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 provideprotectionagainstmetaltoxicityandoxidativestresscounteractingthecelldamagecausedbyessen- tial and non-essential heavy metals. Equally important is the physiological role of MTs in the homeostasis of essentialmetals,whichareinvolvedinawidevarietyofcellularprocesses.Theaimofthisworkwastoinves- tigate the expression and the territorial localization of Paracentrotus lividus MT (Pl-MT) mRNA during sea urchindevelopmentbyQuantitativePolymeraseChainReaction(QPCR)andWholeMountInSituHybridiza- tion(WMISH),aswellasthephylogeneticcomparisonwithselectedMThomologspresentindifferentphyla. WefoundthatPl -MTmRNAisaccumulatedinunfertilizedeggsandconstitutivelyexpressedduringdevelop- ment,withverylow levels ofmaternalmRNA atcleavagestages,followedbyasignificantriseduringgastru- lation 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 immu- nofluorescence(IF)usingaspecificantibodyfortheendodermmarkerEndo1demonstratedaco-localization with the Pl-MT transcripts in the midgut and hindgut after the intestine differentiation occurs and when lar- valfeedingbegins.Ourresultsshowforthefirsttimetheconstitutivetemporalandtissue-specificexpression 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.