynthesis, the amount of GABA was measured in DjGAD-knockdown planarians created by RNAi. We used an RNAi method in which the mRNA level is knocked down in the newly regenerating region. This regeneration-dependent conditional gene knockdown is called Readyknock (meaning ready-toknock down) (Takano et al., 2007). DjGAD immunoreactivity analysis revealed that this DjGAD-knockdown caused a reduction of DjGAD at least from day 7 to day 14 after the injection of DjGAD-dsRNA. As DjGAD-knockdown planarians, we used the animals regenerated from the tail piece at 7 days after amputation. DjGAD-immunoreactivity was clearly detected in the newly regenerated head region of control (vehicleinjected, amputated) planarians (Fig. 6A), while DjGADimmunoreactivity was almost lost in DjGAD-knockdown planarians (Fig. 6B). HPLC analysis revealed that the amount of GABA was significantly decreased in the newly regenerated head region in DjGAD-knockdown planarians
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Fig. 3. Distribution pattern of DjGAD-positive cells in planarians. Expression of DjPC2 mRNA, a pan-neural marker in planarians (A). Expression patterns of DjGAD mRNA (B–E) and protein (F–I). Ventral view of whole body (A, B, F) and head region (C, G, H), and transverse view of head region (D) and pharynx region (E, I). Different focal planes of the same field of view (G, H). DjGAD-expressing cells in the dorsal region are indicated by arrows (D). DjGAD-expressing cells in the ventral region are indicated by arrowheads (D). Scale bars⫽500 m (A, B, F), 100 m (C–E, G–I). The amount of GABA in each body piece analyzed by HPLC (J). Whole bodies (three planarians/HPLC sample) and body parts (i.e. the head, trunk and tail) (each part, nine pieces/HPLC sample). Each value is the mean⫾S.E.M. of three independent samples. Significance (Bonferroni/Dunn test post hoc comparisons after ANOVA): * P⬍0.05; *** P⬍0.001.
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Fig. 4. Process of regeneration of head and pharynx DjGAD-immunopositive cells. Planarians were amputated immediately posterior to the pharynx. The distribution of head DjGAD-immunopositive neurons was visualized at 1, 3, 5 and 7 days after amputation (A–D). The distribution of DjGAD-immunopositive cells in the pharynx was visualized at 5 and 7 days after amputation (E–G). Scale bars⫽100 m. The lower panel shows a schematic drawing of the regeneration process. Blue represents the brain. Magenta represents DjGAD-positive cells. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
compared with that in vehicle-control animals (Fig. 6C). These results clearly suggest that DjGAD is required for GABA biosynthesis in planarians. On the other hand, either the process, speed, or size of head regeneration was not markedly different between vehicle and DjGAD-knockdown planarians (Fig. 6A, B). In addition, a simple feeding assay revealed that DjGAD-knockdown planarians fed normally on chicken liver paste (data not shown). We further examined DjGAD-knockdown planarians using a phototaxis assay (Inoue et al., 2004). Negative phototactic behavior was clearly observed in control animals (Fig. 7B). However, DjGAD-knockdown planarians did not avoid the direction of the light source, and could not move to the dark area (Fig. 7C, D). The total distance of movement did not change in DjGAD-knockdown animals compared with that in vehicle controls (Fig. 7E).
By immunohistochemical analysis using antibodies against Djarrestin (a marker of photoreceptor neurons) and DjSYT (a pan-neural marker), no marked morphological difference of the neural circuit of photoreceptor cells (Fig. 7F, G) or the pan-neural structure (Fig. 7H, I) was detected between DjGAD-knockdown and vehicle-treated planarian brains.
DISCUSSION Distribution of GABAergic neurons in planarian brain Here, we have identified an orthologue of the GAD gene and determined the distribution pattern of its mRNA and protein in a lower invertebrate, the planarian Dugesia japonica. Immunofluorescence analysis revealed that DjGAD-immunopositive neurons were mainly distributed in the
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Fig. 5. Double-immunofluorescence of DjGAD (green) and DjTH (magenta). Dorsal (A, B) and ventral (C, D) regions of the head. (B, D) More highly magnified views of (A) and (C), respectively. Scale bars⫽100 m (A, C), 50 m (B, D). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
head region and the pharynx (Fig. 3F–I). In DjGAD-knockdown planarians, DjGAD-immunopositive neurons and the amount of GABA were significantly reduced (Fig. 6), sug-
gesting that DjGAD is required for GABA biosynthesis in planarians. Thus, DjGAD-immunopositive neurons in the planarian CNS are GABAergic neurons.
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Fig. 6. Immunofluorescence and the amount of GABA in DjGAD-knockdown planarians. Immunoreactivity of DjGAD-immunopositive neurons in heads regenerated from tail pieces that were injected with water (vehicle, A), or DjGAD-dsRNA (B). Scale bar⫽100 m. The amount of GABA in newly regenerated heads in DjGAD-knockdown planarians (C). Five head pieces/sample were analyzed by HPLC. Each value is the mean⫾S.E.M. of three independent samples. Significance (Student’s t-test): ** P⬍0.005 vs. vehicle.
DjGAD-immunopositive neurons consisted of only a small number of cells in both the dorsal and ventral sides of the brain (Fig. 3G, H). The dorsally localized DjTHimmunopositive neurons were also located in this region. However, we never detected doubly DjGAD- and DjTHimmunopositive neurons (Fig. 5). DjTH-immunopositive neural cell bodies and axons existed close to DjGAD-immunopositive neurons (Fig. 5), suggesting that GABAergic neurons may be functionally associated with dopaminergic neurons in the planarian CNS. GABAergic neural regeneration and function After amputation of planarians posterior to the pharynx, DjGAD-immunoreactivity could not be detected in the anterior region of the tail piece until the third day of regeneration (Fig. 4B). Then, newly generated DjGAD-immunopositive neurons were detected in the anterior region on the third day of regeneration (Fig. 4B). This is about the same time at which DjTH-immunopositive neurons are first detected during regeneration (Nishimura et al., 2007a). At this stage, netrin and netrin receptor homologues begin to be expressed in the head region (Cebrià et al., 2002b; Cebrià and Newmark, 2005). Cell adhesion molecules (DjCAMs) are also activated at this time in the head region (Fusaoka et al., 2006). The formation of the neural network of the brain at this stage and the initiation of the expression of neurotransmitter-related genes (DjGAD and DjTH) are likely to be functionally related. During the fifth to seventh days of regeneration, the formation of DjGAD-immunopositive axonal projections and the neural network pro-
gressed (Fig. 4C). On the seventh day of regeneration, the DjGAD-immunopositive neural network was almost completely reconstructed (Fig. 4D). Our finding that the head and pharynx were normally regenerated even in DjGADknockdown planarians implies that DjGAD may not influence planarian regeneration. In recent studies, several factors, including Pitx2 and Lhx7, that contribute to GABAergic neuronal differentiation have been identified in vertebrates (Westmoreland et al., 2001; Bachy and Rétaux, 2006). However, it is unknown whether these factors contribute to GABAergic neural differentiation and regeneration in planarians. Usually, planarians display a distinctive light avoidance behavior known as negative phototaxis. Planarian locomotion is regulated by both the photosensing system and motor system (Inoue et al., 2004; Nishimura et al., 2007a). Regarding the time-course of regeneration of phototactic behavior, although most 4-day regenerants could not recognize light and moved in random directions, most 5-day regenerants moved to the dark side, indicating that the functional connection between the photosensing system and motor system may be reestablished during the fifth day after amputation (Inoue et al., 2004). In our planarian gene-knockdown study, we amputated the body of planarians into which dsRNA had been microinjected 1 day earlier, and then examined the regenerants. Basically, the head and brain seemed to be normally regenerated and developed even in DjGAD-knockdown planarians. Therefore, we further examined the regenerants using the phototaxis assay. DjGAD-knockdown planarians showed ab-
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normal negative phototactic behavior (Fig. 7C). In brief, they could not avoid the light area. However, the total distance of movement did not change in DjGAD-knockdown planarians (Fig. 7E). The neural circuit of photoreceptor cells (photosensing system) was observed to be located extremely close to the GABAergic neural network in the dorsal side of the brain (Fig. 7F). In the DjGADknockdown planarian brain, although almost all DjGADimmunoreactivity was lost, the photosensing system and the brain structure were not morphologically changed (Fig. 7G, I). Recently, we have found that: i) the knockdown of DjDSCAM (a planarian homolog of Down syndrome cell adhesion molecule) causes disorganization of the brain neural network and severe defects in locomotion activity, but normal recognition of light stimulation (Fusaoka et al., 2006); ii) planarians with knockdown of DjSNAP-25 (a planarian homolog of synaptosome associated protein 25kDa) lose both locomotion activity and negative phototaxis, but have normal brain structure (Takano et al., 2007); and iii) DjTH-knockdown planarians show normal brain structure, negative phototaxis and normal locomotion activity (cilia-mediated movement), but show a lack of musclemediated movement (Nishimura et al., 2007a). In the present study, DjGAD-immunopositive neurons were seen to be located extremely close to the neural circuit of photoreceptor cells (photosensing system) and dopaminergic tiara (DA neural network) in the normal planarian brain (Figs. 5, 7F). Based on these observations, we speculate that planarian GABAergic neurons may play key role(s) in light information-processing, recognition, and/or the choice of the direction for negative phototaxis, after the reception of light signals by the photosensing system. Subsequently, GABAergic stimuli may output to or modulate the motor system coordinating the cilia-mediated movement and dopaminergic muscle-mediated movement. We also speculate that DjDSCAM and DjSNAP-25 may promote the formation and maintenance of functional synapses between the photosensing system and their target neurons, including GABAergic and dopaminergic neurons. Thus, GABAergic neurons may mediate between the photosensing system and the motor system, but the detailed regulatory mechanisms remain to be determined in future studies. GABAergic cells in planarian pharynx
Fig. 7. Phototaxis assay and immunofluorescence in DjGAD-knockdown planarians. (A) A schematic drawing of the container compartment in the phototaxis assay. The circle indicates the start area. Distribution of 90-s trajectories representing the movement of vehicle (B) and DjGAD-knockdown (C) animals. (D) The time spent in the target quadrant is given as mean⫾S.E.M. of 10 independent samples. Significance (Student’s t-test): *** P⬍0.001 vs. vehicle. (E) The total distance of movement. Each value is the mean⫾S.E.M. of 10 independent samples. Significance (Student’s t-test): Not significantly different. Immunofluorescence of vehicle (F) and DjGAD-knockdown (G) animals using antibodies against DjGAD (green) and Djarrestin (magenta). Immunofluorescence of vehicle (H) and DjGAD-knockdown (I) animals using anti-DjSYT (synaptotagmin) antibody. No morphological
In previous studies, we did not detect any immunoreactivity for DjTH or DjTPH (dopaminergic and serotonergic markers, respectively) on the pharynx tip (Nishimura et al., 2007a,b). In the present study, DjGAD-immunoreactivity was detected around the pharynx and its tip and the level of GABA was high in the trunk piece (Fig. 3). Although pharynx tip cells that expressed both DjGAD mRNA and protein did not have morphologically typical neural shapes, DjPC2 mRNA (Fig. 3A) and DjSYT immunoreactivity (data
changes were detected in either the brain or photoreceptor neurons in DjGAD-knockdown planarians compared with vehicle-treated planarians. Scale bars⫽100 m (G), 200 m (I). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.
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not shown) were also detected around the pharynx tip. Therefore, pharynx GABAergic cells may be neuron-like cells. Similar to the regeneration of the head, brain and photosensing system, the pharynx was normally regenerated and developed in DjGAD-knockdown planarians. In addition, since DjGAD-knockdown planarians fed normally on chicken liver paste, whether GABAergic function is important in the pharynx is unknown.
CONCLUSION A full-length GAD gene (DjGAD) was newly identified in planarians. Moreover, we clearly showed that DjGAD was expressed in the planarian CNS by both whole-mount in situ hybridization and immunofluorescence. Since planarians have a relatively well-organized CNS, the GABAergic system of the planarian CNS can be considered a prototype of the GABAergic system. Research on the GABAergic nervous system of planarians should yield many insights into primitive GABAergic functions in the animal kingdom. Acknowledgments—We thank Elizabeth Nakajima for critical reading of the manuscript. We also thank Eicom Corporation (Kyoto, Japan) for support with the HPLC analysis, and Eisai Co. Ltd. (Tsukuba, Japan) and KAN Research Institute Inc. (Kobe, Japan) for support with the generation of a mouse monoclonal anti-DjGAD antibody. This study was supported in part by the 21st Century Center of Excellence (COE) Program (Y.K. and T.T.), Frontier Research Program (Y.K. and T.T.), in part by the Global COE Program (K.A.), Grants-in-Aid for Creative Research (K.A.) and Scientific Research on Priority Areas (K.A.), and Grants-inAid from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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(Accepted 10 March 2008) (Available online 22 March 2008)