2. MATERIAL AND METHODS Specimens of the E. vermiculata were obtained from the Istituto Internazionale di Elicicoltura (Cherasco, Italy). were treated before sampling by injecting 1.2 mg succinylcholine in 4 M magnesium chloride solution into the haemocoele. Genomic DNA was extracted by proteinase K digestion and pur- ified by phenol–chloroform extraction. RNA was extracted using Cloning of an olfactory liquid nitrogen and purified following standard procedures with Tri- zol Reagent (GIBCO, Life Technology). cDNA was generated using sensory neuron-specific a kit based on Moloney murine leukaemia virus reverse transcriptase (GIBCO, Life Technology). OMP degenerate primers (Operon protein in the land snail Technologies) were designed based on the mammalian common regions MAED(G/S/R)PR (OMP1 sense 5Ј-ATG GC(T/C/A/G) GA(A/G) GA(T/C) (A/G)G(T/C/A/G) CC(T/C/A/G) CA(A/G)-3Ј) ( vermiculata) and SVVNFQL (OMP2 antisense 5Ј-(C/T)TG (G/A)TT (G/A)AA (C/T/G/A)AC (C/T/G/A)AC (G/A)CT (C/T/G/A)A(A/G)-3Ј) corre- Andrea Mazzatenta1,2*, Paolo Pelosi2 sponding to the first and the last seven amino acids. PCR conditions 3,4 were as follows: first step at 94 °C for 5 min; second step 35 cycles and Alessandro Cellerino at 94 °C for 1 min, 48 °C for 1 min, and 72 °C for 1 min; and third 1Dipartimento di Neurobiologia, Scuola Internazionale Superiore step at 72 °C for 5 min. Electrophoretic bands were purified using a di Studi Avanzati, 34014 Trieste, Italy QIAEX II (Qiagen) kit and ligated into a p-GEM plasmid. The crude 2Dipartimento di Chimica e Biotecnologie Agrarie, Pisa University, ligation product was used to transform Bluescript competent cells. 60124 Pisa, Italy Positive colonies were checked for the presence of the insert by PCR, 3Scuola Normale Superiore, and 4Istituto di Neuroscienze, using the plasmid’s primers SP6 and T7, and DNA was purified with Centro Nazionale delle Richerche, 56100 Pisa, Italy a miniprep QIAquick (Qiagen) kit and sequenced. Sequence analyses * Author for correspondence ([email protected], [email protected]). were performed using the NCBI-BLAST and Expasy Molecular Ser- ver (http://www.ncbi.nln.nih.gov; http://www.au.expasy.org). For in situ hybidization we dissected posterior rhinophores and Recd 01.07.03; Accptd 04.08.03; Online 01.10.03 fixed them in paraformaldehyde 4% in phosphate buffered saline (PBS) for 2 h. After washing, rhinophores were treated with protein- We have isolated a gene encoding for an olfactory ase K (0.25 mg mlϪ1) then incubated overnight at 37 °C with a 5Ј sensory neuron (OSN)-specific protein in an invert- biotinylated oligonucleotide corresponding to the first 51 nucleotides: ebrate, the land snail 5Ј-CTG AAA CGA CTT GTG TGG AGA AGA TTT CAG CTG CGG ACT ATC CTC AGC CAT-3Ј (MWG Biotech AG, (GenBank accession number AY147909.). Using in Germany). Control samples (pedal muscle, salivary and mucipares situ hybridization, we detected expression of its glands) were incubated overnight with biotinylated oligonucleotide mRNA in the dendrite, cell body and axon of OSNs. and 30 times molar excess of unmarked oligonucleotide. Unspecific binding sites were blocked by overnight incubation with 25% goat By neural tracing, using the lipophilic tracer DiI and serum, 1% Triton, 10% bovine serum albumin (BSA), washed in PBS, in situ hybridization, we have revealed the organiza- and incubated in 1% BSA, 0.1% Triton and 1 : 400 avidin–fluorescein tion of OSNs and their connections with olfactory isothiocyanate. Tissue samples were cryoprotected and embedded in µ glomeruli in the land snail. Sequence and expression TissueTek, 25 m slices were sectioned using a cryostat. For histology, tissues were fixed by immersion in saline formalin pattern analogy of land snail protein with olfactory (4% formalin in 0.9% NaCl solution), dehydrated in an ethanol series marker protein (OMP) from vertebrates suggest and embedded in paraffin. Transverse sections and longitudinal sec- that the land snail protein is an OMP-like protein. tions were cut on a microtome (15 µm thickness), dewaxed, and stained with cresyl violet, hematoxilyn and eosin (Pabisch Top- This protein could represent a plesiomorphic Rapidstain). character in the evolution of olfactory proteins. For tracing of OSNs we used DiI C18 (Molecular Probe, NL). Dissected posterior rhinophores were fixed in paraformaldehyde 4%. Keywords: olfactory marker protein; neural tracing; Microinjection was performed under a dissecting microscope using olfactory sensory neuron; olfaction; land snail; mollusc a micromanipulator with 1 µm tip diameter glass microcapillaries filled with saturated solution of DiI in ethanol. Tissues were incu- bated at 37 °C for one week. They were first observed and photo- graphed as whole mounts and then embedded in 5% agarose low 1. INTRODUCTION melting temperature. Cross-sections of 100 µm thickness were The olfactory system in molluscs is poorly characterized obtained using a vibratome. as compared to that in other invertebrates and vertebrates. While the morphology of the olfactory system in land snail 3. RESULTS has been described (Wright 1974; Chase & (a) Molecular cloning Tolloczko 1989, 1993; Zaitseva 1991; Mazzatenta et al. We designed OMP degenerated primers based on the 2003), the neural network and molecular components mammalian common regions and used them to perform remain unknown. a PCR on land snail genomic DNA. A band between 450– In vertebrates, it has been proved that olfactory marker 500 bp was amplified. PCR was, therefore, performed protein (OMP) is the only specific molecular marker of with cDNA prepared from the olfactory sensory epi- olfactory sensory neurons (OSNs) (Margolis 1972; thelium, with pedal muscle tissue as a control. A band Keller & Margolis 1976; Sydor et al. 1986; Krishna et al. of the same size was obtained from the olfactory sensory 1992; Buiakova et al. 1994; Ro¨ssler et al. 1998; Horowitz epithelium, but not from the pedal muscle. This band was et al. 1999; Baldisseri et al. 2002; Celik et al. 2002). purified, cloned and sequenced. The resulting nucleotide In the present study we investigated the presence of sequence is composed of 465 bp and encodes a mature specific proteins in OSNs of the land snail (Eobania protein of 155 amino acids (figure 1). It lacks introns and vermiculata). We report the cloning and sequencing of the the entire sequence is an open reading frame. corresponding cDNA and its expression in OSNs detected by in situ hybridization. Combining the results of in situ (b) Tissue expression hybridization and neural tracing with DiI, we have General anatomy and morphology of the olfactory sen- revealed the organization of OSNs and their connections sory epithelium is revealed with cresyl violet, hematoxylin with olfactory glomeruli in the land snail. and eosin staining (see figure 5 in electronic Appendix A,

Proc. R. Soc. Lond. B (Suppl.) 271, S46–S49 (2004) S46  2003 The Royal Society DOI 10.1098/rsbl.2003.0093 An OSN-specific protein in the land snail A. Mazzatenta and others S47

atggctgaggatagtccgcagctgaaatcttctccacacaagtcgtttcagttctaccaacttctgatctgcatgcgtactttgtgcattgtactg

M A E D S P Q L K S S P H K S F Q F Y Q L L I C M R T L C I V L

gtcctgctggccgccacattcgccatttctgatgctacagaggttgacaagagacttataggaggtcttgacttgagtgccgtttttaaactggtg

V L L A A T F A I S D A T E V D K R L I G G L D L S A V F K L V

gctagcgttcctacgttattacaacaatcagtcaaaagtgttcaggaccttctgcccagtctgttggacgccgtcactagcctggtgcgggatgtc

A S V P T L L Q Q S V K S V Q D L L P S L L D A V T S L V R D V

acgggcgctgccaccggtattgtctacaaagccctagagactgttctaggagtgactaaggcacttctcctggtaggcaataatgtcttggggctg

T G A A T G I V Y K A L E T V L G V T K A L L L V G N N V L G L

atcctttcggctgtaaactccagttctgggctccttactagtcttgtgacatctattctcagcgtcgtgtttaaccagctc

I L S A V N S S S G L L T S L V T S I L S V V F N Q L

Figure 1. Nucleotide and amino acid sequence of an OSN-specific protein in the land snail, Eobania vermiculata. The land snail nucleotide sequence has been cloned by cDNA, using as a template OSN mRNA. The GenBank accession number is AY147909.

ose nse nse g2 ose

g1

g2

osn osn g3 g3 ax

(b)

g4

g1 g2 g3 g5 g4

g5

(a) (c)

Figure 2. (a,b) In situ hybridization of a 25 µm cryosection and (c) a whole mount of the land snail olfactory cup expressing mRNA for a specific OSN protein. (a,b) Posterior rhinophore cut and mounted invaginated for comparison of the olfactory sensory epithelium (ose), where OSNs are specifically labelled, and non-sensory epithelium (nse) covers the rhinophore; ax indicates the OSN axon bundles. (c) A photomicrograph of olfactory glomeruli (g1–g5), positive stain due to terminating OSN axons. The scale bar in (a) is 100 µm, and in (b,c) 200 µm. available on The Royal Society’s Publications Web site). OSNs project their axons to the glomeruli and their den- The olfactory region is localized at the tip of the rhino- drites to the external environment in the spongiform layer. phore, below the eye. It is composed of an olfactory sen- In situ hybridization experiments were performed using sory epithelium, where the bipolar OSNs are located. a5Ј biotinylated 51-mer probe, corresponding to the first

Proc. R. Soc. Lond. B (Suppl.) S48 A. Mazzatenta and others An OSN-specific protein in the land snail

snail (E. vermiculata). This protein shares many distinctive ax g characteristics with vertebrate OMPs. It lacks introns and the entire sequence is an open reading frame as for ver- tebrate OMPs (Margolis et al. 1993; Wang & Reed 1993; Buiakova et al. 1994), a common feature among other olfactory system proteins such as olfactory receptors and odorant binding proteins. Indeed, this may be a peculiar characteristic of proteins involved in chemoreception, bx indicative of an hypothetical common ancestoral protein. Land snail protein is composed of 155 amino acids, while the OMP family ranges in size from 155 amino acids in l (b) fish, to 163 amino acids in mammals (Margolis 1972; Keller & Margolis 1976; Sydor et al. 1986; Krishna et al. 1992; Buiakova et al. 1994; Ro¨ssler et al. 1998; Celik et al. 2002). These little differences in protein length ax l correspond to a progressive reduction of amino acid composition in the transitions among vertebrates and d invertebrates. Further, the calculated molecular weights of the land snail protein and that of the vertebrate OMPs are s comparable: 16.32931 kDa in the land snail and ranging from 14 kDa in fish (Celik et al. 2002) to 18 kDa in mam- mals (Margolis 1972; Keller & Margolis 1976; Sydor et al. 1986; Buiakova et al. 1994). Moreover, the land snail proteins’ calculated isoelectric point is 7.01, which is simi- (a) lar to that of a frog. Sequence identity between the land snail protein and vertebrate OMPs is as follows: 12.9% Figure 3. Land snail OSN: DiI tracing and in situ with fish; 13.5% with Xenopus 2; 16.2% with Xenopus 1; hybridization of specific OSN protein. (a) A cross-section of 18.1% with human; 20% with mouse and rat (figure 4) the olfactory cup where the dye has traced OSNs and the and homology increases when looking at the consensus. olfactory glomerulus. d, dendrite; s, soma; ax, axon; g, In particular, among invertebrate and vertebrate glomerulus; l, lobuli. (b) High magnification of OSN axons sequences there are at least three well-conserved regions. expressing OMP mRNA spread inside the glomerulus, where Furthermore, land snail predicted secondary structure is they synapse with glomerular cells. bx, axon bundles. The scale bar in (a,b)is25µm. similar to that of OMP’s (see figure 6 in electronic Appen- dix A). Moreover, using in situ hybridization experiments, we 51 nucleotides of the E. vermiculata protein sequence. Fig- found that the land snail protein is expressed only in ure 2a,b shows cross-sections of the rinophore and figure OSNs and not in other analysed tissues. The specificity 2c shows a whole-mount preparation. The labelling and selectivity of the land snail protein are two of the prin- observed follows incubation with the biotinylated probe cipal characteristics of OMPs. In contrast to previous and fluorescent streptavidin is localized on OSNs in the work (Chase & Tolloczko 1993), we were able to detect olfactory sensory epithelium: no labelling is in the non- labelled fibres that project only to glomeruli; we were not sensory epithelium, figure 2a. In figure 2b, higher magnifi- able to detect any fibre that projects directly to the cer- cation of the olfactory sensory epithelium, OSNs grouped ebral ganglia. in bundles projecting their axons to glomeruli are labelled by the probe. In figure 2c the positive stain is due to termi- To date, a clear function for the OMP has not been nating OSN axons in the glomeruli. To test for the speci- clearly established (Smith et al. 2002). Therefore, it is not ficity of the labelling, sections were co-incubated with an possible to confirm absolutely the presence of an OMP excess of unlabelled oligonucleotide. No distinct labelling using a functional analysis. Instead, the main character- of OSNs and glomeruli was observed in the presence of an istic of an OMP is its unique expression restricted to excess of unlabelled oligonucleotide and in control tissues OSNs. Thus, our data support the existence of an OMP- (pedal muscle, salivary and mucipares glands). like protein in the land snail and also provide strong To complement the results obtained by in situ hybridiz- evidence that this protein is phylogenetically and func- ation, OSNs were labelled by tracing with the fluorescent tionally conserved among taxa with different evolution- dye, DiI. The lipophilic tracer was micro-injected directly ary histories. into olfactory glomeruli. After one week of incubation, a limited number of OSNs were labelled retrogradely. The Acknowledgements morphology of OSNs labelled by in situ hybridization and The authors would like to thank Professor John Nicholls for his by DiI tracing are compared in figure 3, showing the selec- fundamental contribution, Professor Roger C. Thomas and Dr tivity and specificity of this protein expression. Luciano Domenici for their suggestions, Dr Matteo Caleo and Dr Enrica Strettoi for their laboratory support, and Adriano Tacchi for his excellent technical assistance. Dr Michela Cesco-Gaspere, Dr 4. DISCUSSION Laura Lagostena, Dylan Dean and Christopher Spadaccini are thanked for critical review of this manuscript. Thanks to Dr G. We have isolated a gene encoding for a protein specifi- Avagnina Societa` Italiana di Elicicoltura, Cherasco, Italy, for supply- cally and selectively expressed in the OSNs of the land ing the land snails.

Proc. R. Soc. Lond. B (Suppl.) An OSN-specific protein in the land snail A. Mazzatenta and others S49

Ever MAEDSPQLKSSPHKSFQFYQLLICMRTLCIVLVLLAATFAISDATEVDKRLIGGLDLSAVFKLVASVPTLLQQSVKSVQDLL

hum MAEDRPQQPQLDMPLVLDQGLTRQMRLRVESLKQRGEKRQDGEKLLQPAESVYRLNFTQQQRLQFERWNVVLDKPGKVTITG

Mus MAEDGPQKQQLEMPLVLDQDLTQQMRLRVESLKQRGEKKQDGEKLIRPAESVYRLDFIQQQKLQFDHWNVVLDKPGKVTITG

rat MAEDGPQKQQLDMPLVLDQDLTKQMRLRVESLKQRGEKKQDGEKLLRPAESVYRLDFIQQQKLQFDHWNVVLDKPGKVTITG

Xen1 MASETSEMELPFIEDT---QLTKCMRIRVQTLQQKNAKPQEGEMLLRANEYIYRVDFSKQ-KLRFLWWKVHLKSPGKVMITG

Xen2 MAPETSEMELPFNEDT---QLTKCMRIRVQTLQQKNGKPQEGEMLLRANDYIYRLDFPKQ-KLRFLWWKVHLKTPGKVMITG

fish MSLELTFNPDV------QLTEMMRLRVQSLQQRGQKRQDGERLLKSNEHVYSLDFSEQ-ALHFTCWNICISSPGRLNIIA * * ** * * *

Ever PS---LLDAVTSLVRDVTGAATGIVYKALETVLGVTKALLLV-GNNVLGLILSAVNSSSGLLTSL----VTSILSVVFNQL

hum TSQNWTPDLTNLMTRQLLDPTAIFWRKEDSDAIDWNEADALEFGERLSDLAKIRKVMYFLVTFGEGVEPANLKASVVFNQL

Mus TSQNWTPDLTNLMTRQLLDPAAIFWRKEDSDAMDWNEADALEFGERLSDLAKIRKVMYFLITFGEGVEPANLKASVVFNQL

rat TSQNWTPDLTNLMTRQLLDPAAIFWRKEDSDAMDWNEADALEFGERLSDLAKIRKVMYFLITFGEGVEPANLKASVVFNQL

Xen1 TSQHWTPDLTNLMTRQLLEPSAVFYKKDANDEVECNEADAQEFGERIAELAKIRKVMYFVITFLDGADPATIECSIGFRA

Xen2 TSQHWTPDLTNLMTRQLLEPSAVFYKKDAKDKVECNEADAQEFGERIAELAKIRKVMYFVFTFLDGADPSTVEYSIGFRG

fish TSQLWTPDLTHLMTRQLLEPTGLFWRSADDENIQCYEADAQEFGERIAELAKVRKVMYFLFAFEDGLSPESVECSIEFQTSK * * * * * * * * *

Figure 4. Alignments of land snail OSN-specific protein and vertebrate OMP sequences. The land snail sequence is composed of 155 amino acids as in fish, mammalian OMPs by 163 amino acids and frogs by 158 amino acids. Vertebrate OMPs are indicated as follows: hum, human; Mus, mouse; rat, rat; Xen 1 and 2, frog; fish, zebrafish; Ever, land snail OSN-specific protein. Asterisks indicate identical amino acids among all sequences. Amino acids that are identical to the land snail but not shared among all sequences are highlighted by grey boxes.

Baldisseri, D. M., Margolis, J. W., Weber, D. J., Koo, J. H. & Marg- Margolis, F. L., Kudrycki, K., Stein-Izak, C., Grillo, M. & Akeson, olis, F. L. 2002 Olfactory marker protein (OMP) exhibits a β-clam R. 1993 From genotype to olfactory neuron phenotype: the role fold in solution: implication for target peptide interaction and of the Olf-1-binding site. Ciba. Found. Symp. 179,3–20. olfactory signal transduction. J. Mol. Biol. 319, 823–837. Mazzatenta, A., Baldaccini, N. E. & Bedini, C. 2003 General anat- Buiakova, O. I., Krishna, N. S. R., Getchell, T. V. & Margolis, F. L. omy and morphology of posterior rhinophore, in Pulmonate land 1994 Human and rodent OMP genes: conservation of structural snail Eobania vermiculata. Ital. J. Zool. (In the press.) and regulatory motifs and cellular localization. Genomics 20, Ro¨ssler, P., Mezler, M. & Breer, H. 1998 Two olfactory marker pro- 452–462. teins in Xenopus laevis. J. Comp. Neurol. 395, 273–280. Celik, A., Fuss, S. H. & Korsching, S. I. 2002 Selective targeting of Smith, P. C., Firestein, S. & Hunt, F. 2002 The crystal structure of zebrafish olfactory receptor neurons by the endogenous OMP pro- the olfactory marker protein at 2.3 A˚ resolution. J. Mol. Biol. 319, moter. Eur. J. Neurosci. 15, 798–806. 807–821. Chase, R. & Tolloczko, B. 1989 Interganglionic dendrites constitute Sydor, W., Teitelbaum, Z., Blacher, R., Sun, S., Benz, W. & Marg- an output pathway from the procerebrum of the snail Achatina ful- olis, F. L. 1986 Amino acid sequence of a unique neuronal protein: ica. J. Comp. Neurol. 283, 143–152. rat olfactory marker protein. Arch. Biochem. Biophys. 249, 351–362. Chase, R. & Tolloczko, B. 1993 Tracing neural pathways in snail Wang, M. M. & Reed, R. R. 1993 Molecular cloning of the olfactory olfaction: from the tip of the tentacles to the brain and beyond. neuronal transcription factor Olf-1 by genetic selection in yeast. Microsc. Res. Tech. 24, 214–230. Nature 364, 121–126. Horowitz, L. F., Montmayeur, J. P., Echelard, Y. & Buck, L. B. 1999 Wright, B. R. 1974 Sensory structure of the tentacles of the slug, A genetic approach to trace neural circuits. Proc. Natl Acad. Sci. Arion ater (, ). Cell Tissue Res. 151, 254–257. USA 96, 3194–3199. Zaitseva, O. V. 1991 Structural organisation of the tentacular sensory Keller, A. & Margolis, F. L. 1976 Isolation and characterization of system in land pulmonates. In Studies in neuroscience (ed. S. rat olfactory marker protein. J. Biol. Chem. 251, 6232–6237. Sakarov & W. Winlow), pp. 238–257. Manchester: University Krishna, N. S. R., Getchell, T. V., Margolis, F. L. & Getchell, M. L. Press. 1992 Amphibian olfactory receptor neurons express olfactory marker protein. Brain Res. 593, 295–298. Margolis, F. L. 1972 A brain unique protein to the olfactory bulb. Visit http://www.pubs.royalsoc.ac.uk to see an electronic appendix to Proc. Natl Acad. Sci. USA 69, 1221–1224. this paper.

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