In France, cuttlefish experiment and maintenance are covered under European Union guidelines (Directive 86/609) and the French law (decree 87/848) regulating animal experimentation that does not concerns embryos before hatching. Nonetheless, all experiments were performed according to France and European ethical guidelines in the treatment and handling of all animals used within this study. Fertilized eggs of S. officinalis used in this study come from SMEL (Synergie MEr et Littoral, Blainville, France). The protocols for the staging and the fixation of the embryos are detailed in Buresi et al. (2012).

mRNA fragments of Sof-six1/2, Sof-eya, Sof-dac, and Sof-opsin were characterized in an embryonic EST library of S. officinalis (ADY0AAA48YE16CM1, tc_01401, ADY0AAA73Y015CM1, and tc_01048 respectively; Bassaglia et al., 2012). For the phylogenetic analyzes all alignments were performed using MAFFT and the G-INS-I iterative refinement method (Katoh and Standley, 2013). The best maximum-likelihood trees were inferred using PhyML with the WAG evolutionary model and 100 bootstrap replicates. Each of the genes has been tested in a phylogenetic work including genes from other metazoans. Six is identified as a six1/2, and our opsin sequence shows exactly the same sequence as that of AY450853 and that of O16005, identified as a true Gq-coupled/rhabdomeric photoreceptor opsin in the phylogenetic tree of Yoshida et al. (2015). From these characterized sequences, specific primers were designed for PCR amplification: Sof-rhodopsinF3 (5′-GTACAACCCCACCATGGAGG-3′) and Sof-rhodopsinR3 (5′-CGCCGATGAAGCCGTATACT-3′), Sof-six1/2F3 (5′-CCTCCCATGCTTCCATCGTT-3′) and Sof-six1/2R3 (5′-GAAATTTTCGGCGGACCCTG-3′), Sof-eyaF1 (5′-ACCTACACGAGGTGGTCGTC-3′), and Sof-eyaR1 (5′-CCACGGACTCCAGTTGCTAT-3′), Sof-dacF1: (5′-CGGCCAGAAGCACCAGTTAT-3′) and Sof-dacR1 (5′-CAGTGCTTCACCATTGGGGACT-3′). They were used to amplify respectively a 311, 346, 372, and 400 bp fragment. Sof-dac was cloned as described in the protocol of Buresi et al. (2012). Concerning Sof-six1/2, Sof-eya, and Sof-rhodopsin genes, each of the genes has been synthetized and cloned by Genecust (Dudelange, Luxembourg). The probes for in situ hybridization were synthetized from theses plasmids.

From stage 23 on, it is necessary to observe the expression on sections to localize the expressing cells. Embryos used for cryo-sections in situ hybridization were impregnated in 0.12M phosphate buffered (0.08M di-sodium hydrogen ortho-phosphate, 0.02M sodium dihydrogen phosphate dehydrate) plus 30% sucrose treated with 0.1% DEPC (Diethyl pyrocarbonate) for 48 h at 4°C. Then, they were included in Tissue-Tek and blocks were frozen in isopentane cooled at −80°C for 1 min. Sections of 20 μm were performed with cryostat Leica and used for ISH experiments. From stage 23 to stage 30, the expression patterns of those genes were stained by a cryo-sections in situ hybridization. Note that control negatives were used for each slide as a test for the same embryo selected and each gene studied. Unless otherwise specified, all steps of the experiments were performed in a humid chamber at room temperature. After 30 min at room temperature, the sections were rehydrated 2 times in 1X phosphate buffered saline (1X PBS) with 0.1% DEPC and treated 1 time in standard 5X saline citrate (75 mM tri-sodium citrate, 0.75 M NaCl) each time for 15 min. A prehybridization step was done in hybridization solution (HS) for 2 h at 65°C (50% deionized formamide, 5X standard saline citrate, 40 μg/ml salmon sperm DNA, 5X Denhardt's, 10% Dextran sulfate). Sections were next incubated overnight at 65°C in HS containing 300 ng/ml of probes. Excess probe was removed by 2 rinses in standard 2X saline citrate (30 mM tri-sodium citrate, 0.3 M NaCl) (respectively 30 min then 1 h, 65°C). Slides were washed for 1 h at 65°C in standard 0.1X saline citrate (0.015 mM tri-sodium citrate, 15 mM NaCl). Sections were then treated twice (15 min each) with MABT (100 mM Maleic acid, 150 mM NaCl, 1% Tween20, pH 7.5). Saturation was performed for 1 h in blocking solution (MABT, 4% Blocking powder (Roche), 15% fetal bovine serum), followed by incubation for 1 h at 4°C with anti-digoxigenin antibodies (Roche) coupled to alkaline phosphatase (AP) and diluted at 1:500 in blocking solution (MABT, 1% Blocking powder, 5% fetal bovine serum). Excess antibody was eliminated by 3 rinses (10 min each) in MABT then 3 rinses (5 min each) in PTW (PBS plus 0.10% Tween20). Sections were impregnated for 20 min in AP solution (100 mM tris hydrochloride, 50 mM Magnesium chloride, 0.1% tween20) with 100 μM levamisole hydrochloride (Sigma, France). The revelation of AP activity was conducted in AP solution (100 mM tris hydrochloride, 50 mM Magnesium chloride, 0.1% tween20 plus 1 mM levamisole hydrochloride) containing 165 μg/ml BCIP (5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt) and 330 μg/ml NBT (nitro-blue tetrazolium chloride) (Roche). The reaction was stopped by washing 3 times (10 min each) in 1X PBS. The slides were treated with DAPI (4′,6-diamidino-2-phenylindole; 25 μg/ml).

For fluorescent in situ hybridization, a POD-coupled anti-digoxigenin antibody (Roche) diluted 1:500 was used and bound antibodies were revealed using FITC-tyramide diluted 1:200 in PTW containing 0.001% of hydrogen peroxide, at room temperature for thrice 45 min, in the dark. After washing, the sections were mounted in Mowiol.

A Leica M16 2F binocular stereomicroscope was used to observe the embryos labeled by in situ hybridizations. For cryo-sections ISH, the sections were observed under a Leica DMLB compound microscope. All images were taken by a camera color CoolSnapPro and treated using Adobe Photoshop Elements 9 (Adobe, CA, USA) for contrast and brightness.

The embryonic development of S. officinalis has long been the subject of morphological research (e.g., Naef, 1928; Lemaire, 1971; Boletzky, 1989, 2003; Boletzky et al., 2006; for review Boletzky et al., 2016). The authors have described 30 stages along three main periods. The cephalopod eye is composed of numerous structures, analogous to those of vertebrates (Figure 1) and its development has been described for long time. In S. japonica, the development has been described in 40 stages with four phases of retinal differentiation (Yamamoto, 1985). Here we described the development of the S. officinalis retina based on that of S. japonica. The eye morphogenesis of cuttlefish is characterized by four successive ectodermal folds and begins at stage 15 when the ocular primordium is visible. At stage 16, the invagination of the ocular primordium yields the primary optic vesicle that becomes completely closed at stage 18 (Figure 2). Development of the retina of S. officinalis begins at stage 18, when the primary optic vesicle is closed and appears as a single layer with uniform columnar cells (Figure 2). At stage 21, the primary cornea develops from an outer fold surrounding the retinal thickening and the iris develops from an external second ectodermal fold (Figure 2). The lens starts to form at this stage, first teardrop shaped and becomes subspherical at stage 25 (Figure 2). Between stages 21 to 25 (corresponding to stages 24–29 in S. japonica), differentiation of two cell types begins. At stage 24, the retina begins to be slightly colored with orange in the periphery of the middle retina. From stage 25 on, the eyes are entirely orange and darken until hatching. Further in the development (stages 25 and 26), a third ectodermal fold covering the eye appears to form a secondary cornea (Figure 2). During the process of establishment of the secondary cornea, the photoreceptor cells and supporting cells begin to differentiate. From stage 28 on, the photoreceptors continue to grow and complete their specific differentiation, forming rhabdomeric cells. The end of the embryonic period at stage 29 is marked by the formation of the eyelid (Figure 2). At hatching, the eye is completely functional (Gilbert et al., 1990).

The eye morphogenesis in numerous metazoans is controlled by several important genes that includes pax, six, eya, and dac. They were shown to play key developmental roles in non-retinal structures and tissues of vertebrates and Drosophila. Blast analyses from our S. officinalis EST library revealed the existence of ESTs putatively encoding for Sof-dac, Sof-six, Sof-eya and Sof-opsin. However, since it was previously shown that cephalopods contain several opsins (Yoshida et al., 2015) or more than one six gene (Koenig et al., 2016), we performed Maximum-likelihood tree based analyses to confirm the identity of ESTs studied here. Using a set of chordate, lophotrochozoan, and ecdysozoan published sequences we obtained a phylogenetic position for the eyes absent (EYA) (Figure 3), sine oculis homeobox (SIX) family (Figure 4), dachshund (Figure 5), and opsins (Figure 6) ESTs. Moreover, to strengthen our phylogenetic analyses, we also used unpublished data corresponding to a recent draft assembly of several transcriptomes of juveniles from S. officinalis. Our data demonstrate that our six EST belong to the Six1-Six2 Clade (Figure 4), and that the opsin EST is identical to a previous opsin of the Clade II Gq-coupled/rhabdomeric opsin Yoshida et al., 2015 (Figure 6).

Our expression data show that Sof-eya expression is restricted to the eye area and surrounding tissues at stages 20 and 21. Sof-six1/2 is also expressed in the eye area during the early stages of organogenesis (from stage 20 to stage 21) in S. officinalis. Surprisingly, no Sof-six1/2 and Sof-eya expression is shown in the eye area before stage 20 and after stage 22. Eya and Six have been strongly proposed to have an ancient role in eye development and their orthologs are involved in visual system development in both “invertebrates” and vertebrates (Vopalensky and Kozmik, 2009). The analysis of Eya genes expression in vertebrate eye development has shown that the three Eya genes, Eya1, 2, 3 are differentially expressed in the developing eye (Xu and Saunders, 1997). Indeed, Eya1 is expressed in the lens, optic stalk, and neural retina (Xu et al., 1997). Eya2 is expressed in the neural retina only. Eya3 is present in the optic vesicle and the periocular mesenchyme, but both genes Eya2 and Eya3 are absent in the lens (Xu et al., 1997). In Drosophila, sine oculis has been shown to be required for the development of both, the compound eyes and the ocelli (Cheyette et al., 1994). Indeed, it has a critical role for the development of the entire visual system (Gehring, 2002). In mouse, sine oculis orthologs six1/six2 are known to be expressed in the adult differentiating cells of the retina (Oliver et al., 1995). In protostome, six1/2 homologs are known to be important for the early specification of the visual system (Cheyette et al., 1994; Arendt et al., 2002). The role of six1/2 homologs in early visual system specification has been proposed to be evolutionary conserved outside Bilateria (Stierwald et al., 2004). Recent studies in cephalopods indicate that six2, six3, and eya expressions have been observed in the eye area, the optic lobes and often, at very early stages: in the lip of the placode and edges of the lid in D. pealeii embryo until stage 27. This suggests that six genes and eya are involved in lens formation as it is observed in vertebrates (Koenig et al., 2016). In E. scolopes, six and eya expressions were detected in numerous tissues including, until stage 26, the ventral light organ, with no known “visual function” (Peyer et al., 2014). However, in the results shown in these studies, expressions are presented by whole-mount ISH without showing precisely the staining in the eye fields with convincing histological data. According to our results both on sections and whole-mount, we point out that the expression shown in whole mount ISH in these studies must be confirmed by sections in the areas supposed to be stained. Furthermore, Ogura et al. (2013) have shown that six3/6 is involved in the lens formation and its expression is localized in the lentigenic cells. Studies from vertebrates and Drosophila and even cnidarian, report six3/6 expression in the developing eye (Oliver et al., 1995; Loosli et al., 1999; Zuber et al., 1999). Although we encountered problems and were not able to repeat staining of six1/2 expression on sections, our expression data do not suggest any evolutionary conserved role of Sof-six1/2 in the retinal specification and the eye formation. However, we cannot exclude the expression of another six-subfamily gene such as six3/6 proposed to have an evolutionary conserved role. Sof-dac transcript is expressed from stage 18 to stage 30 in eyes and the optic lobes (Figure 8) but surprisingly its expression is never observed in the retina of cuttlefish. In vertebrates and Drosophila, dac expression is observed in the central nervous system, optic cup and also in the entire neural retina as it is shown on sections (Hammond et al., 1998; Caubit et al., 1999; Heanue et al., 1999; Loosli et al., 2002; Martín-Durán et al., 2012). In molluscs, dac is expressed in eye photoreceptor cell's development in larval chiton (Vöcking et al., 2015). In a cephalopod, E. scolopes, dac transcript expression is observed in the eyes, arms, mantle and light organs by whole mount in situ hybridization (Peyer et al., 2014). Strikingly, this expression is shown only on sections in the light organs but not in the retina. We point out that dac expression at the cellular level of eyes and other tissues must be confirmed by sections. In addition to being expressed in eye-associated tissues, Sof-dac has been also stained in supra-esophageal and sub-esophageal masses, gills, arms, and statocysts, all tissues playing important roles in sensory-motors functions. Our results show that the role of dac transcript is conserved in the central nervous system of metazoans. Nevertheless, Sof-dac seems expressed in optic lobes, precisely in the inner, the outer plexiform layers and also in the medullar zone at late stages until stage 30 before hatching. The plexiform area is also called the “deep retina” due to its similarity with the ganglionic layer of the vertebrate retina (Young, 1962). It is constituted by an inner and outer plexiform layer separated by a complex neuropil zone (Young, 1974). In cephalopods, the plexiform area contains several cell types (amacrine neurons, centrifugal, centrally, and centripetal cells). Furthermore, the retinal photoreceptors connect with the optic lobes by the photoreceptor axons in the plexiform area (Hartline and Lange, 1974). Indeed, the photoreceptors make synaptic contact with the amacrine neurons located in the inner and outer plexiform layers (Young, 1971; Case et al., 1972). Sof-dac expression in these areas could be an indication of the presence of photoreceptive cells in the optic lobes. These results suggest that Sof-dac is involved in the morphogenesis of the eye and visual control structures such as optic lobes in developing S. officinalis. In addition, we evidenced unexpected Sof-dac expression, for the first time: in some cells of the arm cord and in tissue layer surrounding the nerve cord. The histological character of this tissue is not yet identified, and additional studies must be done to determine if it is nervous or muscular cells as shown in some species (Heanue et al., 1999).

In S. officinalis we evidence that the gene network, six1/2, eya, and dac is involved in the early eye area formation but not in the differentiation of the retina. Similar results were found with Pax6, not involved in the retina formation (Navet et al., 2017). Pax6 is considered as a universal master control gene of eye formation and developing photoreceptors in metazoans (Gehring, 2005; see review in Kumar, 2009); it regulates upstream the RDGN, which instructs the formation of the adult eye in Drosophila. In numerous cephalopods (L. opalescens: Tomarev et al., 1997; E. scolopes: Hartmann et al., 2003; Peyer et al., 2014; S. officinalis: Navet et al., 2009, 2017; I. paradoxus: Yoshida et al., 2014; D. pealeii: Koenig et al., 2016), Pax6 expression has been shown in optical areas, eye and optic lobes. In S. officinalis, Pax6 presents a large ectodermic and mesodermic expression in the optic area but never in the retina (Buresi et al., 2016; Navet et al., 2017) and no clear expression in retina cells has been finally shown in the other species. These findings suggest the non-conservation of pax6 in the differentiation of the retinal photoreceptors by the loss of the conserved RDGN upstream regulation: this could explain the loss of expression of six, eya, and dac in the retina. No other pax genes (pax3/7 and pax2/5/8) is expressed in S. officinalis embryo all along the development in the retina cells from the first steps of specification until the rhabdomeric photoreceptors are fully differentiated. Nevertheless, other genes appears to have a role in retina differentiation, such as Sof-otx, which is expressed in the retina as already shown (Buresi et al., 2012).

Indeed, otx2 transcript factor is known to play a role equally in the development of the eye and the photoreceptor cells. In vertebrates, otx orthologs could control all retinal cell types (Chen et al., 1997; Viczian et al., 2003). Otx2 and pax6 are expressed upstream of the opsin: they could act to enhance r-opsin expression for the rhabdomeric photoreceptor; but only otx2 (and not pax6) with Rx activate the expression of c-opsin, characteristic of ciliary photoreceptors (Vopalensky and Kozmik, 2009). Otx2 transcription factor expression is reported in the photoreceptors of the fly ommatidia, in the photoreceptor precursors of larval eyes of T. transversa and the planarian pigment-cup eyes (Umesono et al., 1999; Passamaneck et al., 2011). In Sepia embryos, otx is expressed in the eyes particularly in the retina from early to late developmental stages (from stage 19 to stage 26) when the photoreceptor's differentiation started but its expression is not found from stage 26 when the retinal cell type organization is being achieved (Buresi et al., 2012). Thus, the mystery of the final differentiation of retinal photoreceptors and the genes that control this process remains to be characterized in order to understand what the underlying molecular mechanisms are.

Our results reveal for the first time in S. officinalis embryo the expression patterns of rhodopsin transcript in the developing retina from stage 23 to hatching (Figure 9). Several cephalopod species have both retinal and extraocular photoreceptors located in the light organ, skin, paraolfactory vesicles, epistellar body, and nervous system where a great diversity of opsin proteins can be found (Cobb and Williamson, 1998; Tong et al., 2009). In coleoid cephalopods, the retina only has a single layer containing rhabdomeric photoreceptors, supporting cells and blood vessels (Messenger, 1981). Additionally, the retinal rhabdomeric photoreceptors are known to express a single type of rhodopsin in coleoid cephalopods (Bellingham et al., 1998). Rhodopsin transcript has been evidenced by RT-PCR and immunolabeling not only in the retinas of several adult species of cephalopods such as squid D. pealeii, cuttlefish S. officinalis and Sepia latimanus but also in the skin of the same species (Kingston et al., 2015a,b). These authors indicate that the rhodopsin detected both in the skin and retina of adult cuttlefish is the same. But interestingly, in S. officinalis embryo, unlike adults, we have evidenced rhodopsin expression only in the retina, not in other tissues or structures, such as the skin. As the rhodopsin sequence used is exactly the same described in Kingston et al. (2015a) and Yoshida et al. (2015), and as no other rhodopsin has been found in our EST-library, we propose that rhodopsin is expressed in the skin after hatching, in the juveniles, when the patterns are useful for camouflage. A differential expression of rhodopsin during development has been shown in annelid suggesting a control of the “visual function” in accordance with developmental stage (Randel et al., 2013). Finally, rhodopsin transcript can be expressed so weakly that it cannot be detected by in situ hybridization. The weak expression of Sof-rhodopsin observed between stages 23 and 25 can be explained by the first differentiation step of cells at stage 23, and the beginning of the differentiation into photoreceptor cells and supporting cells from stage 25 on, as observed in Sepiella. This expression might be localized in the cells which differentiate into rhabdomeric photoreceptors at late stages as it is observed in other cephalopod adult species. But, in order to link sof-rhodopsin expression to the retinal cell differentiation process, further studies should be conducted during cuttlefish embryogenesis. Moreover, the expression of Sof-otx in cuttlefish's retina at stage 19, long before the expression of rhodopsin (stage 23) is congruent with the upstream regulation of rhodopsin in vertebrates (Vopalensky and Kozmik, 2009), and the timing of retina cone differentiation in mouse preceding the rhodopsin expression (Rodgers et al., 2016). Nevertheless, as Sof-otx expression stopped at stage 26 (Buresi et al., 2012), we question the control of rhodopsin expression in late developmental stages, i.e., in rhabdomeric photoreceptors. This expression and the production of visual rhodopsin combined with the observation of reactivity of the eye from stage 25 on show that rhodopsin is present before the setting up of the rhabdomeric photoreceptors (Yamamoto et al., 1985). These data strengthen the hypothesis of Romagny et al. (2012) suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, when the first retinal pigments appear, a stage when the rhabdomes are not totally differentiated. As a consequence, a fully differentiated rhabdomeric photoreceptor is not necessary to have a “basic” answer to light stimulus. Nevertheless, Romagny et al. (2012) have shown, by behavioral experiments of answer to light, that the habituation to light, the memory process is evidenced only at late stages of development, when the retinal rhabdomeric photoreceptors are totally differentiated and when the rhodopsin expression is very high (Figure 9). It is linked to the final maturation of the analysis centers (brain and optic lobes).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.