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SUPPLEMENTAL INFORMATION
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Identification and expression of Acetylcholinesterase in Octopus vulgaris arm development and regeneration: a
conserved role?
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Sara Maria Fossati1, Simona Candiani2, Marie-Therese Nödl1, Luca Maragliano1, Maria Pennuto1,3, Pedro
Domingues4, Fabio Benfenati1,5, Mario Pestarino2, Letizia Zullo1
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Istituto Italiano di Tecnologia, Department of Neuroscience and Brain Technologies, Genova, Italy
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University of Genova, DISTAV, Genova, Italy
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Dulbecco Telethon Institute, Lab of Neurodegenerative Diseases, CIBIO, University of Trento, Italy
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Instituto Español de Oceanografía, Centro Oceanográfico de Vigo, Vigo, Spain
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Department of Experimental Medicine, University of Genova, Genova, Itally
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Corresponding author: Sara Maria Fossati, Via Morego 30, 16163, Genoa, Italy, Telephone number: +39
3475752235, Fax number: +39 010 71781230, e-mail: sara.fossati@iit.it
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Contents: Supplemental Experimental Procedures (Adult animal treatment, O. vulgaris ACHE gene cloning and
identification, Synthesis of DIG-labeled ACHE RNA probe for in situ hybridization, In situ hybridization, RT-PCR,
Molecular Dynamics Simulations), Supplemental References, Supplemental Figure Captures.
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES
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Adult animal treatment
Following capture, adult animals were each placed in a 120x50x45 cm marine aquarium. The tanks were filled
with artificial sea water (ASW) and kept at a temperature of 18°C on 12 h light/dark cycle. Water cleaning and
oxygenation was ensured by a pump-filter and aeration system which continuously circulated the water through
biological filters and all relevant chemo/physical water parameters were constantly checked. Animals were left to adapt
to captivity for at least 10 days before experimentation.
The experimental animals were selected on the basis of the following criteria: healthy shape (all the arms and
body parts intact), the animal showed normal reflexes and voluntary movements (e.g., arm extension, walking, etc.),
regular eating and motivation to attack prey.
Following surgery, each animal was replaced in the experimental tank where it slowly recovered from
anesthesia (about 2-5 minutes). All the animals employed in this study did not display behavioral modifications after the
operation, and their amputated arms regenerated as in the natural environment [1].
At the end of the experiments, the possibility of returning the animals to their natural environment was
evaluated on the basis of the animal’s health. Animals were then either released into the wild at suitable habitats far
from the collecting sites or received a terminal overdose of anesthetic.
Our research conformed to the ethical principles of replacement, reduction, refinement and minimization of
suffering [2]. Particular attention was given to the method of capture from the wild, the housing, the animal care and
health monitoring program as well as to identifying signs of pain or distress in the animals. Animal experimentation was
conducted following the guidelines reported in the European Directive 86/609/EEC.
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In situ hybridization
Both sample sections and embryos were incubated with a DIG-labeled ACHE probe at 60°C overnight.
Endogenous peroxydase inactivation and SSC washes were not performed.
O. vulgaris ACHE gene cloning and identification
Given the absence of an O. vulgaris Genebank database, the cloning of the ACHE full length cDNA from O.
vulgaris arm was obtained from degenerate primers whose design was based on the deposited sequences from Loligo
vulgaris (GenBank Accession No. AF065384; [3]) and Electrophorus electricus (GenBank Accession No. AF030422;
[4]).
The degenerate primers were designed on the basis of three highly conserved regions (EDCLYLN, FGESAG
and WPEWVG) (Fig. S3).
The sequencing of the full length ACHE cDNA was continued by performing 5’ and 3’ RACE-PCR. The 5'
RACE reaction was carried out according to the instruction manual of the 5’ RACE System (Invitrogen) using AAP and
GSP2 primers (FIGURE S2). The 3' RACE reaction was carried out according to the instruction manual of the 3’ RACE
System (Invitrogen). The 3' end of cDNA was amplified using UAP and GSP primers (Fig. S2).
Synthesis of DIG-labeled ACHE RNA probe for in situ hybridization
The cDNA obtained by cloning the PCR product of about 1400 bp was used as a template for in vitro
transcription by a Boehringer Mannheim DIG RNA labeling Kit, according to the supplier's instructions. The
restriction site used for the construction of the probe for in situ hybridization is shown in Fig. S3. The insert was
linearized with BamHI and the probe was synthesized using SP6 RNA polymerase.
RT-PCR
Each sample was replicated three times in each real-time PCR experiment to capture intra-assay variability.
PCR amplifications were performed with Power SYBR Green Master Mix (Qiagen) in a 7900HT Fast Real-Time PCR
System (Applied Biosystems). Each assay included a no-template control for each primer pair and a dissociation curve
analysis was performed to detect the presence of non-specific products. All data were analyzed using the 2-DDCT method.
Molecular Dynamics Simulations
The system to be modeled comprised the ACHE protein model, discussed above, immersed in a box of TIP3P
model water molecules [5] and counter ions to neutralize the total charge, making a total of 85,618 atoms. Residues’
protonation states were determined using HHpred [6]. Periodic boundary conditions were used to replicate the system
and remove box surface effects [7]. Short-range non-bonded interactions were cut off at 12 Å, while long-range
electrostatic interactions were computed using the Particle Mesh Ewald (PME) method [8]. Chemical bonds connecting
hydrogen atoms to heavy atoms were kept fixed using SHAKE [9]. The integration time step was 1 fs to ensure stability
of the dynamics algorithm. The proper size for the simulation box corresponding to a pressure of 1 atm was obtained by
simulating the system in the constant pressure and temperature (NPT) ensemble using the Nosé-Hoover Langevin piston
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method for 0.5 ns [10]. The simulation ensemble was then switched to constant volume and temperature (NVT) for the
rest of the simulation by keeping the temperature stationary around 300K using Langevin dynamics. The full simulation
run lasted 60 ns.
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Fig. S1 Percent identity and divergence were calculated by using the ClustalW algorithm provided in the MEGALIGN
module. Percent identity compares sequences directly without accounting for phylogenetic relationships. Percent
divergence is calculated by comparing sequence pairs in relation to the phylogeny reconstructed by MegAlign
(Neighbor Joining method). Sequences used in the phylogenetic analysis with the corresponding accession numbers are
also reported.
Fig. S2 Sequences (FW: forward; REV: reverse) of primers used for cloning and RT-qPCR experiments. According to
GenBank (http://www.ncbi.nlm.nih.gov/genbank/) for each gene is listed the accession number and the product size.
Fig. S3 Nucleotide and deduced amino acid sequences of ACHE cDNA from O. vulgaris. Red asterisk indicates the
stop codon. The regions where primers were designed are underlined. Red underlined: primers corresponding to the
EDCLYLN, FGESAG and WPEWVG regions; orange underlined, Gene Specific Primer used for 5’RACE-PCR; green
underlined, Gene Specific Primer used for 3’RACE-PCR; dashed blue underlined, BamH1 restriction site used for the
construction of the probe for in situ hybridization.
Fig. S4 Volume representation of the catalytic gorge and protein stability. A) Volume representation of the catalytic
gorge (gray). The PAS (orange, top left) and CAT (red, bottom right) residues are shown as sticks. B) Backbone RMSD
of O. vulgaris ACHE from the starting conformation along the simulated trajectory for the full protein (black), the CAT
(red) and the PAS (orange) residues.
Fig. S5 Confocal stacks through the midline of developing arm buds stained for Phallacidin (a-d) and DAPI (e-h). Arm
buds are shown in a top view, except for stage 29, which is a side view (proximal to the left). a, e: stage 21; b, f: stage
23; c, g: stage 25; d, h: stage 29. Scale bars: 100 µm. lm: longitudinal muscle; tm: transversal muscle; m: future muscle
layer; e: epithelium; anc: axial nerve cord.
Movie S1 Movie showing O. vulgaris ACHE protein conformation and the localization of the active residues.
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SUPPLEMENTAL FIGURE CAPTURES
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