Identificazione e caratterizzazione di FoxP nel sistema nervoso centrale di Octopus vulgaris (Mollusca, Cephalopoda)

In this study, I searched and was able to identify FoxP in the transcriptome of the\ncephalopod mollusc Octopus vulgaris, an invertebrate. In addition, I attempted to analyze\nthe expression of Ov-FoxP in the brain of this animal. The results of this analysis are\npreliminary at this stage.\nFox proteins are a set of transcription factors highly conserved in metazoans. They are\ncharacterized by a typical DNA binding domain (Forkhead) that, among others, allows to\nidentify 15 different classes of Fox genes. Fox proteins are reported to act as\nactivators/repressors of transcription during both development (including differentiation)\nand the adult life (e.g. lung, brain, etc.). In vertebrates, FoxP2 (together with FoxP1), in\nparticular, are known to be involved in the development of the neural circuit controlling\nbird-song and human speech.\nOur interest for the octopus derives from the fact that this animal, together with other\ncephalopods, is considered as the most evolved among molluscs. The complexity of the\narchitecture and wiring of the cephalopod nervous system stems from the simpler nervous\nsystems of other taxa belonging to the phylum. In addition, cephalopods show a highly rich\nbehavioral repertoire including the unique capability of changing the appearance of their\nbody (through body patterning) in fractions of seconds and for both mimetic and\ncommunicative purposes. Taken all together, these features allow these animals to be\nconsidered analogous to higher vertebrates.\nIn the first part of my project, a detailed analysis of the aminoacidic and nucleotidic\nsequences available for FoxP2 (vertebrates) and FoxP (invertebrates), allowed us to design\nFoxP in Octopus vulgaris\n1\nappropriate oligos that were utilized in subsequent PCR experiments to identify the gene of\ninterest in the transcriptome of the brain of O. vulgaris. FoxP resulted in a fragment of 220\nbp that corresponded to the Forkhead domain. Further efforts allowed us to identify a\n1111bp mRNA sequence of Ov-FoxP corresponding to almost the entire part of the mature\nmRNA codifying for this protein (the 5’ extremity of the gene results unidentified at this\nstage).\nDuring the second part of my project, I attempted to analyze the expression pattern of Ov-\nFoxP in the octopus brain using Real Time qPCR and in-situ hybridization. This was\ncarried out with the aim of investigating the possible variability of expression of the gene in\ndifferent parts of the brain (i.e. supra-, sub-esophageal masses and optic lobes) relative to\nanother tissue (muscular tissue of the mantle) here considered as control. Other genes (16S,\ntubulin, actin) were also cloned for the aims of this project and their expression was taken\nas reference; an analysis that is carried out for the first time in O. vulgaris.\nBy Real-Time qPCR I was able to recognize a different pattern of expression in different\nparts of the brain (N = 10). The data allowed to identify a gradient in the expression levels\nof FoxP (relative to reference genes) in the subesophageal mass, when the smallest\nindividual of my sample (30 g body weight) was compared with the others (150-2100 g\nbody weight).\nIn situ hybridization (N=6) allowed to localize the expression of FoxP in the lobes of the\noctopus brain. Ov-FoxP transcripts were identified in neurons of: i. the optic lobes (several\nsparse cells possibly related with visual input processing); ii. the superior buccal and the\nlateral part of the basal lobes (high-order motor centers of the supraesophageal mass), and\niii. the pedal tracts and anterior and posterior chromatophore lobes (subesophageal mass).\nFoxP in Octopus vulgaris\n2\nAn elevated number of cells was revealed through in-situ hybridization in the last two\nlobes. It is noteworthy to mention that these structures are known to play a key role in the\nneural control of the chromatic expression of the skin of O. vulgaris (and other\ncephalopods): namely the animal’s body pattern.\nOur data seems to suggest that Ov-FoxP is expressed during different phases of the life of\nthe octopus. In addition the localized expression in definite lobes and the variability among\nindividuals of its expression in the same brain parts allows us to formulate the working\nhypothesis of the role of Ov-FoxP in the plasticity and/or maintainance of neural networks.\nMy project in O. vulgaris confirms similar results deduced from other studies in both\ninvertebrates (i.e. motor neurons in C. elegans) and vertebrates (i.e. song-birds, mouse,\netc).