Tissue-specificity and phylogenetics of Pl-MT mRNA during Paracentrotus lividus embryogenesis

Metallothioneins (MTs) constitute a family of cysteine-rich, low molecular weight proteins, which generally provide protection against metal toxicity and oxidative stress counteracting the cell damage caused by essential and non-essential heavy metals. Equally important is the physiological role of MTs in the homeostasis of essential metals, which are involved in a wide variety of cellular processes. The aim of this work was to investigate the expression and the territorial localization of Paracentrotus lividus MT (Pl-MT) mRNA during sea urchin development by Quantitative Polymerase Chain Reaction (QPCR) and Whole Mount In Situ Hybridization (WMISH), as well as the phylogenetic comparison with selected MT homologs present in different phyla. We found that Pl-MT mRNA is accumulated in unfertilized eggs and constitutively expressed during development, with very low levels of maternal mRNA at cleavage stages, followed by a significant rise during gastrulation with a peak at the prism stage. Pl-MT mRNA was expressed in the vegetative plate at mesenchyme blastula, later restricted to the endoderm of gastrula embryos and finally to the gut of plutei. Indirect immunofluorescence (IF) using a specific antibody for the endoderm marker Endo1 demonstrated a co-localization with the Pl-MT transcripts in the midgut and hindgut after the intestine differentiation occurs and when larval feeding begins. Our results show for the first time the constitutive temporal and tissue-specific expression of MT in P. lividus embryos, providing new information for studies on the mechanisms controlling basal and induced MT gene expression. The analysis of the phylogenetic relationship of Pl-MT with homologs from different phyla, ranging from yeast to vertebrates, suggests the evolutionary process of these proteins, which could have been selected not only on the basis of their ability to bind metals but also by their tissue-specificity.