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Expression of Asn-d-Trp-Phe-NH2 in the brain of the terrestrial slug Limax valentianus
Comparative Biochemistry and Physiology, Part B 160 (2011) 89–93
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
Expression of Asn-d-Trp-Phe-NH2 in the brain of the terrestrial slug Limax valentianus Ryota Matsuo a,⁎, Suguru Kobayashi a, Fumihiro Morishita b, Etsuro Ito a a b
Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Japan Department of Biological Science, Hiroshima University, Japan
a r t i c l e
i n f o
Article history: Received 7 June 2011 Received in revised form 24 June 2011 Accepted 27 June 2011 Available online 3 July 2011 Keywords: Giant neuron Limax NdWFamide Slug
a b s t r a c t The tripeptide Asn-d-Trp-Phe-NH2 (NdWFamide) is a D-amino acid-containing cardioexcitatory peptide initially isolated from Aplysia. Previously we detected NdWFamide immunoreactivity in the visceral giant cells, the largest neurons in the brain of the terrestrial slug Limax located at the dorsal surface of the visceral ganglia. In the present study, we further analyzed the morphological features of these neurons by an intracellular injection of Lucifer yellow, and found that these neurons extend neurites out of the brain through at least 5 nerve bundles. We then isolated a gene and a cDNA clone potentially encoding a NdWFamide precursor, and investigated expression at the levels of mRNA and protein in Limax. The NdWFamide gene consists of 5 exons spanning at least 17 kb of the genome, and its open reading frame extends over 3 exons. The spatial expression pattern of NdWFamide mRNA was almost identical to that of the NdWFamide peptide, with some minor discrepancies in between. Although the most remarkable expression was evident in the visceral giant cells, we also found the expression of NdWFamide mRNA and peptide in the cerebral and pedal ganglia. These results suggest the involvement of NdWFamide in the regulation of a broad area of the slug's body. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The gastropod mollusks are useful model animals in neuroscience because of their simple nervous system with large identifiable neurons, higher learning capacity, and feasibility of in vitro physiological studies. In comparison to the wealth of knowledge about neuronal networks and electrophysiological properties, accumulation of the molecular data has long been behind. However, several recent comprehensive screening studies are uncovering the molecules expressed in the nervous system of the gastropods (Moroz et al., 2006; Feng et al., 2009), especially in terms of higher nervous functions (Lee et al., 2008a, 2008b; Rosenegger et al., 2010; Silverman-Gavrila, et al., 2011). Given such large molecular databases in hand, we are now able to study the function of genes at the levels of from the genome to the protein, and to the neuronal functions. Asn-d-Trp-Phe-NH2 (NdWFamide) may be one example. NdWFamide has been isolated biochemically as a potent cardioactive neuropeptide in the marine gastropod Aplysia (Morishita et al., 1997). This tri-amino-acid peptide contains D-amino acid, and exerts its effect at as low as sub-nano molar concentration (Morishita et al., 1997, 2001, 2003a, 2003b). The distribution of NdWFamide in the central
nervous system (CNS) of Aplysia (Morishita et al., 2003a) and the land snail Euhadra (Morishita et al., 2003b) has been investigated by immunohistochemistry. Recently we identified a pair of the largest neurons, named visceral giant cells (VGCs), on the dorsal surface of the visceral ganglion of the terrestrial slug Limax, and found that VGCs exhibit NdWFamide-like immunoreactivity (Yamagishi et al., 2011). The volume of the VGCs got larger as the slugs gained weight, further reinforcing the notion that the NdWFamide plays some role in body size-related functions such as hemolymph circulation (Morishita et al., 2001). However, the immunoreactivity does not necessarily confirm the presence of NdWFamide, taking into account the fact that the specificity of the antibody might be compromised because of the presence of an enormous variety of peptides with C-terminally amidated phenylalanine expressed in the CNS of mollusks (Veenstra, 2010), which may potentially react with the antibody in a non-specific manner (Greenberg et al., 1988; Santama and Benjamin, 2000). In the present study, we isolated the cDNA and the genomic clones of NdWFamide, and thoroughly analyzed expression at the levels of peptide and mRNA within the brain of Limax. 2. Materials and Methods
⁎ Corresponding author at: Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki, Kagawa 769– 2193, Japan. Tel.: + 81 87 894 5111x6601; fax: + 81 87 894 0181. E-mail addresses: [email protected] (R. Matsuo), [email protected] (S. Kobayashi), [email protected] (F. Morishita), [email protected] (E. Ito). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.06.007
2.1. Animals The terrestrial slugs Limax valentianus were maintained in our laboratory at 19 °C for at least 17 generations as a closed colony. They were fed on a diet of humidified powder mixture consisting of 521 g of
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rat chow (Oriental Yeast, Tokyo, Japan), 500 g of wheat starch (Wako, Osaka, Japan) and 21 g of vitamins (Oriental Yeast). 2.2. Molecular cloning The cDNA potentially encoding NdWFamide was identified in our λ-ZAP-cDNA library (Kobayashi et al., 2010). The 3’ part of the genome clone of NdWFamide was isolated by screening the genomic library (Matsuo et al., 2008) using a full length NdWFamide cDNA as a probe. The genome of the 5’-UTR region was cloned using a Genome Walker Universal kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. The nucleotide sequences of the 1st gene-specific primer and 2nd gene-specific nested primer follow: 1st: 5’-CGGATAGCAGTTGAGCTGTGGGGGTAGTCGAC-3’ 2nd: 5’-GAGCACGTTCTCTTGATAGGACTTACTTCAGCG-3’. The amplified PCR fragment was cloned into the vector pCRII-TOPO (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and the nucleotide sequence was determined by sequencing. The nucleotide sequence information for NdWFamide cDNA has been deposited in GenBank (AB635583). The immunohistochemistry for NdWFamide was performed as described previously (Yamagishi et al., 2011). For in situ hybridization, a part (1–168 bp) of the NdWFamide cDNA was transcribed to generate digoxigenin-labeled cRNA probes. The working concentrations of the cRNA probes were determined by titration to ensure the equivalence of the digoxigenin incorporation into the antisense and sense probes. Hybridization and wash were performed as described previously (Matsuo et al., 2001). 2.3. Injection of Lucifer Yellow The isolated Limax brain was placed in a dish filled with Limax physiological saline (70 mM NaCl, 2.0 mM KCl, 4.7 mM MgCl2, 4.9 mM CaCl2, 5.0 mM glucose, 5.0 mM HEPES, pH 7.0). Lucifer yellow (5% w/v) was dissolved in 0.5 M LiCl, 50 mM Tris (pH 7.0), and was injected iontophoretically into the cell body of either of two VGCs (for 20 min with 1 – 5 nA, 500 ms duration pulses of 1 Hz). The brain was then incubated in Limax physiological saline at 20 °C overnight, and was observed under a MZ16F fluorescent stereo microscope (Leica, Nussloch, Germany) with an attached DP-70 CCD camera (Olympus, Tokyo, Japan).
observations suggest that the two largest neurons VGC1 and VGC2 have essentially symmetrical projections, and send nerves out from multiple thick nerve bundles to play their functional roles. We are currently unable to decisively determine the homologues of VGCs in the other pulmonate species. The Achatina dorsal-right parietal large neuron (d-RPLN)/dorsal-visceral large neuron (d-VLN) (Goto et al., 1986) and the Lymnaea visceral dorsal 1 (VD1)/right parietal dorsal 2 (RPD2) (Boer et al., 1979) are the two candidate large neuron pairs. The Achatina d-RPLN/d-VLN were, however, NdWFamide-ir negative (R.M. unpublished observation). On the other hand, the Lymnaea VD1/RPD2 were also NdWFamide-ir negative according to our previous immunohistochemical data (Morishita et al., 2003b). It would be noteworthy that there is a pair of large NdWFamide-ir neurons located at the dorsal surface of the visceral ganglion of the land snail Euhadra (Fig. 4A, D in Morishita et al., 2003b). They may be the homologues of the Limax VGCs although further analyses are required to compare the morphological and functional features between the both neuron pairs. In the next section, we investigated the distribution of other NdWFamidergic neurons in the brain of Limax. 3.2. Isolation of the NdWFamide cDNA To identify a cDNA clone of NdWFamide, we searched in our λ-ZAP-cDNA library constructed from mRNAs derived from the brains and tentacles of Limax valentianus (Kobayashi et al., 2010). Among approximately 80 cDNA clones randomly isolated from the λ-phage plaques, we found a single clone whose potential open reading frame (ORF) encodes an amino acid sequence containing NWF followed by a canonical cleavage site, GKR (Fig. 2). The N-terminal 24 amino acids were predicted to be a signal peptide that is to be removed after translation. Therefore, a tripeptide NWF was expected to be produced as a result of protein processing. An alignment of the protein sequences translated from the putative homologues in Genbank (Aplysia kurodai, EY423228; Tritonia diomedea, EV286992; Lottia gigantea, FC642682) is shown in Fig. 2. The positions of the signal sequence cleavage are predicted to be the same, and all four proteins have the conserved “GKR” sequence that is a cleavage substrate of the prohormone convertase, to generate a carboxyamidated peptide where the glycine just preceding the dibasic amino acids can serve as an amide donor (Eipper, 1992). These facts further strengthened the notion that the identified Limax clone encodes NdWFamide. 3.3. Distribution of the peptide and mRNA of NdWFamide
3. Results and Discussion 3.1. Morphological features of the VGCs Because the VGCs are the most prominent NdWFamideimmunoreactive neurons in the brain of Limax (Yamagishi et al., 2011), we firstly analyzed the morphological features of the two VGCs by an injection of Lucifer yellow, and found that they extended axons out from several nerve bundles in a very similar fashion (Fig. 1A, C). Here we have named the VGC neuron located at the dorsal surface of the visceral ganglion the VGC1, and that located just to the ventro-posterior side of the VGC1 the VGC2 (Fig. 1E). Both VGC1 and VGC2 extended nerves out in the left and right anterior palial nerves (“a” in Fig. 1A, C), and the posterior pedal nerves (“b” in Fig. 1C). They also sent a projection in either one of the intestinal, anal, or right posterior palial nerves (“c” in Fig. 1A, C), although we could not determine precisely which of them was stained because these three nerves are protrude in very close proximity to one another (Fig. 1E, Takeuchi et al., 1996). We also observed nerve sending to the cerebral ganglia via the cerebropleuro connectives, although we failed to trace these neurons to the targets of these projections (“d” in Fig. 1C). There was no dye coupling between VGC1 and VGC2 (Fig. 1A, C). Taken together, our
Identification of a putative transcript among as few cDNAs as 80 library clones implies that this mRNA is highly expressed in the CNS. We thus tried to investigate the spatial expression pattern of the identified transcript by in situ hybridization in parallel with the immunohistochemistry by anti-NdWFamide antibody, in order to confirm that the isolated cDNA is actually that of NdWFamide. The antisense probe exhibited strong signals in a subset of neurons whereas the sense probe did not (inset in Fig. 3). As shown in Fig. 3, the distribution patterns of the positive signals were almost identical between the immunohistochemistry (upper, white arrowheads) and the in situ hybridization (middle, green circles). In the cerebral and pedal ganglia, however, there were some discrepancies between the results of the two methods. A few immunopositive signals were not stained in the in situ hybridization (yellow arrows), whereas a few positive neurons in the in situ hybridization were not detected by the immunohistochemistry (yellow circles “b” and “e”). The former discrepancy can be explained by the cross-reaction of the antibody with the other similar peptides (Greenberg et al., 1988), or closely related NdWFamide family peptides as demonstrated in Lottia (Veenstra, 2010). The latter may reflect the presence of other splice variants sharing the exons corresponding to the cRNA probe in the in situ hybridization but lacking the same protein coding exons.
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Fig. 1. Tracing of Lucifer Yellow injected into VGC1 or VGC2. (A) Fluorescence of Lucifer Yellow injected into VGC1. (B) A bright field image of (A). (C) Fluorescence of Lucifer Yellow injected into VGC2. (D) A bright field image of (C). (E) A schematic drawing of nerve projections from VGC1 (red) and VGC2 (blue). a, anterior palial nerves; b, posterior pedal nerve; c, intestinal or anal or right posterior palial nerve; d, cerebro-pleuro connectives. A, anterior; P, posterior; R, right; L, left. The approximate size of the brains is 1.5 mm.
Fig. 2. An alignment of amino acid sequences of putative NdWFamide of Limax, Aplysia, Tritonia, and Lottia. Asterisks indicate the amino acids conserved among the four species. An arrow indicates the predicted signal peptide cleavage sites of all four protein precursors. Note that the predicted mature peptide of Lottia is WWF instead of NWF. The accession numbers are as follows: Limax, AB635583; Aplysia, EY423228; Tritonia, EV286992; Lottia, FC642682.
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Fig. 3. Distribution of positive neurons in the coronal sections stained by immunohistochemistry (upper) and in situ hybridization (middle). The neurons positively stained by both techniques are indicated by white arrowheads (upper) and green circles (middle). The neurons stained only by immunohistochemistry are indicated by yellow arrows, whereas those stained only by in situ hybridization are marked by yellow circles. The neurons stained by in situ hybridization are shown as red dots in the lower drawing. Inset: coronal sections dissecting VGCs stained by antisense and sense probes. A, anterior; P, posterior; R, right; L, left; D, dorsal; V, ventral. Scale bar: 250 μm.
3.4. The genomic structure of NdWFamide gene To investigate the possibility that there are other alternative splice variants of the NdWFamide gene transcript, we analyzed the genomic structure of the NdWFamide gene (Fig. 4). The gene consists of 5 exons, with an ORF spanning 3 exons. Exonic sequences of 3 of 4 splice junctions terminate as the dinucleotide “AG”, which is the common tendency, as in the case of the Limax nitric oxide synthase 1 gene (Matsuo et al., 2008). The gene extends over at least 17 kb. Because the region for the in situ hybridization probe corresponded to the 5’-terminal 168 bases of the cDNA (included in the first exon), the presence of a splice junction at the 5’-UTR conformed to the possible existence of splice variants sharing the first exon but differing in some of the following exons, compared to the presently identified NdWFamide cDNA clone. 4. Conclusion Our present study suggests that the largest neurons VGCs have extensive arborization within as well as out of the brain of Limax,
implying that these neurons exert their influence over a broad area of the body. Nearly total coincidence of the signals of immunohistochemistry and in situ hybridization strongly supports the validity of the use of the anti-NdWFamide antibody for analysis of the spatial distribution of this peptide. Although cautious interpretation will be required because the antibody seemed to cross-react with other antigens in some minor cases, our present data strongly suggest that Limax VGC neurons express NdWFamide, which is released as a signal molecule.
5. Acknowledgments This study was partly supported by Grants-in-Aid for KAKENHI from the Japan Society for the Promotion of Science (22570077 to RM, 21657022 to EI) and by a Grant from Tokushima Bunri University for Educational Reform and Collaborative Research (No. TBU2011-23). We thank Miki Yamagishi for providing a cartoon of the Limax brain.
R. Matsuo et al. / Comparative Biochemistry and Physiology, Part B 160 (2011) 89–93
~4 kb
>8 kb
1.5 kb
93
2.2 kb AAAAAAAA
ORF
Fig. 4. Structure of the NdWFamide gene. Exons are expressed as rectangles, and the ORF is indicated by shading (upper). The structure of NdWFamide is shown below, where the ORF is highlighted by underlined bold letters. The region corresponding to the in situ hybridization probe is underlined by a wavy line. The positions of the splice junctions are indicated by arrowheads. A putative poly-A signal is circumscribed by a rectangle.
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Morishita, F., Nakanishi, Y., Kaku, S., Furukawa, Y., Ohta, S., Hirata, T., Ohtani, M., Fujisawa, Y., Muneoka, Y., Matsushima, O., 1997. A novel D-amino-acid-containing peptide isolated from Aplysia heart. Biochem. Biophys. Res. Commun. 240, 354–358. Morishita, F., Sasaki, K., Kanemaru, K., Nakanishi, Y., Matsushima, O., Furukawa, Y., 2001. NdWFamide: a novel excitatory peptide involved in cardiovascular regulation of Aplysia. Peptides 22, 183–189. Morishita, F., Nakanishi, Y., Sasaki, K., Kanemaru, K., Furukawa, Y., Matsushima, O., 2003a. Distribution of the Aplysia cardioexcitatory peptide, NdWFamide, in the central and peripheral nervous system of Aplysia. Cell Tissue Res. 312, 95–111. Morishita, F., Minakata, H., Sasaki, K., Tada, K., Furukawa, Y., Matsushima, O., Mukai, S.T., Saleuddin, A.S.M., 2003b. Distribution and function of an Aplysia cardioexcitatory peptide, NdWFamide, in pulmonate snails. Peptides 24, 1533–1544. Moroz, L.L., Edwards, J.R., Puthanveettil, S.V., Kohn, A.B., Ha, T., Heyland, A., Knudsen, B., Sahni, A., Yu, F., Liu, L., Jezzini, S., Lovell, P., Iannucculli, W., Chen, M., Nguyen, T., Sheng, H., Shaw, R., Kalachikov, S., Panchin, Y.V., Farmerie, W., Russo, J.J., Ju, J., Kandel, E.R., 2006. Neuronal trascriptome of Aplysia: neuronal compartments and cirsuitry. Cell 127, 1453–1467. Rosenegger, D., Wright, C., Lukowiak, K., 2010. A quantitative proteomic analysis of long-term memory. Mol. Brain 3, 9. Santama, N., Benjamin, P.R., 2000. Gene expression and function of FMRFamide-related neuropeptides in the snail Lymnaea. Microsc. Res. Tech. 49, 547–556. Silverman-Gavrila, L.B., Senzel, A.G., Charlton, M.P., Feng, Z.P., 2011. Expression, phosphorylation, and glycosylation of CNS proteins in aversive operant conditioning associated memory in Lymnaea stagnalis. Neuroscience 186, 94–109. Takeuchi, H., Araki, Y., Emaduddin, M., Zhang, W., Han, X.Y., Salunga, T.L., Wong, S.M., 1996. Identifiable Achatina giant neurons: their localizations in ganglia, axonal pathways and pharmacological features. Gen. Pharmacol. 27, 3–32. Veenstra, J.A., 2010. Neurohormones and neuropeptides encoded by the genome of Lottia gigantea, with reference to other mollusks and insects. Gen. Comp. Endocrinol. 167, 86–103. Yamagishi, M., Ito, E., Matsuo, R., 2011. DNA endoreplication in the brain neurons during body growth of an adult slug. J. Neurosci. 31, 5596–5604.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b
Expression of Asn-d-Trp-Phe-NH2 in the brain of the terrestrial slug Limax valentianus Ryota Matsuo a,⁎, Suguru Kobayashi a, Fumihiro Morishita b, Etsuro Ito a a b
Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Japan Department of Biological Science, Hiroshima University, Japan
a r t i c l e
i n f o
Article history: Received 7 June 2011 Received in revised form 24 June 2011 Accepted 27 June 2011 Available online 3 July 2011 Keywords: Giant neuron Limax NdWFamide Slug
a b s t r a c t The tripeptide Asn-d-Trp-Phe-NH2 (NdWFamide) is a D-amino acid-containing cardioexcitatory peptide initially isolated from Aplysia. Previously we detected NdWFamide immunoreactivity in the visceral giant cells, the largest neurons in the brain of the terrestrial slug Limax located at the dorsal surface of the visceral ganglia. In the present study, we further analyzed the morphological features of these neurons by an intracellular injection of Lucifer yellow, and found that these neurons extend neurites out of the brain through at least 5 nerve bundles. We then isolated a gene and a cDNA clone potentially encoding a NdWFamide precursor, and investigated expression at the levels of mRNA and protein in Limax. The NdWFamide gene consists of 5 exons spanning at least 17 kb of the genome, and its open reading frame extends over 3 exons. The spatial expression pattern of NdWFamide mRNA was almost identical to that of the NdWFamide peptide, with some minor discrepancies in between. Although the most remarkable expression was evident in the visceral giant cells, we also found the expression of NdWFamide mRNA and peptide in the cerebral and pedal ganglia. These results suggest the involvement of NdWFamide in the regulation of a broad area of the slug's body. © 2011 Elsevier Inc. All rights reserved.
1. Introduction The gastropod mollusks are useful model animals in neuroscience because of their simple nervous system with large identifiable neurons, higher learning capacity, and feasibility of in vitro physiological studies. In comparison to the wealth of knowledge about neuronal networks and electrophysiological properties, accumulation of the molecular data has long been behind. However, several recent comprehensive screening studies are uncovering the molecules expressed in the nervous system of the gastropods (Moroz et al., 2006; Feng et al., 2009), especially in terms of higher nervous functions (Lee et al., 2008a, 2008b; Rosenegger et al., 2010; Silverman-Gavrila, et al., 2011). Given such large molecular databases in hand, we are now able to study the function of genes at the levels of from the genome to the protein, and to the neuronal functions. Asn-d-Trp-Phe-NH2 (NdWFamide) may be one example. NdWFamide has been isolated biochemically as a potent cardioactive neuropeptide in the marine gastropod Aplysia (Morishita et al., 1997). This tri-amino-acid peptide contains D-amino acid, and exerts its effect at as low as sub-nano molar concentration (Morishita et al., 1997, 2001, 2003a, 2003b). The distribution of NdWFamide in the central
nervous system (CNS) of Aplysia (Morishita et al., 2003a) and the land snail Euhadra (Morishita et al., 2003b) has been investigated by immunohistochemistry. Recently we identified a pair of the largest neurons, named visceral giant cells (VGCs), on the dorsal surface of the visceral ganglion of the terrestrial slug Limax, and found that VGCs exhibit NdWFamide-like immunoreactivity (Yamagishi et al., 2011). The volume of the VGCs got larger as the slugs gained weight, further reinforcing the notion that the NdWFamide plays some role in body size-related functions such as hemolymph circulation (Morishita et al., 2001). However, the immunoreactivity does not necessarily confirm the presence of NdWFamide, taking into account the fact that the specificity of the antibody might be compromised because of the presence of an enormous variety of peptides with C-terminally amidated phenylalanine expressed in the CNS of mollusks (Veenstra, 2010), which may potentially react with the antibody in a non-specific manner (Greenberg et al., 1988; Santama and Benjamin, 2000). In the present study, we isolated the cDNA and the genomic clones of NdWFamide, and thoroughly analyzed expression at the levels of peptide and mRNA within the brain of Limax. 2. Materials and Methods
⁎ Corresponding author at: Laboratory of Functional Biology, Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki, Kagawa 769– 2193, Japan. Tel.: + 81 87 894 5111x6601; fax: + 81 87 894 0181. E-mail addresses: [email protected] (R. Matsuo), [email protected] (S. Kobayashi), [email protected] (F. Morishita), [email protected] (E. Ito). 1096-4959/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2011.06.007
2.1. Animals The terrestrial slugs Limax valentianus were maintained in our laboratory at 19 °C for at least 17 generations as a closed colony. They were fed on a diet of humidified powder mixture consisting of 521 g of
90
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rat chow (Oriental Yeast, Tokyo, Japan), 500 g of wheat starch (Wako, Osaka, Japan) and 21 g of vitamins (Oriental Yeast). 2.2. Molecular cloning The cDNA potentially encoding NdWFamide was identified in our λ-ZAP-cDNA library (Kobayashi et al., 2010). The 3’ part of the genome clone of NdWFamide was isolated by screening the genomic library (Matsuo et al., 2008) using a full length NdWFamide cDNA as a probe. The genome of the 5’-UTR region was cloned using a Genome Walker Universal kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. The nucleotide sequences of the 1st gene-specific primer and 2nd gene-specific nested primer follow: 1st: 5’-CGGATAGCAGTTGAGCTGTGGGGGTAGTCGAC-3’ 2nd: 5’-GAGCACGTTCTCTTGATAGGACTTACTTCAGCG-3’. The amplified PCR fragment was cloned into the vector pCRII-TOPO (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and the nucleotide sequence was determined by sequencing. The nucleotide sequence information for NdWFamide cDNA has been deposited in GenBank (AB635583). The immunohistochemistry for NdWFamide was performed as described previously (Yamagishi et al., 2011). For in situ hybridization, a part (1–168 bp) of the NdWFamide cDNA was transcribed to generate digoxigenin-labeled cRNA probes. The working concentrations of the cRNA probes were determined by titration to ensure the equivalence of the digoxigenin incorporation into the antisense and sense probes. Hybridization and wash were performed as described previously (Matsuo et al., 2001). 2.3. Injection of Lucifer Yellow The isolated Limax brain was placed in a dish filled with Limax physiological saline (70 mM NaCl, 2.0 mM KCl, 4.7 mM MgCl2, 4.9 mM CaCl2, 5.0 mM glucose, 5.0 mM HEPES, pH 7.0). Lucifer yellow (5% w/v) was dissolved in 0.5 M LiCl, 50 mM Tris (pH 7.0), and was injected iontophoretically into the cell body of either of two VGCs (for 20 min with 1 – 5 nA, 500 ms duration pulses of 1 Hz). The brain was then incubated in Limax physiological saline at 20 °C overnight, and was observed under a MZ16F fluorescent stereo microscope (Leica, Nussloch, Germany) with an attached DP-70 CCD camera (Olympus, Tokyo, Japan).
observations suggest that the two largest neurons VGC1 and VGC2 have essentially symmetrical projections, and send nerves out from multiple thick nerve bundles to play their functional roles. We are currently unable to decisively determine the homologues of VGCs in the other pulmonate species. The Achatina dorsal-right parietal large neuron (d-RPLN)/dorsal-visceral large neuron (d-VLN) (Goto et al., 1986) and the Lymnaea visceral dorsal 1 (VD1)/right parietal dorsal 2 (RPD2) (Boer et al., 1979) are the two candidate large neuron pairs. The Achatina d-RPLN/d-VLN were, however, NdWFamide-ir negative (R.M. unpublished observation). On the other hand, the Lymnaea VD1/RPD2 were also NdWFamide-ir negative according to our previous immunohistochemical data (Morishita et al., 2003b). It would be noteworthy that there is a pair of large NdWFamide-ir neurons located at the dorsal surface of the visceral ganglion of the land snail Euhadra (Fig. 4A, D in Morishita et al., 2003b). They may be the homologues of the Limax VGCs although further analyses are required to compare the morphological and functional features between the both neuron pairs. In the next section, we investigated the distribution of other NdWFamidergic neurons in the brain of Limax. 3.2. Isolation of the NdWFamide cDNA To identify a cDNA clone of NdWFamide, we searched in our λ-ZAP-cDNA library constructed from mRNAs derived from the brains and tentacles of Limax valentianus (Kobayashi et al., 2010). Among approximately 80 cDNA clones randomly isolated from the λ-phage plaques, we found a single clone whose potential open reading frame (ORF) encodes an amino acid sequence containing NWF followed by a canonical cleavage site, GKR (Fig. 2). The N-terminal 24 amino acids were predicted to be a signal peptide that is to be removed after translation. Therefore, a tripeptide NWF was expected to be produced as a result of protein processing. An alignment of the protein sequences translated from the putative homologues in Genbank (Aplysia kurodai, EY423228; Tritonia diomedea, EV286992; Lottia gigantea, FC642682) is shown in Fig. 2. The positions of the signal sequence cleavage are predicted to be the same, and all four proteins have the conserved “GKR” sequence that is a cleavage substrate of the prohormone convertase, to generate a carboxyamidated peptide where the glycine just preceding the dibasic amino acids can serve as an amide donor (Eipper, 1992). These facts further strengthened the notion that the identified Limax clone encodes NdWFamide. 3.3. Distribution of the peptide and mRNA of NdWFamide
3. Results and Discussion 3.1. Morphological features of the VGCs Because the VGCs are the most prominent NdWFamideimmunoreactive neurons in the brain of Limax (Yamagishi et al., 2011), we firstly analyzed the morphological features of the two VGCs by an injection of Lucifer yellow, and found that they extended axons out from several nerve bundles in a very similar fashion (Fig. 1A, C). Here we have named the VGC neuron located at the dorsal surface of the visceral ganglion the VGC1, and that located just to the ventro-posterior side of the VGC1 the VGC2 (Fig. 1E). Both VGC1 and VGC2 extended nerves out in the left and right anterior palial nerves (“a” in Fig. 1A, C), and the posterior pedal nerves (“b” in Fig. 1C). They also sent a projection in either one of the intestinal, anal, or right posterior palial nerves (“c” in Fig. 1A, C), although we could not determine precisely which of them was stained because these three nerves are protrude in very close proximity to one another (Fig. 1E, Takeuchi et al., 1996). We also observed nerve sending to the cerebral ganglia via the cerebropleuro connectives, although we failed to trace these neurons to the targets of these projections (“d” in Fig. 1C). There was no dye coupling between VGC1 and VGC2 (Fig. 1A, C). Taken together, our
Identification of a putative transcript among as few cDNAs as 80 library clones implies that this mRNA is highly expressed in the CNS. We thus tried to investigate the spatial expression pattern of the identified transcript by in situ hybridization in parallel with the immunohistochemistry by anti-NdWFamide antibody, in order to confirm that the isolated cDNA is actually that of NdWFamide. The antisense probe exhibited strong signals in a subset of neurons whereas the sense probe did not (inset in Fig. 3). As shown in Fig. 3, the distribution patterns of the positive signals were almost identical between the immunohistochemistry (upper, white arrowheads) and the in situ hybridization (middle, green circles). In the cerebral and pedal ganglia, however, there were some discrepancies between the results of the two methods. A few immunopositive signals were not stained in the in situ hybridization (yellow arrows), whereas a few positive neurons in the in situ hybridization were not detected by the immunohistochemistry (yellow circles “b” and “e”). The former discrepancy can be explained by the cross-reaction of the antibody with the other similar peptides (Greenberg et al., 1988), or closely related NdWFamide family peptides as demonstrated in Lottia (Veenstra, 2010). The latter may reflect the presence of other splice variants sharing the exons corresponding to the cRNA probe in the in situ hybridization but lacking the same protein coding exons.
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Fig. 1. Tracing of Lucifer Yellow injected into VGC1 or VGC2. (A) Fluorescence of Lucifer Yellow injected into VGC1. (B) A bright field image of (A). (C) Fluorescence of Lucifer Yellow injected into VGC2. (D) A bright field image of (C). (E) A schematic drawing of nerve projections from VGC1 (red) and VGC2 (blue). a, anterior palial nerves; b, posterior pedal nerve; c, intestinal or anal or right posterior palial nerve; d, cerebro-pleuro connectives. A, anterior; P, posterior; R, right; L, left. The approximate size of the brains is 1.5 mm.
Fig. 2. An alignment of amino acid sequences of putative NdWFamide of Limax, Aplysia, Tritonia, and Lottia. Asterisks indicate the amino acids conserved among the four species. An arrow indicates the predicted signal peptide cleavage sites of all four protein precursors. Note that the predicted mature peptide of Lottia is WWF instead of NWF. The accession numbers are as follows: Limax, AB635583; Aplysia, EY423228; Tritonia, EV286992; Lottia, FC642682.
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Fig. 3. Distribution of positive neurons in the coronal sections stained by immunohistochemistry (upper) and in situ hybridization (middle). The neurons positively stained by both techniques are indicated by white arrowheads (upper) and green circles (middle). The neurons stained only by immunohistochemistry are indicated by yellow arrows, whereas those stained only by in situ hybridization are marked by yellow circles. The neurons stained by in situ hybridization are shown as red dots in the lower drawing. Inset: coronal sections dissecting VGCs stained by antisense and sense probes. A, anterior; P, posterior; R, right; L, left; D, dorsal; V, ventral. Scale bar: 250 μm.
3.4. The genomic structure of NdWFamide gene To investigate the possibility that there are other alternative splice variants of the NdWFamide gene transcript, we analyzed the genomic structure of the NdWFamide gene (Fig. 4). The gene consists of 5 exons, with an ORF spanning 3 exons. Exonic sequences of 3 of 4 splice junctions terminate as the dinucleotide “AG”, which is the common tendency, as in the case of the Limax nitric oxide synthase 1 gene (Matsuo et al., 2008). The gene extends over at least 17 kb. Because the region for the in situ hybridization probe corresponded to the 5’-terminal 168 bases of the cDNA (included in the first exon), the presence of a splice junction at the 5’-UTR conformed to the possible existence of splice variants sharing the first exon but differing in some of the following exons, compared to the presently identified NdWFamide cDNA clone. 4. Conclusion Our present study suggests that the largest neurons VGCs have extensive arborization within as well as out of the brain of Limax,
implying that these neurons exert their influence over a broad area of the body. Nearly total coincidence of the signals of immunohistochemistry and in situ hybridization strongly supports the validity of the use of the anti-NdWFamide antibody for analysis of the spatial distribution of this peptide. Although cautious interpretation will be required because the antibody seemed to cross-react with other antigens in some minor cases, our present data strongly suggest that Limax VGC neurons express NdWFamide, which is released as a signal molecule.
5. Acknowledgments This study was partly supported by Grants-in-Aid for KAKENHI from the Japan Society for the Promotion of Science (22570077 to RM, 21657022 to EI) and by a Grant from Tokushima Bunri University for Educational Reform and Collaborative Research (No. TBU2011-23). We thank Miki Yamagishi for providing a cartoon of the Limax brain.
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~4 kb
>8 kb
1.5 kb
93
2.2 kb AAAAAAAA
ORF
Fig. 4. Structure of the NdWFamide gene. Exons are expressed as rectangles, and the ORF is indicated by shading (upper). The structure of NdWFamide is shown below, where the ORF is highlighted by underlined bold letters. The region corresponding to the in situ hybridization probe is underlined by a wavy line. The positions of the splice junctions are indicated by arrowheads. A putative poly-A signal is circumscribed by a rectangle.
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