Final Report Summary - SEPIACARTILAGE (Molecular characterization of cartilage development in the cephalopod mollusk Sepia officinalis) Background Cephalopods offer enormous opportunities for studies of the developmental genetics of complex organ systems within an invertebrate model. Data derived from cephalopod studies can be used to compare and contrast known mechanisms underlying the development of analogous vertebrate structures. For example, the European cuttlefish, sepia officinalis, possesses cartilage tissues with an unparalleled histological similarity with vertebrate cartilages. The presence of these cellular cartilaginous tissues outside the chordate lineage may indicate a common origin of cartilage as a metazoan tissue type (homology), or alternatively highlights constraints that animals face in the construction of internal cellular endoskeletons (convergence). If a genetic programme for specifying cartilage cells arose only once during metazoan evolution, elements of a shared molecular fingerprint will be present in both cephalopods and vertebrates, despite their long independent evolutionary history. However, knowledge of the molecular control of the development in cephalopods molluscs is scarce; molecular studies are difficult due to the absence of genome sequence data for any cephalopod. Our work directly addresses this gap in our knowledge for the cephalopod mollusk, sepia officinalis. We focus our attention on three families of transcription factors that are known to have roles in vertebrate chondrogenesis: Sox and the Ant- and Prd- class families of the homeodomain proteins. In addition we have investigated components of the retinoic acid and hedgehog signalling pathways, both of which have relevant roles in vertebrate chondrogenesis. Methodology The absence of genomic resources renders the search for paralogous cuttlefish genes dependent upon the presence of highly conserved protein motifs within genes of interest, thus we used a degenerate primer strategy to isolate partial coding sequence from the conserved domains specific to these families. We then extended these sequences in both directions using rapid-amplification of complementary deoxyribonucleic acid (c-DNA) ends (RACE) to obtain full length transcripts, all of which will be deposited in the public database GenBank. Gene identity was determined initially based upon BLAST similarity and confirmed by phylogenetic analysis. Relative levels of gene expression throughout development were determined by quantitative polymerase chain reaction (PCR) analysis and in cases where sufficient specific gene sequence was acquired, ribonucleic acid (RNA) probes were generated to evaluate the spatial expression patterns during the period of organogenesis. Results and discussion Once considered a unique feature of vertebrate patterning, components of the retinoic acid (RA) signalling pathway, including both RA and the retinoid (RX) class of receptors, have been found within invertebrate genomes. RA signalling in vertebrates is important for axial patterning of all germ layers, including involvement in the formation of cartilage and excessive exposure to RA from the environment often leads to morphological defects particularly evident in the skeleton. RA receptor (RAR)-gamma expression is essential for vertebrate chondrocyte differentiation. The RA class of the receptors has also been identified within available sequenced molluscan genomes; however no data has been collected regarding their expression or function. We isolated partial coding sequence for a retinoid X receptor (RXR) receptor (Sof-RXR : JN101950) and an aldehyde dehydrogenase (ALDH) (Sof-ALDH : JN101949) homologue. Both predicted proteins are highly conserved with other known homologous sequences, with the exception of two small domains present only in the Sof-RXR. Spatial expression analysis of both genes revealed that Sof-RXR is expressed ubiquitously in all cells of the embryo, including the chondrocytes, whereas Sof-ALDH was expressed only in a sub-set of epithelial cells and no expression at the level of chondrocytes was observed. These genes were not pursued further and no other components of the RA signalling pathway were obtained with our cloning strategy. These data are presented in an undergraduate thesis at the Università degli Studi del Sannio for the candidate Silvia Voso who contributed to this project. 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, including vertebrate chondrogenesis. Members of the SoxE and SoxD families play an important, well documented role in the earliest phases of vertebrate chondrogenesis. Members of the remaining Sox families have also been identified in microarray studies of vertebrate chondrogenesis, but have thus far remained uninvestigated. We isolated the conserved high mobility group (HMG) domains of three Sox family transcription factors: Sof-SoxE, Sof-SoxB and Sof-SoxB2, which were classified by both analysis of family-specific residues and phylogenetic analysis together with other known Sox family genes. We only managed to isolate these three Sox genes with our cloning strategy; however other family members may be present in the genome. Analysis of predicted protein structure reveals conserved domains outside of the HMG domain. Temporal and spatial expression analysis of Sof-SoxB1 and Sof-SoxE demonstrated that both genes express from the onset of organogenesis (stage 18) in non-overlapping domains; Sof-SoxB1expresses in the presumptive ectodermal tissues overlying most of the embryonic surface whereas Sof-SoxE expressed in the presumptive mesodermal tissues. Sof-SoxB1 continues to express in the head epithelium and finally is restricted to sensory epithelium as development proceeds, whereas Sof-SoxE expression is retained within the mesenchyme of the abdominal cavity and show elevated expression in the developing closed peripheral circulatory system - a novel expression domain for this gene family. Both Sof-SoxB1 and Sof-SoxE also show neural expression in regions of the brain in newly hatched individuals. Neither gene was found to express in regions of cartilage formation. These data were presented at Canadian Society of Zoologists (18 to 22 May 2010, Vancouver BC, Canada) and EuroCeph (7 to 10 April 2011, Vico Equenze Italy), have been submitted for publication in Evolution and Development and will be presented in a doctoral thesis at the Università degli Studi del Sannio for the candidate Laura Focareta, who contributed to this project. Ant-class homeodomain genes include the well-known HOX genes, which are well studied for their role in body patterning and are known to influence shape and positioning of vertebrate cartilage bodies. Prd-class homeodomain genes, in particular members of the ARX-group, are involved in vertebrate chondrogenesis; members of the prd-class gene family have not been well investigated in any lophatrochozoan lineage. We isolated the conserved homeobox domains of seven Ant- and four Prd-class homeodomain transcription factors, in addition to one parahox gene (Sof-xLox) and one Lim-class gene (Sof-Lim3/4,: Sof-Hox1, Sof-Hox3, Sof-Hox4, Sof-Hox5, Sof-Hox6, Sof-Hox8 and Sof-Gbx (ANT-class); Sof-Arx, Sof-Drg, Sof-Prop and Sof-Vsx (Prd-class). Temporal analysis of gene expression demonstrates that all genes show moderately elevated expression levels throughout organogenesis. Eight of these gene fragments were expanded and spatial analysis of gene expression is ongoing. These data are presented in an undergraduate thesis at the Università degli Studi del Sannio for the candidate Salvatore Sesso, who contributed to this project. These data were presented at EuroCeph (7 to 10 April 2011, Vico Equenze Italy) and are under preparation for further publication. Conclusions Cephalopod molluscs are morphologically intriguing invertebrates that have many parallels with vertebrates in terms of complexity of morphology and neural capacity; so much so that they are now regulated together under the same European Union (EU) directive on animal care (2010/63/EU). Our knowledge of cephalopod biology and the genetics underlying their development is only now being addressed in a systematic manner. The studies completed under this grant have helped progress this knowledge in terms of advancement in technical know-how and analysis of specific gene families that play a role in patterning of the embryonic layers in order to create these highly sophisticated animals.