Exploration of chemosensory ionotropic receptors in cephalopods: the IR25 gene is expressed in the olfactory organs, suckers, and fins of Sepia officinalis.

While they are mostly renowned for their visual capacities, cephalopods are also good at olfaction for prey, predator, and conspecific detection. The olfactory organs and olfactory cells are well described but olfactory receptors-genes and proteins-are still undescribed in cephalopods. We conducted a broad phylogenetic analysis of the ionotropic glutamate receptor family in mollusks (iGluR), especially to identify IR members (Ionotropic Receptors), a variant subfamily whose involvement in chemosensory functions has been shown in most studied protostomes. A total of 312 iGluRs sequences (including 111 IRs) from gastropods, bivalves, and cephalopods were identified and annotated. One orthologue of the gene coding for the chemosensory IR25 co-receptor has been found in Sepia officinalis (Soff-IR25). We searched for Soff-IR25 expression at the cellular level by in situ hybridization in whole embryos at late stages before hatching. Expression was observed in the olfactory organs, which strongly validates the chemosensory function of this receptor in cephalopods. Soff-IR25 was also detected in the developing suckers, which suggests that the unique « taste by touch ¼ behavior that cephalopods execute with their arms and suckers share features with olfaction. Finally, Soff-IR25 positive cells were unexpectedly found in fins, the two posterior appendages of cephalopods, mostly involved in locomotory functions. This result opens new avenues of investigation to confirm fins as additional chemosensory organs in cephalopods.

Histamine and histidine decarboxylase in the olfactory system and brain of the common cuttlefish Sepia officinalis (Linnaeus, 1758).

Cephalopods are radically different from any other invertebrate. Their molluscan heritage, innovative nervous system, and specialized behaviors create a unique blend of characteristics that are sometimes reminiscent of vertebrate features. For example, despite differences in the organization and development of their nervous systems, both vertebrates and cephalopods use many of the same neurotransmitters. One neurotransmitter, histamine (HA), has been well studied in both vertebrates and invertebrates, including molluscs. While HA was previously suggested to be present in the cephalopod central nervous system (CNS), Scaros, Croll, and Baratte only recently described the localization of HA in the olfactory system of the cuttlefish Sepia officinalis. Here, we describe the location of HA using an anti-HA antibody and a probe for histidine decarboxylase (HDC), a synthetic enzyme for HA. We extended previous descriptions of HA in the olfactory organ, nerve, and lobe, and describe HDC staining in the same regions. We found HDC-positive cell populations throughout the CNS, including the optic gland and the peduncle, optic, dorso-lateral, basal, subvertical, frontal, magnocellular, and buccal lobes. The distribution of HA in the olfactory system of S. officinalis is similar to the presence of HA in the chemosensory organs of gastropods but is different than the sensory systems in vertebrates or arthropods. However, HA's widespread abundance throughout the rest of the CNS of Sepia is a similarity shared with gastropods, vertebrates, and arthropods. Its widespread use with differing functions across Animalia provokes questions regarding the evolutionary history and adaptability of HA as a transmitter.

Eye Development in Sepia officinalis Embryo: What the Uncommon Gene Expression Profiles Tell Us about Eye Evolution.

In metazoans, there is a remarkable diversity of photosensitive structures; their shapes, physiology, optical properties, and development are different. To approach the evolution of photosensitive structures and visual function, cephalopods are particularly interesting organisms due to their most highly centralized nervous system and their camerular eyes which constitute a convergence with those of vertebrates. The eye morphogenesis in numerous metazoans is controlled mainly by a conserved Retinal Determination Gene Network (RDGN) including pax, six, eya, and dac playing also key developmental roles in non-retinal structures and tissues of vertebrates and Drosophila. Here we have identified and explored the role of Sof-dac, Sof-six1/2, Sof-eya in eye morphogenesis, and nervous structures controlling the visual function in Sepia officinalis. We compare that with the already shown expressions in eye development of Sof-otx and Sof-pax genes. Rhodopsin is the pigment responsible for light sensitivity in metazoan, which correlate to correlate visual function and eye development. We studied Sof-rhodopsin expression during retina differentiation. By in situ hybridization, we show that (1) all of the RDGN genes, including Sof-pax6, are expressed in the eye area during the early developmental stages but they are not expressed in the retina, unlike Sof-otx, which could have a role in retina differentiation; (2) Sof-rhodopsin is expressed in the retina just before vision gets functional, from stage 23 to hatching. Our results evidence a role of Sof-six1/2, Sof-eya, and Sof-dac in eye development. However, the gene network involved in the retinal photoreceptor differentiation remains to be determined. Moreover, for the first time, Sof-rhodopsin expression is shown in the embryonic retina of cuttlefish suggesting the evolutionary conservation of the role of rhodopsin in visual phototransduction within metazoans. These findings are correlated with the physiological and behavioral observations suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, as soon as the first retinal pigments appear.

The Pax gene family: Highlights from cephalopods.

Pax genes play important roles in Metazoan development. Their evolution has been extensively studied but Lophotrochozoa are usually omitted. We addressed the question of Pax paralog diversity in Lophotrochozoa by a thorough review of available databases. The existence of six Pax families (Pax1/9, Pax2/5/8, Pax3/7, Pax4/6, Paxß, PoxNeuro) was confirmed and the lophotrochozoan Paxß subfamily was further characterized. Contrary to the pattern reported in chordates, the Pax2/5/8 family is devoid of homeodomain in Lophotrochozoa. Expression patterns of the three main pax classes (pax2/5/8, pax3/7, pax4/6) during Sepia officinalis development showed that Pax roles taken as ancestral and common in metazoans are modified in S. officinalis, most likely due to either the morphological specificities of cephalopods or to their direct development. Some expected expression patterns were missing (e.g. pax6 in the developing retina), and some expressions in unexpected tissues have been found (e.g. pax2/5/8 in dermal tissue and in gills). This study underlines the diversity and functional plasticity of Pax genes and illustrates the difficulty of using probable gene homology as strict indicator of homology between biological structures.

Reflectin genes and development of iridophore patterns in Sepia officinalis embryos (Mollusca, Cephalopoda).

BACKGROUND: In the cuttlefish Sepia officinalis, iridescence is known to play a role in patterning and communication. In iridophores, iridosomes are composed of reflectins, a protein family, which show great diversity in all cephalopod species. Iridosomes are established before hatching, but very little is known about how these cells are established, their distribution in embryos, or the contribution of each reflectin gene to iridosome structures. RESULTS: Six reflectin genes are expressed during the development of iridosomes in Sepia officinalis. We show that they are expressed in numerous parts of the body before hatching. Evidence of the colocalization of two different genes of reflectin was found. Curiously, reflectin mRNA expression was no longer detectable at the time of hatchling, while reflectin proteins were present and gave rise to visible iridescence. CONCLUSION: These data suggest that several different forms of reflectins are simultaneously used to produce iridescence in S. officinalis and that mRNA production and translation are decoupled in time during iridosome development.

ESTs library from embryonic stages reveals tubulin and reflectin diversity in Sepia officinalis (Mollusca ­ Cephalopoda).

New molecular resources regarding the so-called "non-standard models" in biology extend the present knowledge and are essential for molecular evolution and diversity studies (especially during the development) and evolutionary inferences about these zoological groups, or more practically for their fruitful management. Sepia officinalis, an economically important cephalopod species, is emerging as a new lophotrochozoan developmental model. We developed a large set of expressed sequence tags (ESTs) from embryonic stages of S. officinalis, yielding 19,780 non-redundant sequences (NRS). Around 75% of these sequences have no homologs in existing available databases. This set is the first developmental ESTs library in cephalopods. By exploring these NRS for tubulin, a generic protein family, and reflectin, a cephalopod specific protein family,we point out for both families a striking molecular diversity in S. officinalis.

The dynamic nitric oxide pattern in developing cuttlefish Sepia officinalis.

BACKGROUND: Nitric oxide (NO) is implied in many important biological processes in all metazoans from porifera to chordates. In the cuttlefish Sepia officinalis NO plays a key role in the defense system and neurotransmission. RESULTS: Here, we detected for the first time NO, NO synthase (NOS) and transcript levels during the development of S. officinalis. The spatial pattern of NO and NOS is very dynamic, it begins during organogenesis in ganglia and epithelial tissues, as well as in sensory cells. At later stages, NO and NOS appear in organs and/or structures, including Hoyle organ, gills and suckers. Temporal expression of NOS, followed by real-time PCR, changes during development reaching the maximum level of expression at stage 26. CONCLUSIONS: Overall these data suggest the involvement of NO during cuttlefish development in different fundamental processes, such as differentiation of neural and nonneural structures, ciliary beating, sensory cell maintaining, and organ functioning.

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda).

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the 'receptive areas' via the CNS to the chromatophore effectors.

Shh and Pax6 have unconventional expression patterns in embryonic morphogenesis in Sepia officinalis (Cephalopoda).

Cephalopods show a very complex nervous system, particularly derived when compared to other molluscs. In vertebrates, the setting up of the nervous system depends on genes such as Shh and Pax6. In this paper we assess Shh and Pax6 expression patterns during Sepia officinalis development by whole-mount in situ hybridization. In vertebrates, Shh has been shown to indirectly inhibit Pax6. This seems to be the case in cephalopods as the expression patterns of these genes do not overlap during S. officinalis development. Pax6 is expressed in the optic region and brain and Shh in gut structures, as already seen in vertebrates and Drosophila. Thus, both genes show expression in analogous structures in vertebrates. Surprisingly, they also exhibit unconventional expressions such as in gills for Pax6 and ganglia borders for Shh. They are also expressed in many cephalopods' derived characters among molluscs as in arm suckers for Pax6 and beak producing tissues, nuchal organ and neural cord of the arms for Shh. This new data supports the fact that molecular control patterns have evolved with the appearance of morphological novelties in cephalopods as shown in this new model, S. officinalis.