EFFECT OF Γ-AMINOBUTYRRIC ACID (GABA) EXPOSURE ON EMBRYOGENESIS OF PARACENTROTUS LIVIDUS AND IDENTIFICATION OF GABA-RECEPTOR GENES IN SEA URCHINS

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Abstract

Developmental processes are controlled by regulatory genes encoding for transcription factorsand signaling molecules. Functional relationships between these genes are described by generegulatory networks (GRN), models which allow integration of various levels of information.The sea urchin embryo is an experimental model system which offers many advantages for theanalysis of GRN. Recently, the GRN that governs the biomineralization of the sea urchinembryonic skeleton has begun to be deciphered. Preliminary evidence suggest that the γ-aminobutyric acid (GABA) signaling pathway is involved in skeletal morphogenesis duringdevelopment of the sea urchin. GABA is a molecule synthesized by nearly all organism, frombacteria to humans, and it acts through ionotropic and metabotropic receptors (GABAA-Rs andGABAB-Rs, respectively).We report that Paracentrotus lividus embryos exposed to GABA at concentrations ranging from0.01 to 1.0 mM showed aberrations in axial patterning, with a dose dependent effect.Washout experiments allowed to determine that the period of sensitivity is restricted from theblastula to the gastrula stage.In order to identify GABA-R genes we performed a comprehensive in silico analysis in selectedsea urchin species (P. lividus, Strongylocentrotus purpuratus, and Lytechinus variegatus), and inphylogenetically related organisms, such as the hemichordate Saccoglossus kowalevskii, thechordate Ciona intestinalis, and the nematode Caenorhabditis elegans.By combining iteration of ab initio predictions and pairwise comparative methods, we identifiedthe orthologous genes encoding for GABAB1 and GABAB2, the two subunits which assembleGABAB-R, and we confirmed that all of these organisms possess a unique α/β GABAA-R genepair clustered in the genome. Furthermore, we have observed that the reciprocal disposition ofGABAA-R genes is also evolutionarily conserved.Interestingly, in adjacent position to these genes, we have identified an additional gene, whichshows significant sequence similarity to a invertebrate-specific GABAA-R gene. Indeed, such agene has been only identified in C. elegans, Drosophila melanogaster, and Nematostellavectensis.We also retrieved several cDNA sequences from staged EST databases of the three sea urchinspecies inspected, indicating that these genes are actively transcribed during development. Someselected cDNA plasmids were also isolated from P. lividus total RNA samples and fullysequenced.Hypothetical proteins were deduced and used for phylogenetic analysis, including a selection ofvertebrate and invertebrate GABAA-R subunit sequences. The resulting phylogenetic treestrongly support the hypothesis that the sea urchins contain genes encoding for bothcanonical and invertebrate-specific GABAA-R subunits. Such a collection of data should providea support to better understand the involvement of GABA-signalling pathway in the skeletal GRN
Original languageEnglish
Number of pages2
JournalJOURNAL OF BIOLOGICAL RESEARCH
Volume88
Publication statusPublished - 2015

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