Identification developmental expression and phylogenetic analysis of genes involved in biomineralization of the sea urchin embryo P. lividus

The objective of this study was to monitor the expression of genes involved in the formation of the magnesium calcite skeleton of Paracentrotus lividus (P. lividus) sea urchin embryos. Transcripts were quantified during development from fertilization to the late pluteus stage and localized within the embryo at the late gastrula and pluteus stages. The group includes genes encoding carbonic anhydrase (ca), tetraspanin (tetr), p16 (p16) as well as the C-type lectin advillin (adv). The coding sequences were obtained by RT-PCR using total RNA and oligonuclotide primers designed on the basis of in silico analysis and phylogenetic comparison with orthologous sequences. Methods used in developmental biology, such as whole mount in situ hybridization (WMISH) using Dig-labeled anti-sense DNA or RNA probes and qPCR using primers designed to amplify fragments within the ORFs, were employed in this study. Results demonstrated a gradual increase in the transcription of ca as the embryo develops and skeletogenesis initiates, starting at the blastula stage and reaching an 18-fold increase at the pluteus stage. In parallel, ca, adv and p16 were specifically expressed in one cellular population, the primary mesenchyme cells (PMC), while the transcripts of tetr were localized within the ectoderm cell layer in the region facing the growing spicules. This report supports evidence of specific expression of genes identified for the first time in P. lividus. The time and site-specific expression of ca, adv and p16 in the PMCs suggests their involvement in skeletogenesis. In addition, a molecular tool-set of genes is provided for further exploration of the biomineralization processes in the sea urchin embryo model. *This project has been funded by the EU Marie Curie ITN of the 7th FP. The first author is an international PhD student, fully sponsored by the above mentioned grant.