Preliminary study on toxicological mechanism of golden cuttlefish (Sepia esculenta) larvae exposed to cd.

BACKGROUND: Cadmium (Cd) flows into the ocean with industrial and agricultural pollution and significantly affects the growth and development of economic cephalopods such as Sepia esculenta, Amphioctopus fangsiao, and Loligo japonica. As of now, the reasons why Cd affects the growth and development of S. esculenta are not yet clear. RESULTS: In this study, transcriptome and four oxidation and toxicity indicators are used to analyze the toxicological mechanism of Cd-exposed S. esculenta larvae. Indicator results indicate that Cd induces oxidative stress and metal toxicity. Functional enrichment analysis results suggest that larval ion transport, cell adhesion, and some digestion and absorption processes are inhibited, and the cell function is damaged. Comprehensive analysis of protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to explore S. esculenta larval toxicological mechanisms, and we find that among the 20 identified key genes, 14 genes are associated with neurotoxicity. Most of them are down-regulated and enriched to the neuroactive ligand-receptor interaction signaling pathway, suggesting that larval nervous system might be destroyed, and the growth, development, and movement process are significantly affected after Cd exposure. CONCLUSIONS: S. esculenta larvae suffered severe oxidative damage after Cd exposure, which may inhibit digestion and absorption functions, and disrupt the stability of the nervous system. Our results lay a function for understanding larval toxicological mechanisms exposed to heavy metals, promoting the development of invertebrate environmental toxicology, and providing theoretical support for S. esculenta artificial culture.

Acute effects of polystyrene nanoplastics on the immune response in Sepia esculenta larvae.

With extensive use of plastic products, microplastics (MPs, < 5 mm) and nanoplastics (NPs, < 1 µm) have become major pollutants in ecosystem, especially in marine environment. In recent years, researches on the impact of NPs on organisms have gradually increased. However, studies on the influence of NPs on cephalopods are still limited. Golden cuttlefish (Sepia esculenta), an important economic cephalopod, is a shallow marine benthic organism. In this study, the effect of acute exposure (4 h) to 50-nm polystyrene nanoplastics (PS-NPs, 100 µg/L) on the immune response of S. esculenta larvae was analyzed via transcriptome data. A total of 1260 DEGs were obtained in the gene expression analysis. The analyses of GO, KEGG signaling pathway enrichment, and protein-protein interaction (PPI) network were then performed to explore the potential molecular mechanisms of the immune response. Finally, 16 key immune-related DEGs were obtained according to the number of KEGG signaling pathways involved and the PPI number. This study not only confirmed that NPs had an impact on cephalopod immune response, but also provided novel insights for further unmasking the toxicological mechanisms of NPs.

Effect of polycyclic aromatic hydrocarbons on homeobox gene expression during embryonic development of cuttlefish, Sepia officinalis.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants (POPs) commonly found in marine environments. Their bioaccumulation can cause harm to aquatic organisms, including invertebrates, particularly during the early stages of embryonic development. In this study, we evaluated, for the first time, the patterns of PAH accumulation in both capsule and embryo of common cuttlefish (Sepia officinalis). In addition, we explored the effects of PAHs by analysing the expression profiles of seven homeobox genes [i.e., gastrulation brain homeobox (GBX), paralogy group labial/Hox1 (HOX1), paralogy group Hox3 (HOX3), dorsal root ganglia homeobox (DRGX), visual system homeobox (VSX), aristaless-like homeobox (ARX) and LIM-homeodomain transcription factor (LHX3/4)]. We found that PAH levels in egg capsules were higher than those observed in chorion membranes (35.1 ± 13.3 ng/g vs 16.4 ± 5.9 ng/g). Furthermore, PAHs were also found in perivitellin fluid (11.5 ± 5.0 ng/ml). Naphthalene and acenaphthene were the congeners present at highest concentrations in each analysed egg component suggesting higher bioaccumulation rates. Embryos with high concentrations of PAHs also showed a significant increase in mRNA expression for each of the analysed homeobox genes. In particular, we observed a 15-fold increase in the ARX expression levels. Additionally, the statistically significant variation in homeobox gene expression patterns was accompanied by a concomitant increase in mRNA levels of both aryl hydrocarbon receptor (AhR) and estrogen receptor (ER). These findings suggest that bioaccumulation of PAHs may modulate developmental processes of cuttlefish embryos by targeting homeobox gene-mediated transcriptional outcomes. Mechanisms underlying the upregulation of homeobox genes could be related to the ability of PAHs to directly activate AhR- or ER-related signaling pathways.

Transcriptome analysis preliminary reveals the immune response mechanism of golden cuttlefish (Sepia esculenta) larvae exposed to Cd.

As a well-known marine metal element, Cd can significantly affect bivalve mollusk life processes such as growth and development. However, the effects of Cd on the molecular mechanisms of the economically important cephalopod species Sepia esculenta remain unclear. In this study, S. esculenta larval immunity exposed to Cd is explored based on RNA-Seq. The analyses of GO, KEGG, and protein-protein interaction (PPI) network of 1,471 differentially expressed genes (DEGs) reveal that multiple immune processes are affected by exposure such as inflammatory reaction and cell adhesion. Comprehensive analyses of KEGG signaling pathways and the PPI network are first used to explore Cd-exposed S. esculenta larval immunity, revealing the presence of 16 immune-related key and hub genes involved in exposure response. Results of gene and pathway functional analyses increase our understanding of Cd-exposed S. esculenta larval immunity and improve our overall understanding of mollusk immune functions.

Time-dependent immune injury induced by short-term exposure to nanoplastics in the Sepia esculenta larvae.

Marine organisms are threatened by various environmental contaminants, and nanoplastics (NPs) is one of the most concerned. Studied have shown that NPs has a certain impact on marine organisms, but the specific molecular mechanism is still unclear. At present, researches on the effect of NPs on marine life mostly focus on crustaceans, gastropods, and bivalves. In this study, cephalopod Sepia esculenta larvae were first used to investigate the potential immune response molecular mechanisms caused by PS-NPs (50 nm, 50 mg/L) short-term exposure (4 and 24 h). Through S. esculenta larvae transcriptome profile of gene expression analysis, 548 and 1990 genes showed differential expression at 4 and 24 h after NPs exposure, respectively. GO and KEGG enrichment analysis were performed to find immune related DEGs. Then, the interaction relationship between the immune related DEGs after NPs exposure was known through the constructed protein-protein interaction network. 20 hub genes were found on the base of KEGG pathway numbers involved and protein-protein interaction numbers. This research supply valuable genes for the study of cephalopod immune response caused by NPs, which can help us further uncover the molecular mechanisms of organism against NPs.

Exploration of immune response mechanisms in cadmium and copper co-exposed juvenile golden cuttlefish (Sepia esculenta) based on transcriptome profiling.

Sepia esculenta is a popular economic cephalopod with high yield, delicious meat, and rich nutrition. With the rapid development of heavy industry and medical industry, a large amount of waste has been released into the ocean recklessly in recent years, inducing a significant increase in the content of heavy metals, especially cadmium (Cd) and copper (Cu), in the ocean. This phenomenon significantly affects the growth and development of S. esculenta, causing a serious blow to its artificial breeding. In this study, transcriptome analysis is used to initially explore immune response mechanisms of Cd and Cu co-exposed juvenile S. esculenta. The results show that 1,088 differentially expressed genes (DEGs) are identified. And DEGs functional enrichment analysis results suggests that co-exposure may promote inflammatory and innate immune responses in juvenile S. esculenta. Fifteen key genes that might regulate the immunity of S. esculenta are identified using protein-protein interaction (PPI) network and KEGG enrichment analyses, of which the three genes with the highest number of interactions or involve in more KEGG pathways are identified as hub genes that might significantly affect the immune response processes. Comprehensive analysis of PPI network and KEGG signaling pathway is used for the first time to explore co-exposed S. esculenta juvenile immune response processes. Our results preliminarily reveal immune response mechanisms of cephalopods exposed to heavy metals and provide a valuable resource for further understanding of mollusk immunity.

Effect of acute Cu exposure on immune response mechanisms of golden cuttlefish (Sepia esculenta).

Sepia esculenta is a common economic cephalopod that has received extensive attention due to the tender meat, rich protein content and certain medicinal value thereof. Over the past decade, multiple industries have discharged waste into the ocean in large quantities, thereby significantly increasing the concentration of heavy metals in the ocean. Copper (Cu) is a common heavy metal in the ocean. The increase of Cu content will affect numerous biological processes such as immunity and metabolism of marine organisms. High concentrations of Cu may inhibit S. esculenta growth, development, swimming, and other processes, which would significantly affect its culture. In this research, transcriptome analysis is used to initially explore Cu-exposed S. esculenta larval immune response mechanisms. And compared to control group with normally growing larvae, 2056 differentially expressed genes (DEGs) are identified in experimental group with Cu-exposed larvae. The results of DEGs functional enrichment analyses including GO and KEGG indicate that Cu exposure might promote inflammatory and innate immune responses in cuttlefish larvae. Then, 10 key genes that might regulate larval immunity are identified using a comprehensive analysis that combines protein-protein interaction (PPI) network and KEGG functional enrichment analyses, of which three genes with the highest number of protein interactions or involve in more KEGG signaling pathways are identified as hub genes that might significantly affect larval immune response processes. Comprehensive analysis of PPI network and KEGG signaling pathway are used for the first time to explore Cu-exposed S. esculenta larval immune response mechanisms. Our results preliminarily reveal immune response mechanisms of cephalopods exposed to heavy metals and provide valuable resources for further understanding mollusk immunity.

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.

Transcriptome analysis of the Sepia pharaonis: Identification of low salinity stress-related information and microsatellite markers.

Sepia pharaonis has great commercial value for aquaculture. However, it is sensitive to salinity fluctuations and lacking in genomic information. The present work utilized high-throughput transcriptome sequencing to assess the effect of low salinity (22.0 ppt) on gills of S. pharaonis. 6153 genes were identified as differentially expressed (p < 0.05), of which 3340 were increased and 2813 were decreased in low salinity group (22.0 ppt) relative to the control group (29.0 ppt). Subsequently, these DEGs were allocated to 226 KEGG pathways and 491 GO terms. Analysis of the transcriptome sequences and DEGs identified several unigenes and pathways involved in salt stress regulation. Moreover, the S. pharaonis carried 101,576 simple sequence repeats (SSRs). This is the first time osmoregulation in S. pharaonis has been explored by transcriptome sequencing. The data presented here reveals key insights into the genetic markers of salt stress in S. pharaonis.

An FMRFamide Neuropeptide in Cuttlefish Sepia pharaonis: Identification, Characterization, and Potential Function.

Neuropeptides are released by neurons that are involved in a wide range of brain functions, such as food intake, metabolism, reproduction, and learning and memory. A full-length cDNA sequence of an FMRFamide gene isolated from the cuttlefish Sepia pharaonis (designated as SpFMRFamide) was cloned. The predicted precursor protein contains one putative signal peptide and four FMRFamide-related peptides. Multiple amino acid and nucleotide sequence alignments showed that it shares 97% similarity with the precursor FMRFamides of Sepiella japonica and Sepia officinalis and shares 93% and 92% similarity with the SpFMRFamide gene of the two cuttlefish species, respectively. Moreover, the phylogenetic analysis also suggested that SpFMRFamide and FMRFamides from S. japonica and S. officinalis belong to the same sub-branch. Tissue expression analysis confirmed that SpFMRFamide was widely distributed among tissues and predominantly expressed in the brain at the three development stages. The combined effects of SpFMRFamide+SpGnRH and SpFLRFamide+SpGnRH showed a marked decrease in the level of the total proteins released in the CHO-K1 cells. This is the first report of SpFMRFamide in S. pharaonis and the results may contribute to future studies of neuropeptide evolution or may prove useful for the development of aquaculture methods for this cuttlefish species.

Transcriptome analysis for identifying possible causes of post-reproductive death of Sepia esculenta based on brain tissue.

BACKGROUND: The subpeduncle lobe/olfactory lobe-optic gland axis is called the endocrine regulation center of cephalopods. However, little is known about the mechanism of the subpeduncle lobe/olfactory lobe-optic gland axis regulate the sexual maturation and post-reproductive death of Sepia esculenta Hoyle. OBJECTIVES: The primary objective of this study was to provide basic information for revealing the mechanism of the subpeduncle lobe/olfactory lobe-optic axis regulating the rapid post-reproductive death of S. esculenta. METHODS: In this paper, Illumina sequencing based transcriptome analysis was performed on the brain tissue of female S. esculenta in the three key developmental stages: growth stage (BG), spawning stage (BS), and post-reproductive death stage (BA). RESULTS: A total of 66.19 Gb Illumina sequencing data were obtained. A comparative analysis of the three stages showed 2609, 3333, and 170 differentially expressed genes (DEGs) in BG-vs-BA, BG-vs-BA, and BS-vs-BA, respectively. The Gene Ontology (GO) enrichment analysis of DEGs revealed that the regulation of cyclin-dependent protein serine/threonine kinase activity, oxidative phosphorylation, and respiratory chain were significantly enriched. The significant enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway identified pathways associated with the regulation of death, such as the mammalian target of rapamycin (mTOR) signaling pathway, AMPK signaling pathway, oxidative phosphorylation, and cell cycle. CONCLUSION: The post-reproductive death of S. esculenta was found to be a complex energy steady-state regulation network system. The mTOR acted as an energy receptor and had a key role in regulating energy homeostasis.

Crustacean cardioactive peptides: Expression, localization, structure, and a possible involvement in regulation of egg-laying in the cuttlefish Sepia officinalis.

The cuttlefish (Sepia officinalis) is a cephalopod mollusk distributed on the western European coast, in the West African Ocean and in the Mediterranean Sea. On the Normandy coast (France), cuttlefish is a target species of professional fishermen, so its reproduction strategy is of particular interest in the context of stock management. Egg-laying, which is coastal, is controlled by several types of regulators among which neuropeptides. The cuttlefish neuropeptidome was recently identified by Zatylny-Gaudin et al. (2016). Among the 38 neuropeptide families identified, some were significantly overexpressed in egg-laying females as compared to mature males. This study is focused on crustacean cardioactive peptides (CCAPs), a highly expressed neuropeptide family strongly suspected of being involved in the control of egg-laying. We investigated the functional and structural characterization and tissue mapping of CCAPs, as well as the expression patterns of their receptors. CCAPs appeared to be involved in oocyte transport through the oviduct and in mechanical secretion of capsular products. Immunocytochemistry revealed that the neuropeptides were localized throughout the central nervous system (CNS) and in the nerve endings of the glands involved in egg-capsule synthesis and secretion, i.e. the oviduct gland and the main nidamental glands. The CCAP receptor was expressed in these glands and in the subesophageal mass of the CNS. Multiple sequence alignments revealed a high level of conservation of CCAP protein precursors in Sepia officinalis and Loligo pealei, two cephalopod decapods. Primary sequences of CCAPs from the two species were fully conserved, and cryptic peptides detected in the nerve endings were also partially conserved, suggesting biological activity that remains unknown for the time being.

Transcriptome Sequencing and De Novo Assembly of Golden Cuttlefish Sepia esculenta Hoyle.

Golden cuttlefish Sepia esculenta Hoyle is an economically important cephalopod species. However, artificial hatching is currently challenged by low survival rate of larvae due to abnormal embryonic development. Dissecting the genetic foundation and regulatory mechanisms in embryonic development requires genomic background knowledge. Therefore, we carried out a transcriptome sequencing on Sepia embryos and larvae via mRNA-Seq. 32,597,241 raw reads were filtered and assembled into 98,615 unigenes (N50 length at 911 bp) which were annotated in NR database, GO and KEGG databases respectively. Digital gene expression analysis was carried out on cleavage stage embryos, healthy larvae and malformed larvae. Unigenes functioning in cell proliferation exhibited higher transcriptional levels at cleavage stage while those related to animal disease and organ development showed increased transcription in malformed larvae. Homologs of key genes in regulatory pathways related to early development of animals were identified in Sepia. Most of them exhibit higher transcriptional levels in cleavage stage than larvae, suggesting their potential roles in embryonic development of Sepia. The de novo assembly of Sepia transcriptome is fundamental genetic background for further exploration in Sepia research. Our demonstration on the transcriptional variations of genes in three developmental stages will provide new perspectives in understanding the molecular mechanisms in early embryonic development of cuttlefish.

Analyses of Sox-B and Sox-E Family Genes in the Cephalopod Sepia officinalis: Revealing the Conserved and the Unusual.

Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.

A transcriptome resource for pharaoh cuttlefish (Sepia pharaonis) after ink ejection by brief pressing.

Ink ejection is one of the most important defense mechanisms against external stimuli for pharaoh cuttlefish (Sepia pharaonis). The molecular changes during this process remain unknown. To understand the transcriptome changes after ink ejection by brief pressing, two cDNA libraries of pharaoh cuttlefish, from the inkjet group and control group were sequenced using Illumina HiSeq™ 2000. A total of 9,255,502,440nt bases were obtained and by de novo assembly, 73,298 unigenes were generated, which first provided numerous expressed sequence tags from pharaoh cuttlefish. By comparing the expression levels between the two groups, we identified 7064 up-regulated and 2024 down-regulated genes after ink ejection. These differentially-expressed genes included genes related to immunity, cancer, and blood coagulation, which indicated the various effects after ink ejection by brief pressing. These results provide new valuable resources for functional genomic and genetic studies on pharaoh cuttlefish.

Nervous system development in cephalopods: How egg yolk-richness modifies the topology of the mediolateral patterning system.

Cephalopods possess the most complex centralized nervous system among molluscs and the molecular determinants of its development have only begun to be explored. To better understand how evolved their brain and body axes, we studied Sepia officinalis embryos and investigated the expression patterns of neural regionalization genes involved in the mediolateral patterning of the neuroectoderm in model species. SoxB1 expression reveals that the embryonic neuroectoderm is made of several distinct territories that constitute a large part of the animal pole disc. Concentric nkx2.1, pax6/gsx, and pax3/7/msx/pax2/5/8 positive domains subdivide this neuroectoderm. Looking from dorsal to ventral sides, the sequence of these expressions is reminiscent of the mediolateral subdivision in model species, which provides good evidence for "mediolateral patterning" conservation in cephalopods. A specific feature of cephalopod development, however, includes an unconventional orientation to this mediolateral sequence: median markers (like nkx2.1) are unexpectedly expressed at the periphery of the cuttlefish embryo and lateral markers (like Pax3/7) are expressed centrally. As the egg is rich with yolk, the lips of the blastopore (that classically organizes the neural midline) remain unclosed at the lateral side of the animal pole until late stages of organogenesis, therefore reversing the whole embryo topology. These findings confirm - by means of molecular tools - the location of both ventral and dorsal poles in cephalopod embryos.

Complete mitochondrial genome of the needle cuttlefish Sepia aculeata (Sepioidea, Sepiidae).

In this study, we determined the complete mitochondrial genome of the needle cuttlefish Sepia aculeata. The genome was 16,219 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 2 main non-coding regions [both are control regions (CR)]. The composition and order of genes, for the mitogenome found in S. aculeate, were similar to most other invertebrates. The overall base composition of S. aculeata is T 34.0%, C 17.0%, A 40.5% and G 8.5%, with a highly A + T bias of 74.5%. Two control regions (CR) both contain termination-associated sequences and conserved sequence blocks. This mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the Sepiidae.

The Toll/NF-κB pathway in cuttlefish symbiotic accessory nidamental gland.

The female genital apparatus of decapod cephalopods contains a symbiotic accessory nidamental gland (ANG) that harbors bacterial symbionts. Although the ANG bacterial consortium is now well described, the impact of symbiosis on Sepia officinalis innate immunity pathways remains unknown. In silico analysis of the de novo transcriptome of ANG highlighted for the first time the existence of the NF-κB pathway in S. officinalis. Several signaling components were identified, i.e. five Toll-like receptors, eight signaling cascade features, and the immune response target gene iNOS, previously described as being involved in the initiation of bacterial symbiosis in a cephalopod gland. This work provides a first key for studying bacterial symbiosis and its impact on innate immunity in S. officinalis ANG.

Genetic diversity and population structure of Sepia officinalis from the Tunisian cost revealed by mitochondrial COI sequences.

Population substructure of Sepia officinalis sampled along the Tunisian coastline was studied. We have scored the genetic variation of the mitochondrial gene cytochrome oxidase 1. A total of 20 specimens from four sampling sites were analysed and revealed 12 different haplotypes. Haplotype diversity showed a decreasing north to south gradient which may be explained by the hydrogeography of the study area. The overall estimate of genetic divergence (FST) revealed significant genetic differentiation between the pair-wise population comparisons supported by the AMOVA analysis which reveals significant genetic divergence. Finally, populations showed an excess of rare haplotypes. The mismatch distribution and several population genetic statistics indicate that the excess of rare variants is due to a recent expansion for Djerba and Kelibia populations. For Rades and Bizerte populations a constant population size was detected. These findings are important for fisheries management to preserve this marine resource for long-term utilization.

Characterization of homeobox genes reveals sophisticated regionalization of the central nervous system in the European cuttlefish Sepia officinalis.

Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morphological complexity, including a centralized nervous system with sophisticated cognitive functionality. Very little is known about the genetic mechanisms underlying patterning of the cephalopod embryo to arrive at this anatomical structure. Homeodomain (HD) genes are transcription factors that regulate transcription of downstream genes through DNA binding, and as such are integral parts of gene regulatory networks controlling the specification and patterning of body parts across lineages. We have used a degenerate primer strategy to isolate homeobox genes active during late-organogenesis from the European cuttlefish Sepia officinalis. With this approach we have isolated fourteen HD gene fragments and examine the expression profiles of five of these genes during late stage (E24-28) embryonic development (Sof-Gbx, Sof-Hox3, Sof-Arx, Sof-Lhx3/4, Sof-Vsx). All five genes are expressed within the developing central nervous system in spatially restricted and largely non-overlapping domains. Our data provide a first glimpse into the diversity of HD genes in one of the largest, yet least studied, metazoan clades and illustrate how HD gene expression patterns reflect the functional partitioning of the cephalopod brain.