Emergence of sensory structures in the developing

R E S EA R C H A R T I C L E

Emergence of Sensory Structures in the Developing Epidermis in Sepia officinalis and Other Coleoid Cephalopods Auxane Buresi,1,2 Roger P. Croll,3 Stefano Tiozzo,4 Laure Bonnaud,1,5 and Sebastien Baratte1,6* 1

Museum National d’Histoire Naturelle (MNHN), DMPA, UMR Biologie des Organismes et Ecosyste`mes Aquatiques (BOREA), MNHN CNRS 7208, IRD 207, UPMC CP51 75005 Paris, France 2 Universite Pierre et Marie Curie—Paris, Paris 6, France 3 Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4R2 4 CNRS, Sorbonne Universites, UPMC Universite Paris 6, Laboratoire de Biologie du Developpement de Villefranche-sur-mer, Observatoire Oceanographique, 06230, Villefranche-sur-mer, France 5 Universite Paris Diderot—Sorbonne Paris Cite, Paris 4, France 6 Universite Paris Sorbonne—Paris 4, Paris France

ABSTRACT Embryonic cuttlefish can first respond to a variety of sensory stimuli during early development in the egg capsule. To examine the neural basis of this ability, we investigated the emergence of sensory structures within the developing epidermis. We show that the skin facing the outer environment (not the skin lining the mantle cavity, for example) is derived from embryonic domains expressing the Sepia officinalis ortholog of pax3/7, a gene involved in epidermis specification in vertebrates. On the head, they are confined to discrete brachial regions referred to as “arm pillars” that expand and cover Sof-pax3/7-negative head ectodermal tissues. As revealed by the expression of the S. officinalis ortholog of elav1, an early marker of neural differentiation, the olfactory organs first differentiate at about stage 16 within Sof-pax3/7-negative ectodermal regions before they are covered by the definitive Sof-pax3/7-positive

outer epithelium. In contrast, the eight mechanosensory lateral lines running over the head surface and the numerous other putative sensory cells in the epidermis, differentiate in the Sof-pax3/7-positive tissues at stages 24–25, after they have extended over the entire outer surfaces of the head and arms. Locations and morphologies of the various sensory cells in the olfactory organs and skin were examined using antibodies against acetylated tubulin during the development of S. officinalis and were compared with those in hatchlings of two other cephalopod species. The early differentiation of olfactory structures and the peculiar development of the epidermis with its sensory cells provide new perspectives for comparisons of developmental processes among molluscs. J. Comp. Neurol. 000:000–000, 2014. C 2014 Wiley Periodicals, Inc. V

INDEXING TERMS: sensory neurons; elav; pax3/7; epidermis development; Sepia officinalis; Euprymna scolopes; Doryteuthis opalescens; cephalopod

Comparisons of extant and fossil body plans, developmental processes, gene sequences, and gene functions provide the foundation for understanding how evolution has shaped biodiversity. For some taxa, however, evolutionary events have been so numerous and so drastic that such comparisons turn out to be difficult or confusing. Cephalopods, for instance, belong to the phylum Mollusca, but the numerous evolutionary novelties that make them efficient marine predators have led to both sizeable divergences with the typical molluscan body plan and puzzling convergences with vertebrates C 2014 Wiley Periodicals, Inc. V

(Shigeno et al., 2010). Thus, in coleoid species (i.e., all cephalopods except nautiluses), the external protective shell, typical of most molluscs, has been internalized and the mantle has become a powerful organ for

*CORRESPONDENCE TO: Sebastien Baratte, Museum National d’Histoire Naturelle (MNHN), DMPA, UMR Biologie des Organismes et Ecosyste`mes Aquatiques (BOREA), MNHN CNRS 7208, IRD 207, UPMC, 55 rue Buffon, CP51 75005 Paris, France. E-mail: [email protected] Received October 27, 2013; Revised February 10, 2014; Accepted February 10, 2014. DOI 10.1002/cne.23562 Published online February 18 in Wiley Online (wileyonlinelibrary.com)

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locomotion, camouflage, and sensory functions. The ancestral molluscan foot is hypothesized to have evolved into prehensile arms (Boletzky, 1988). In addition, the nervous system has reached an incomparable degree of centralization and the sensory organs, such as eyes and “lateral lines,” show striking convergences with those of vertebrates (Young, 1971; Budelmann, 1995, 1996). As a final, but not the least, divergence from other molluscs, cephalopods have lost their larval phase of development. Most molluscan species hatch as free-living and autonomous larvae (at the trochophore or veliger stage) that later metamorphose into a juvenile form. Even in pulmonate species, in which hatching gives rise to adult-like juveniles, encapsulated larval stages can be recognized in the egg where they undergo metamorphosis (see, e.g., Voronezhskaya et al., 2004). In the eggs of cephalopods, however, embryos possess a large amount of yolk and directly develop into adult-like juveniles without passing through any recognizable larval stage and without any metamorphosis event (Boletzky, 1988). The evolution of a true direct development in cephalopods is thus unique among molluscs and provides a challenging evolutionary issue, because it is often difficult to homologize structures between cephalopod embryos and the embryos or larvae of other molluscs. Modern applications of molecular biology and other specific staining techniques appear, however, to be on the brink of finally overcoming this hurdle, and the development of the nervous system is a convenient starting point to perform comparisons within molluscs. A body of evidence now clearly establishes that many other molluscs possess larval nervous systems that predate the first formation of the adult nervous system and are far more extensive than previously thought (Croll and Voronezhskaya, 1996; Voronezhskaya et al., 2002, 2008; Dickinson et al., 1999; Wanninger and Haszprunar, 2003). These larval nervous systems often consist of an apical sensory organ (ASO), posterior FMRFamide-immunoreactive neurons, and catecholamine-containing peripheral neurons (Leise et al., 2004; Croll and Dickinson, 2004; Croll, 2006) and include early sensory neurons that may mediate the detection of external chemical cues inducing metamorphosis (Pires et al., 2000; Pechenik et al., 2007). For cephalopod embryos, it is generally assumed that the ancestral larval nervous system and its sensory components have been lost in the course of the evolution toward direct development: the typical ganglia of molluscs directly develop into the juvenile nervous system, either becoming peripheral ganglia (brachial, gastric, cardiac, and stellate) or fusing into brain lobes (cerebral, palliovisceral, pedal; Shigeno et al., 2001;

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Marquis, 1989; Yamamoto et al., 2003). Recently, a few studies have indicated that some ganglia and neurons differentiate in very early stages during organogenesis of Sepia officinalis, a nectobenthic coleoid species. As they are forming, most ganglia contain large numbers of catecholaminergic terminals, which appear to originate from peripheral sensory neurons (Baratte and Bonnaud, 2009), and both palliovisceral ganglia, in particular, show massive numbers of differentiated neurons before any other ganglia and long before hatching (Wollesen et al., 2010; Aroua et al., 2011; Buresi et al., 2013). These data suggest that cephalopod embryos develop the first components of their nervous system earlier than previously demonstrated and are consistent with recent studies that have revealed that S. officinalis embryos are able to react to tactile, chemical, or visual stimuli (Darmaillacq et al., 2008; Romagny et al., 2012). To examine further the early nervous system and correlate its development with the known sensory capacities of embryos, we decided to investigate the development of the juvenile epidermis in S. officinalis and the emergence of potential sensory structures in it. We focused our attention on the embryonic development of the outer epidermis, i.e., the epidermis that faces the outer environment (and not, for instance, lining the pallial cavity). The main tactile and chemical sensory structures of S. officinalis juveniles are located in this tissue and include (see Budelmann, 1996) the numerous isolated sensory neurons all over the body surface (Graziadei, 1964; Sundermann-Meister, 1978; Boletzky, 1989; Fioroni, 1990; Mackie, 2008; Baratte and Bonnaud, 2009); the olfactory organs, which correspond to two pits on the cheek hills (Woodhams and Messenger, 1974; Wildenbourg and Fioroni, 1989); and the eight lateral lines, which comprise epidermal ciliated cells along the head and arms (Sundermann, 1983). The latter structures are analogous to the mechanoreceptive lateral lines of fish and aquatic amphibians and able to sense local water movements (Budelmann and Bleckmann, 1988; Komak et al., 2005). We specifically searched for early expression of the S. officinalis homolog of pax3 and pax7 (Sof-pax3/7) as a potential marker of adult epidermis specification. In vertebrates, pax3 and pax7 code for transcription factors that intervene in the development of the neural crest cells, in particular, the progenitor cells of melanocytes and the future peripheral neurons (Le Douarin and Kalcheim, 1999). In addition, we examined the expression of the S. officinalis homolog of elav1 (Sof-elav1) in the embryo ectoderm, because this gene is one of the first markers of differentiating and differentiated neurons in most metazoans (Campos et al., 1987; Sakakibara

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et al., 1996; Denes et al., 2007; Marlow et al., 2008), including S. officinalis (Buresi et al., 2013). The differentiated sensory cells were finally described via confocal imaging in late embryos of S. officinalis and in hatchlings of other coleoid species for comparisons, in the nectobenthic species Euprymna scolopes (Sepiolidae) and in the pelagic species Doryteuthis opalescens (Teuthidae).

MATERIALS AND METHODS Animal care and staging of embryos Fertilized clutches of Sepia officinalis eggs were collected in the English Channel from the marine stations at Luc-sur-Mer (Universite de Caen Basse-Normandie) or at Roscoff (Universite Pierre et Marie Curie) between April and September and kept at 18 C in oxygenated sea water in the laboratory. Specimens were sampled daily to obtain a complete collection of different stages over the approximately 30 days of development. The darkly pigmented egg capsule and chorion were removed in sea water, and embryos were fixed in 3.7% paraformaldehyde (PFA) in phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4, pH 7.4) overnight at room temperature. After viewing with a stereo dissecting microscope to determine stages of development according to Lemaire, (1970; see Fig. 1 for the main stages of organogenesis). Embryos were washed for 3 3 5 minutes in PBS, dehydrated in baths of increasing glycerol concentration, and then stored at 220 C in 30% glycerol in PBS. Hatchlings of Euprymna scolopes and Doryteuthis opalescens were generously supplied from the laboratories of Margaret McFall-Ngai (University of Wisconsin) and William Gilly (Stanford University), respectively, where they were initially fixed in 4% paraformaldehyde overnight and then transferred to PBS for shipment and storage. All animal procedures were in compliance with the guidelines of the European Union (directive 86/ 609) and the French law (decree 87/848) regulating animal experimentation. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Sof-pax3/7 and Sof-elav1 cloning and probe synthesis Fragments of elav1 and pax3/7 sequences have been characterized in an embryonic EST library of S. officinalis (Bassaglia et al., 2012). The 687-bp fragment of Sof-elav1 was found to be orthologous to the neurogenic member of the elav/hu family (accession No. HE956712.1; see Buresi et al., 2013). The 783-bp fragment of Sof-pax3/7 (accession No. KF739402) con-

tained both the paired domain of the pax family and a homeodomain (with, respectively, 92% and 91% nucleotide identity with that of the Octopus bimaculoides pax37, FJ876143). For probe synthesis, total RNA of S. officinalis was extracted from a pool of embryos from stages 15 to 23 using Tri reagent (MRC, Cincinnati, OH). mRNAs were isolated with the gDNA Eliminator column and then converted into cDNA by Omniscript reverse transcriptase (Qiagen, Valencia, CA). Initial amplification primers for polymerase chain reaction were synthesized: for Sof-elav1, forward 50 AACTATCTTCCACAAACAAT-30 (NYLPQTM) and reverse 50 -GCCCCTTTAATGCTTTCACT-30 (SESIKGA); for Sofpax3/7, forward 50 -GTCTCACGGATGCGTCAGTA-30 and reverse 50 -TTCCGAGTCAGCGTCTGAGAT-30 . PCR conditions were 95 C for 5 minutes 1 (95 C for 30 seconds, 58 C for 30 seconds, 72 C for 1 minute) for 35 cycles 1 72 C for 10 minutes. The PCR products (of 347 bp and 427 bp, respectively) were cloned into TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by GATC Biotech (Konstanz, Germany). RNA probes were obtained with the digoxigenin (DIG) RNA labeling mix kit from Roche (Mannheim, Germany). According to the sense of PCR product insertion into the vector, the antisense probe was obtained with T3 polymerase (Roche), and the sense probe for control was obtained with T7 polymerase (Roche).

Sof-pax3/7 and Sof-elav1 in situ hybridization In situ hybridizations employing either the Sof-pax3/ 7 or the Sof-elav1 probes were performed, using at least three embryos at each of stages 17–30 (hatchlings). Embryos were treated in PTW (PBS plus 0.10% Tween 20) with proteinase K (10 lg/ml, 20–45 minutes, depending on the stage of the embryos). They were then fixed again for 1 hour in 3.7% PFA in PTW. A prehybridization step was performed in hybridization solution (HS; 50% formamide, 35 standard saline citrate, 0.5% sodium dodecyl sulfate, 1% Tween 20) with 33 lg/ml heparin and 400 lg/ml tRNA over 6 hours at 65 C. Embryos were next incubated overnight at 65 C in HS with the riboprobes together with 25 lg/ml heparin and 100 lg/ml tRNA. Excess probe was eliminated by four rinses (30 minutes each, 55 C) in HS and by progressive impregnation in standard 23 saline citrate (30 mM trisodium citrate, 0.3 M NaCl). Embryos were then bathed in MABT (100 mM maleic acid, 150 mM NaCl, 1% Tween 20, pH 7.5). Saturation was accomplished in blocking solution (MABT, 4% Blocking powder [Roche], 15% fetal bovine serum, for 1 hour at room temperature), followed by incubation

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Figure 1. Patterns of ectodermal Sof-pax3/7 expression throughout the organogenesis of S. officinalis, as revealed by in situ hybridization (see the stage number above the scale bars). A–C: Apical views of embryos showing Sof-pax3/7 expression patterns at stages 16, 18, and 20, respectively. Labeled and schematic representations are shown beneath each photograph in A–C. Three colors have been arbitrarily associated with the stained structures (pink for the “h” territory; blue for the mantle, the funnel, and arms 4 and 5; green for arms 1–3). D–F: Ventral views of embryos showing Sof-pax3/7 expression patterns on mantle, funnel, and ventral arms 4 and 5 at stages 22, 24, and 26, respectively, with photographs at left and labeled schematics at right. Arrows illustrate the progression of the ventral arm pillars (ap) and of the “h” zone on the diagrams in E,F. G–I: Dorsal views of embryos showing Sof-pax3/7 expression patterns on mantle, dorsal and lateral arms (1–3), and “h” territory at stages 22, 24, and 28, respectively, with photographs at left and schematics at right. Arrows illustrate the progression of the ventral arm pillars (ap) and of the “h” territory. J–M: Thin sections through the developing funnel (J, stage 22), the dorsal arm pillars (K, stage 22; L, stage 24), and the dorsal side of the mantle (M, stage 24). Stained tissues are indicated by solid arrowheads and unstained ectoderms by open arrowheads. Orientation: for apical views (A–C), dorsal is upward, animal right is right; for ventral views (D–F), posterior is upward, animal right is right; for dorsal views (G–I), posterior is upward, animal right is left. The letters mark various regions on the developing embryo. a1, a2, a3, a4, a5, arms 1–5; ap, arm pillar; ft, funnel tube; E, eye; M, mantle; MO, mouth; og, optic ganglion; oo, olfactory organ; ss, shell sac; Y, yolk. Scale bars 5 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Skin sensory development in cephalopods

overnight at 4 C with antidigoxigenin antibodies (Roche) coupled to alkaline phosphatase (AP) and diluted at 1:2,000 in blocking solution (MABT, 2.4% Blocking powder, 20% fetal bovine serum). AP activity was revealed by using 370 lg/ml nitroblue tetrazolium chloride (NBT) and 185 lg/ml 5-bromo-4-chloro-30 -indolyphosphate ptoluidine salt (BCIP; Roche). The reaction was stopped by washing in PTW solution. Embryos were then fixed again in 3.7% PFA in PBS for 24 hours. To visualize internal staining, some S. officinalis embryos were incubated in 15% sucrose, 7.5% gelatin in a 0.12 M phosphate buffer (pH 7.2) for 24 hours before being frozen in isopentane at 280 C and finally cut into 20-lm cryostat sections. Specimens used for negative controls (DIGlabeled sense probe and antidigoxigenin antibody conjugated to AP) did not show any pink staining.

Immunocytochemistry After storage (see above), specimens of all species were rinsed several times in fresh PBS and then quartered by a midsagittal cut and then another cut to separate the head and mantle regions. These parts were then incubated overnight in a blocking solution of PBS containing 1% bovine serum albumin, 1% dimethyl sulfoxide (DMSO), and 0.5% Triton X-100. The tissues were then incubated for 7–10 days in a 1:200 dilution of primary monoclonal antibody (6–11-B1; Sigma, St. Louis, MO) in blocking solution at 4 C. This monoclonal mouse antiserum was raised against an interphyletically conserved epitope of sea urchin a-tubulin and has been used previously to label axons and cilia in a variety of species, including S. officinalis (Baratte and Bonnaud, 2009) and other cephalopods (Wollesen et al., 2009). Tissues were then washed and incubated for 5–7 days in a 1:100 dilution of goat anti-mouse antibody conjugated to Alexa Fluor 488 (Invitrogen). Finally, the tissues were washed another four times for 20 minutes each in PBS and then mounted on microscope slides in a solution of three parts glycerol to one part 0.1 M Tris buffer (pH 8.0). Specimens processed as described above, but without incubation in the primary antibody, served as negative controls and did not show any staining.

Microscopy and image processing Embryos labeled by in situ hybridizations were observed with a Leica M16 2F binocular stereomicroscope and a Leica DMLB compound microscope. For detailed studies of tubulin immunoreactivity, specimens were viewed with a Leica SP5 confocal microscope. Stacks of optical sections were made at intervals of 0.5–1.0 lm. Maximum intensity projections were generated in ImageJ (http://rsbweb.nih.gov/ij/). All images were adjusted for contrast and brightness and

assembled into plates in Adobe Photoshop 8 or CS4 (Adobe, San Jose, CA).

RESULTS Main morphological features of development in S. officinalis In cephalopods, embryogenesis starts with a telolecithal and discoidal segmentation, and gastrulation gives rise to an animal pole shaped as an apical disc lying on a large yolk mass (Naef, 1928; Boletzky, 1988). In early organogenesis (stages 15–18 according to the staging of Lemaire, 1970), the 10 future arms, the mouth, and both eye primordia start to form at the periphery of this disc, whereas the mantle and the funnel primordial emerge in the center (Fig. 1A,B). Between stages 20 and 22, the arm crown and the mouth start to shrink; the optic areas swells, and the mantle starts to protrude (see the apical view in Fig. 1C). From stage 23 to hatching, the embryo takes on the general appearance of the juvenile, with the arm crown and the mouth (facing the yolk mass) corresponding to the anterior pole of the animal and the mantle occupying posterior regions. The dorsal and ventral sides become clearly distinguishable. On the developing ventral side (see Fig. 1D–F), the funnel primordial fuse to form the definitive funnel, and the pallial cavity differentiates with both gills inside. During this phase, the shape of the head drastically changes, and tissues from the arms expand to form the eyelids (see the dorsal side development, Fig. 1G–I).

Development of the juvenile epidermis and Sof-pax3/7 expression The embryonic ectoderm corresponds to the most external embryonic layer and is surrounded by a perivitellin liquid within the chorion. Sof-pax3/7 is first expressed from stage 16 in only a portion of the ectodermal tissues and not over the whole ectodermal surface. Only discrete ectodermal territories are Sof-pax3/ 7 positive, namely, the upper surface of the developing arms, the mantle and the funnel ectoderm, and an area located between the eyes and the mantle (arbitrarily labeled h; see labels in Fig. 1). As development proceeds, Sof-pax3/7-positive tissues expand to cover the entire outer surface of the prehatching embryo at stage 30 (Fig. 1D–F for the ventral side and Fig. 1G–I for the dorsal side of the embryo). Thin sections confirmed that these Sof-pax3/7-positive tissues correspond to an ectodermal layer and to the final outer juvenile epidermis (Fig. 1J–M), i.e., the epidermis facing the outer environment. Surfaces corresponding to future inner

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juvenile epidermis (inside the pallial cavity and inside the funnel tube) remained unstained (Fig. 1J,M). Focusing on the future head region (optic and cerebral areas), we observed that most of the early ectodermal tissues are Sof-pax3/7 negative (those arbitrarily labelled a–d, f, g, and i in Fig.1) but are eventually covered by overgrowing Sof-pax3/7-positive ectodermal tissues (h zones and arm tissues). The unstained tissues correspond to the various future cerebral and optic tissues within the juvenile head. The Sof-pax3/7-positive tissues of the embryonic arms correspond to their future outer surfaces (opposite the sucker side). From stages 21 to 22, these arm tissues give rise to ectodermal areas of Sof-pax3/7 expression that are called “arm pillars” (ap; Naef, 1928) and that progressed over the head surface (see “ap” and arrows in Fig. 1F–H). Specifically, on the dorsal side of the head, left arms 1, 2, and 3 give rise to a left dorsal Sof-pax3/7 arm pillar, and right arms 1, 2, and 3 give rise to a right dorsal Sof-pax3/7 arm pillar (see green areas in Fig. 1G–I,K). Similarly on the ventral side of the head, arms 4 and 5 give rise to two Sofpax3/7 ventral arm pillars (blue areas in Fig. 1E,F). The four arm pillars expand laterally over the Sof-pax3/7-negative ectodermal tissues and finally cover the eyes (Fig. 1E–H). Above each eye, the ispilateral ventral and dorsal arm pillars fuse and form a final outer eye layer, the superior lid (or “cornea”). The positive “h” zone corresponds to the posterior connective region of the eyelid (Naef, 1928). This tissue grows in a posterior-to-anterior sequence and finally joins the two lateral arm pillars to finalize the eyelid (Fig. 1I). As a result, all Sof-pax3/7negative ectodermal tissues of the head region (areas a– d, f, g, i, Fig. 1E–H,L) are ultimately covered by the Sofpax3/7-positive tissues derived from either the arms or the posterior eyelid (the “h” zone). Sof-pax3/7 is also expressed in the mantle ectodermal layer at stages 15–16 with the exception of the region that eventually forms the internal shell sac (Fig. 1A). Once the shell sac has been internalized (at about stage 18), Sof-pax3/7 expression persists in the mantle epidermis until hatching (Fig. 1A–I,M), with isolated cells showing a more intense staining at the anterior edge of the mantle surface from stage 19 (see Fig. 1D). The epidermis of the pallial cavity shows no staining (see open arrowhead in Fig. 1M). Sof-pax3/7 is also expressed in the outer surface of the developing funnel tube (Fig. 1A– F), whereas the inner epidermis of the funnel tube shows no Sof-pax3/7 expression (open arrowheads in Fig. 1J).

Development of the future juvenile epidermis and Sof-elav1 expression In early stages of organogenesis (stage 16), two small clusters of Sof-elav1-positive ectodermal cells can

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be observed within a head region that is otherwise Sofpax3/7 negative (labeled c in Fig. 1) and located posterior to the eye vesicles (Fig. 2A,B). While the optic areas of the head develops and changes form, these clusters grow and remain Sof-elav1 positive (Fig. 1C–E). At this stage, other isolated Sof-elav1-positive cells could be detected on the mantle edge (Fig. 2A,B). At stages 23 and 24, the embryo had reaches a juvenile morphology, and the two positive clusters occupy the definitive locations of the olfactory organs, located ventral and posterior to the eyes (Fig. 2F,G). Sof-elav1 expression persists in the olfactory organs until hatching (Fig. 2H,I). As the arm pillars (Sof-pax3/7-positive tissues) develop above the Sof-pax3/7-negative ectoderm, an ectodermal opening to the olfactory pit can be delineated (see below). The first developing lateral lines revealed by the Sofelav1 expression are observed on arms 2 at stage 24 (Fig. 3A), followed soon after by those on arms 3 (Fig. 3B,C). The developing lateral lines of arms 1 appear at stage 25, when developing lateral lines 2 and 3 show strong Sof-elav1 expression (Fig. 3D–F). On the ventral side of the head, the lateral lines of arms 4 appear at stage 24 (Fig. 3G). No developing lateral line are seen along arms 5. From stage 26, the arm pillars achieve their expansion over the eyes, and the lines 2–4 reach their final location on each side of the head (Fig. 3H,I). Isolated cells scattered over the developing epidermis of the whole embryo start expressing Sof-elav1 at about stages 24–25. On the mantle, the first cells to express Sof-elav1 appear on the edge at stage 24 (Fig. 4A) and then develop over the rest of the mantle epidermis (Fig. 4B–D). On the head, positive Sof-elav1 cells first appear on the arm pillars surface (Fig. 4E). Then, as the arm pillars cover the head and begin to form the juvenile epidermis, Sof-elav1-positive cells could be found scattered all over the head (Figs. 3E, 4F). Positive cells are also found on the sucker rings when they start expressing Sof-elav1 at about stage 28 (Fig. 4G–I).

Details of sensory elements in late embryos and hatchlings of Sepia and other coleoids Confocal microscopy provided detailed views of the various sensory cells labeled with the antibody against acetylated tubulin; peripheral sensory cells could be detected in the olfactory organs, in the lateral lines, in the suckers, and scattered over the rest of the outer epidermis of S. officinalis. The locations and numbers of these cells correspond well with the expression patterns of Sof-elav1, as reported above. By stage 25 (the earliest time examined), the olfactory organs could be detected as concentrations of

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Figure 2. Whole-mount and section views of the developing olfactory organ in S. officinalis embryos from stage 16 to stage 30 labeled by Sof-elav1 in situ hybridization (see the stage number above the scale bars). A: Apical view of stage 16 with the developing olfactory organ expressing Sof-elav1 (arrow). B: Apical views of stage 17 embryo (on the left) and magnified view (inset at right) showing Sof-elav1-positive cells in the developing olfactory organ (arrow). C: Developing olfactory organ expressing Sof-elav1 at stage 20 (arrow). D: Ventral view of the developing olfactory organ at stage 23 showing a cluster of Sof-elav1-positive cells (arrow). E: Section view through the olfactory organ at stage 23. F: Ventral view of the olfactory organ expressing Sof-elav1 at stage 24 (arrow). G: Section view through the olfactory organ at stage 24. H,I: Views of embryonic olfactory organs (arrows) expressing Sof-elav1 at stages 26 and 28, respectively. Orientation: for apical views (A–C), future dorsal side is upward, future ventral side is downward; for lateral views (D–I), anterior is downward. The letters mark various regions on the developing embryo (see Fig. 1); a1, a2, a3, a4, and a5, arms 1–5; E, eye; fp, funnel pouch; ft, funnel tube; M, mantle; MO, mouth; opsi, optic sinus; pvg, palliovisceral ganglion; st, statocyst. Scale bars 5 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

dimly immunoreactive elements lying beneath the epidermis on the ventrolateral surface of the head, anterior to the edge of the mantle. By stage 27, the

immunoreactivity had become more intense, and details could be better observed. The olfactory organ is a pit with an opening to the outside environment through the

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Figure 3. Whole-mount and section views of the developing lateral lines of S. officinalis embryos from stage 24 to stage 30, labeled by Sof-elav1in situ hybridization (see the stage number above the scale bars). A: Emerging lateral lines 2 on the dorsal side of the head of a stage 24 embryo. B: Emerging lateral lines 3 on the dorsal side of the head of a late stage 24 embryo. The lateral lines 2 continue along the upper surface of the arms 2. C: Section (see A for location) through the left arm pillar (ap) and the developing lateral lines 2 and 3 at stage 24. D: Dorsal head surface at stage 25 showing the well-developed lateral lines 2 and 3 and both emerging lateral lines 1, facing arms 1. E: Higher magnification of the left lateral lines 2 and 3 at stage 25. Isolated positive cells are also visible between the lateral lines (arrowheads). F: Section (see D for location) through the left arm pillar and the developing lateral lines 2 and 3 at stage 25. G: ventral head surface at stage 24 showing the right developing lateral line 4, continuing along the upper surface of the arm 4. H,I: Lateral view (anterior is right) of the head of embryos at stages 26 and 30, respectively, showing lateral lines 1–4. Orientation: for ventral and dorsal view (A–G), posterior is upward, anterior is downward. The letters mark various regions on the developing embryo (see Fig. 1); a1, a2, a3, a4, a5, arms 1–5; ap, arm pillar; E, eye; M, mantle; og, optic ganglion; oo, olfactory organ. Scale bars 5 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

epidermis and is covered with numerous tufts of long cilia (Fig. 5A). The lumen of the pit contains numerous cilia that appear to originate from hundreds of underlying round or slightly elongated, immunoreactive profiles as viewed from above (Fig. 5B,C). A stout olfactory nerve exits from the base of the olfactory organ. The lateral lines were also first detected at stage 25 as dimly stained structures. Closer examination revealed dimly immunoreactive cells one or two abreast along the length of each line. By stages 26–27 these

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cells had become more intensely immunoreactive and appeared to become more numerous, often appearing four or five abreast in the lines as they extended from head regions around the eyes into the arms (Fig. 5D). Closer examination revealed numerous nerves associated with each line (Fig. 5E), and even higher magnifications revealed that each sensory cell bears a short tuft of cilia from its apical pole and a single axon from its basal pole (Fig. 5F). The epidermis between the lateral lines on the head and arms, and that over the mantle,

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Figure 4. Whole-mount and section views of the epidermis of S. officinalis embryos from stage 24 to stage 30, labeled by Sof-elav1 in situ hybridization (see the stage number above the scale bars). A: Ventral side of the mantle of a stage 24 embryo with the first positive Sofelav1 cells, located at the mantle edge. B: Ventral side of the mantle of a stage 25 embryo with numerous Sof-elav1 positive epithelial cells. C: Magnified view of an area next to the mantle edge at stage 24 (see B for location) and numerous positive Sof-elav1 cells (arrowheads). D: Section through the lateral side of the mantle at stage 25 with a few Sof-elav1-positive cells (arrowheads). E: Section through arm 1 and head epidermis (above) at stage 25, with epithelial cells expressing Sof-elav1 (arrowheads). F: Magnification on the dorsal head epidermis (between lateral lines 2 and 3) of a stage 30 embryo with epithelial cells expressing Sof-elav1 (arrowheads). G–I: Whole-mount views of the developing arm 3 suckers in an S. officinalis embryo at stage 28, labeled by Sof-elav1in situ hybridization and showing isolated positive cells (arrowhead). Orientation: posterior is upward, anterior is downward. a1, arm 1; ft, funnel tube; M, mantle; me, mantle edge; oo, olfactory organ. Scale bars 5 200 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

possesses numerous large tufts of long cilia (Fig. 5D,G,H). Scattered between these tufts are numerous cells with a morphology similar to that of the cells of the lateral lines of S. officinalis; each cell bears both a small tuft of short cilia extending above the epidermal surface and also an axon from its base. As they developed, the suckers also possess numerous sensory cells that are particularly abundant around the rim of the adherent surface (Fig. 5I). The morphology of these cells is distinct from that of the sensory

cells of the epidermis described above. These cells possess a much longer apical process that extends from the cell body. Cilia project from the ends of these processes (Fig. 5J). Finally, confocal microscopy of acetylated tubulin immunoreactivity also allowed us to determine whether cells types observed in S. officinalis were generalized to hatchling specimens of other cephalopods. Essentially, all observations in Doryteuthis opalescens and Euprymna scolopes were consistent with the findings in

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Figure 5. Confocal micrographs of sensory structures on S. officinalis embryos stained with antiacetylated tubulin antibody (see the stage number above the scale bars). A: Right olfactory organ in a stage 27 embryo. The opening of the olfactory pit is lined with numerous, tightly packed immunoreactive profiles. The epidermis surrounding the pit bears large tufts of cilia (examples indicated by arrows). B: Higher magnification view of numerous cilia projecting into the lumen of the olfactory pit of a stage 27 embryo. These cilia (numerous fibrous profiles toward top left) appear to exist singly or in smaller bundles than the cilia in tufts on the surrounding epidermis (examples indicated by arrows). C: A stout olfactory nerve (Olf N) exits the olfactory organ. D: Lateral lines 2–4 around the right eye. Numerous tufts of long cilia provide a scattered covering over the epidermis between the lateral lines. E: Numerous sensory cells along a lateral line (indicated by brackets) send axons into a peripheral nerve (large arrow) of a stage 30 embryo. Tufts of long cilia are plentiful away from the lateral lines (small arrows) and are similar to the tufts located over much of the rest of the outer surface of the embryo. F: Higher magnification of cells along a lateral line of a stage 27 embryo show both the axons projecting from the bases of peripheral sensory cells and also the smaller tufts of short cilia projecting from the apical surface of each of the cells. G: Sensory neurons on the mantle epidermis. Numerous sensory cells (examples indicated by arrows) are scattered among the large tufts of cilia. H: Higher magnification view of the boxed area in G. The trio of sensory cells in the center possesses prominent axons and short cilia, which project to the surface of the epidermis. I: Sensory neurons surrounding the rim of the suckers on an arm. J: Higher magnification of immunoreactive cells with long apical projections (example indicated by large arrow) bearing cilia (small arrows) on the sucker along an arm. Scale bars 5 100 lm in A; 50 lm in B,C,E; 300 lm in D; 25 lm in F–I; 10 lm in J.

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Figure 6. Sensory structures in hatchlings of Doryteuthis opalescens (A–D) and Euprymna scolopes (E–G) stained with antiacetylated tubulin antibody. A: Immunoreactive elements in the olfactory organ. Although this high-magnification view shows numerous round profiles of some immunoreactive elements (toward top left) other elements appear as elongated cones projecting from more dimly fluorescent underlying cell bodies (arrows), which in turn give rise to the fibers of the olfactory nerve (Olf N). B: axons from cells along the lateral lines fasciculate to form stout nerves (arrows). C: Suckers each possess numerous sensory cells with axons and apical processes bearing cilia. Additional sensory cells along the arm are also visible at the bottom of the image. D: In addition to the cells forming the extensions of the lateral lines along the midlines of the arms (indicated by bracket), these appendages also possess many more sensory cells along their sides (examples indicated by arrows). Autofluorescent mouth parts flank the central arm. E: Olfactory organ of E. scolopes. The numerous cilia projecting into the lumen of the olfactory pit appear as a fibrous covering over large and round profiles. F: Two examples of sensory cells in the epidermis. Larger arrows indicate tufts of short cilia projecting through the surface of the epidermis. Both cells possess axons from their basal surfaces. The small arrow indicates what appears to be a bifurcation of one of the axons. G: The large arrow indicates an example of one class of immunoreactive cells with a long apical projection bearing cilia on the adherent surface of the sucker. The small arrows indicate cells thata appear to have shorter apical projections, which terminate on the sides of the suckers. Scale bars 5 25 lm in A,B,D,G; 50 lm in C,E; 10 lm in F.

S. officinalis described above, although additional details could occasionally be better observed in these other species. In particular, because of its smaller body at hatching, D. opalescens generally afforded better reagent penetration and microscopic visualization than the larger species. Figure 6A shows details of the cells of the olfactory organs in D. opalescens. Although intensely immunoreactive elements appeared to be generally round in profile when viewed from above, as in S. officinalis, a lateral perspective of these elements revealed elongated profiles, which appeared to originate from dimly immunoreactive, nucleated cell bodies. The axons of

the olfactory nerve appeared to arise from these underlying cell bodies. As with S. officinalis, each of the lateral lines is associated with peripheral nerves, which appeared to be composed of afferent axons (Fig. 6B). Each of the suckers of D. opalescens contains numerous axons and immunoreactive cells bodies, many of which possess long apical processes that project toward the adherent surface (Fig. 6C). Each arm contains a midline row of sensory cells, constituting the termination of each lateral line on its upper surface and numerous additional sensory cells along each side. Figure 6E shows numerous cilia projecting into the lumen of the olfactory pit and generally round profiles

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of underlying immunoreactive elements in E. scolopes. Figure 6F shows immunoreactive sensory cells in the epidermis with their apical tufts of cilia and basal axons. Occasionally the axons appear to branch and may contribute to a peripheral neural network lying beneath the epithelium. Details of the sucker of E. scolopes are shown in Figure 6G. Some cells with long apical processes terminate with tufts of short cilia on the adherent surface, whereas cells with shorter apical projections are located on the sides of the suckers and are more typical of the types of sensory cells located elsewhere in the epidermis of E. scolopes and the other cephalopods examined.

DISCUSSION Early tissue specification during development of the juvenile epidermis in S. officinalis Sof-pax3/7 expression provided a specific marker to follow the ontogeny of the epidermis that covers the outer surface of juvenile of S. officinalis and a novel view of the developmental dynamics of this tissue. Specifically, we initially detected Sof-pax3/7 transcripts in multiple, distinct ectodermal territories rather than in the whole of the early embryonic ectoderm. These domains ultimately expanded to form the entire outer epidermis of the juvenile (Fig. 1). Our findings are thus consistent with previous reports that the epidermis that eventually covers the head of the juvenile could be derived from different embryonic territories, in particular, from the arms (i.e., from a pedal origin; Arnold, 1984; Boletzky, 2003; Shigeno et al., 2008). In contrast, the inner skin (that lines the pallial cavity, the shell sac, and the internal surface of the funnel) appears to arise from regions of the early ectoderm that do not express Sof-pax3/7. In addition to differences in their ontogeny, the outer skin and the inner skin also possess several other distinguishing characteristics. For example, chromatophores develop only in the outer epidermis of the juvenile cuttlefish (Hanlon and Messenger, 1988). Chromatophores are specific pigmentary–muscular structures involved in camouflage and intraspecific communication in coleoids, and the motoneurons that control their expansion create a dense nervous network under the outer epidermis (Dubas et al., 1986; Mackie, 2008). It is also in the outer epidermis that a majority of peripheral sensory nerve cells develop, as shown by Mackie (2008) and in this report. Conversely, no corresponding sensory cells have been reported for the inner skin within the mantle cavity or inside the funnel tube (Mackie, 2008). These observations of pigment cells

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and nerve cells derived from domains expressing Sofpax3/7 find parallels in vertebrates, in which pax3 and pax7 have been shown to be expressed in the cells of the neural crest, which develop into both melanocyte pigment cells and peripheral sensory neurons (Mansouri et al., 1996; Le Douarin and Kalcheim, 1999; Lacosta et al., 2007), suggesting commonalities in the developmental programmes of these otherwise very different groups of animals. The olfactory organs differentiate within an ectodermal territory that does not express Sof-pax3/7, although the organs themselves do show a slight Sofpax3/7 expression (see Fig. 1E), which could imply a role in the development of these sensory structures. Ultimately, as the arm pillars invade this area and cover the head, the olfactory organs are overgrown by outer epidermis, although two small epidermal pores (or nostrils) are formed that provide an access for the olfactory cells to the outer environment (Fig. 5A).

Sof-elav1 as marker for early differentiation of the olfactory organs in cephalopods Sof-pax3/7 expression provided new insights into the differentiation of the epidermis, and the expression of Sof-elav1, a member of the elav/hu family and one of the first genetic markers of postmitotic neural cells in metazoans (Campos et al., 1987; Sakakibara et al., 1996; Buresi et al., 2013), permitted early labelling of developing neurons in S. officinalis. Expression patterns of Sof-elav1 thus provided a new perspective of the early development of the peripheral nervous system, as they did previously for the developing central nervous system of this organism (Buresi et al., 2013). Here, Sofelav1 expression demonstrates that the two developing olfactory organs were the first ectodermal sensory structures in which differentiating neurons were detected, at about stage 16 (Fig. 2A). In this regard, Sepia appears to be similar to other molluscs, such as the gastropods, in which chemosensory tentacles and rhinophores also appear early in development (Croll et al., 2003; Dickinson and Croll, 2003; Wollensen et al., 2007). This comparison suggests a conserved reliance on olfaction from early stages of development across the molluscs, but further research is obviously warranted, because homology between the olfactory organs of cuttlefish and the rhinophores and tentacles of gastropods has not been established. Also, caution must be exercised when inferring the functionality of the early olfactory system based solely on the initial detection of the peripheral sensory somata. For example, in adult Octopus vulgaris, afferent axons from each of the olfactory organs project to the ipsilateral

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olfactory lobe, located between the optic lobes and the brain (Boycott and Young, 1956; Woodhams and Messenger, 1974). At the time of olfactory organ differentiation in Sepia, only the palliovisceral gangliogenic territories show massive neuronal differentiation (as indicated by massive Sof-elav1 expression [Fig. 2A]; Buresi et al., 2013), and the embryonic origins of the olfactory lobes are unknown. It is, therefore, not clear when the second order neurons are in place to receive the afferent information from olfactory sensory neurons. Further research is also needed to elucidate the development of functional olfactory pathways in cephalopods. Our results nonetheless indicate that the olfaction system develops early and are consistent with the previous finding that early olfactory abilities are exhibited from stage 23 onward in S. officinalis (Romagny et al., 2012). In addition to rhinophores and tentacles, other molluscs, such as scaphopods, polyplacophores, gastropods, and bivalves, also have chemosensory apical organs from very early stages of development (Croll, 2009). This organ has been implicated in the detection of environmental cues that trigger settlement and metamorphosis, but additional sensory roles have also been indicated (Kuang et al., 2002; Voronezhskaya et al., 2004). Moreover, the sensory cells in apical organs of other molluscs are similar in morphology to those of olfactory organs in cephalopods (see below), with their deep internal pockets containing bundles of cilia (Bonar, 1978; Dickinson and Croll, 2003; Kempf and Page, 2005). For Sepia embryos, however, we found no evidence for any unpaired, medial Sof-elav1 expression or tubulin-like immunoreactivity near the mouth or developing cerebral region that could indicate vestiges of an apical organ. However, technical difficulties (e.g., chorion adherence and poor fixation of yolk) precluded examinations of species before stage 15 in the present study.

Differentiation of sensory neurones in lateral lines and other regions of the epidermis Because they are located in the developing outer epidermis, the lateral lines and isolated sensory cells scattered over the body are associated with the development of tissues expressing Sof-pax3/7. For instance, the sensory neurons of the lateral lines run all along the head, from the arms to the neck region, and they acquire their final “lateral” location as soon as the arm pillars reach their ultimate extension over the eyes. As revealed by Sof-elav1 expression, the lateral lines start differentiation from stage 24, as the arm pillars of arms 1–4 start their extension (Fig. 3). With regard to

the developing isolated sensory cells of the epidermis, the first detectable Sof-elav1-positive cells were observed at stage 16 at the mantle edge (Fig. 1A,B) and probably correspond to early developing neurons previously reported (Baratte and Bonnaud, 2009). Later, from stage 24, positive cells also appear on the funnel and on the arm pillars (Fig. 4). The Sof-pax3/7 expression in these areas is first ubiquitous on the outer epidermis and then becomes restricted to isolated cells (see Fig. 1D) at the anterior edge of the mantle, reinforcing the possibility that Sof-pax3/7 specifically determines the development of sensory cells.

Anatomy and development of peripheral sensory neurons Although the expression of Sof-elav1 provides a sensitive early marker for the differentiation of peripheral neurons, tubulin-like immunoreactivity revealed details of the sensory cells in S. officinalis. Double labeling was not performed to demonstrate localization of Sof-elav1 transcripts in the sensory cells with tubulin immunoreactivity, but the locations and numbers of all such cells corresponded well. In addition, immunocytochemistry also provided a basis for morphological comparisons of those peripheral neurons with the ones in Euprymna scolopes and Doryteuthis opalescens and in other cephalopods (Mackie, 2008). Although no concerted effort was made here to determine the earliest time at which the cells could be detected during ontogeny, the dimmer staining and lower numbers of cells relative to later stages suggest that stage 25 was near the time when the cells first stained sufficiently to be recognizable as neurons. For sensory neurons in the lateral lines, in the suckers, and scattered over much of the outer surfaces of the head, this time is consistent with the first expression of Sofelav1; however, future studies must determine when the peripheral sensory neurons, especially those of the olfactory organs, first differentiate. The description of the olfactory neurons of all three species studied here is consistent with previous studies in cephalopods that used classical histology and/or electron microscopy and described several classes of sensory neurons in the olfactory organs (Woodhams and Messenger, 1974; Emery 1975, 1976; Wildenburg and Fioroni, 1989; Mobley et al., 2008). One class possessed cilia projecting above the surface of the epithelium, and the existence of these cells likely explains, at least in part, the numerous cilia that we observed projecting into the lumen of the olfactory pit. Other classes of neurons possess deep pits containing tight bundles of partially internalized cilia, consistent with our observations of intensely tubulin-immunoreactive elements

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between the epithelia surface and an underlying layer of dimly fluorescent cell bodies. This study thus provides a framework and suggests tools for a focused examination of the early ontogeny of the olfactory organs in cephalopods correlating this development with behavioral assays for chemoreception in the embryos of these animals (Romagny et al., 2012). As with the olfactory organs, our observations of sensory neurons in the lateral lines and scattered in the epidermis covering the head and arms of all three species are also consistent with previous descriptions for other cephalopods (Sundermann-Meister, 1978; Sundermann, 1983; Budelmann 1996; Mackie, 2008). The neurons that we observed in these regions possessed a common morphology, with a small, short bundle of cilia projecting above the surface of the epidermis from the apical pole of these cells and what appears to be a centrally projecting axon from the basal pole of the cells. Apparent branching of that axon, however, is also consistent with the possibility that, in addition to providing afferent input to the developing brain, these sensory cells might also contribute to a peripheral nerve net as previously discussed for cephalopods (Mackie, 2008) and other molluscs (Croll et al., 2003). Although sensory cells in the lateral lines of cephalopods have been shown to be mechanosensory (responding to water-borne vibrations; Budelmann and Bleckmann, 1988), the sensory modality of those scattered through the rest of the epidermis has yet to be determined. We also detected what appeared to be a different class of peripheral sensory neurons around the outer rim of each sucker. These cells possessed a long apical process interposed between the underlying soma and the surface of the epidermis, where a small, short bundle of cilia was borne. These cells are thus distinct from the sensory cells found elsewhere in the epidermis covering the head and arms and are very similar to those found in the rim of Octopus vulgaris suckers (Graziadei and Gagne, 1976). As with the sensory cells found scattered in the epidermis covering other regions of the body, the sensory modality of the tubulin-immunoreactive cells in the suckers is presently undetermined, especially in light of the fact that the suckers appear to be sensitive to many types of stimuli, including chemicals, vibrations, and textures (Wells et al., 1965).

CONCLUSIONS AND EVOLUTIONARY PERSPECTIVES We demonstrate here that peripheral sensory structures of the juvenile S. officinalis have two distinct embryonic origins; the olfactory organs develop early

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within a primordial head ectoderm that does not express Sof-pax3/7 (Fig. 2), whereas other classes of sensory cells develop from Sof-pax3/7-positive domains that form the outer epidermis of the juvenile. This outer epidermis derives from discrete ectodermal territories that expand from two main domains: 1) the mantle ectoderm, which is already at its final location and in which neurons differentiate in early stages of organogenesis (Baratte and Bonnaud, 2009), and 2) the arm pillar ectoderm, which spreads and extends over the head surface from stage 22 and in which sensory cells develop between stage 25 to hatching, as do several other epidermal structures, including iridophores (Andouche et al., 2013) and chromatophores (Bassaglia et al., 2013). This process is similar to an event observed during the metamorphosis of gastropods, i.e., the spreading of epidermis from the larval foot over the entire body (Bonar, 1972, 1976). The cephalopod arms are hypothesized to have a pedal origin (Boletzky, 2003), so the arm pillar extension may represent an ancestral molluscan ontogenetic feature conserved in cephalopods. This finding, together with the differentiation of early sensory neurons (see also Baratte and Bonnaud, 2009) and particularly the early differentiation of olfactory organs, provides interesting parallels between the direct development of cephalopods and the development of other molluscs that occurs through a larval stage. Such results open new and exciting perspectives on the evolution of the developmental processes in molluscs and lophotrochozoans.

ACKNOWLEDGMENTS We thank J. Henry and L. Dickel (University of Caen) and S. Henry, L. Leve`que, and X. Bailly (Roscoff Biology Station, UPMC) for providing specimens of Sepia. We also thank Judit Pungor and Danna Staaf in the laboratory of William Gilly (Hopkins Marine Laboratory, Stanford University) and Nell Bekiares in the laboratory of Margaret McFall-Ngai (University of Wisconsin) for providing specimens of Doryteuthis and Euprymna, respectively. Charlotte Farquharson procured specimens of various species from different laboratories and performed initial immunocytochemical staining as her honors research project in biology at Dalhousie University. Finally, R.P.C. wishes to thank the students and staff of the CNRS Unit UMR7009 at the Observatoire Oceanologique de Villefranche-surMer for hosting a sabbatical visit during which a portion of this work was performed.

CONFLICT OF INTEREST STATEMENT We confirm that there are no known conflicts of interest associated with this publication.

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ROLE OF AUTHORS The manuscript has been read and approved by all named authors, and there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Acquisition of data: AB, RPC, ST; Analysis and interpretation of data: AB, SB, RPC; Drafting of the manuscript: SB, RPC; Critical revision of the manuscript for important intellectual content: LB, ST.

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