Evaluation of Epithelial to Mesenchymal Transition in P. lividus embryo

Epithelial to Mesenchymal Transition (EMT) is an evolutionarily conserved developmental process which is essential for shaping embryos, but it is also a key step in activating carcinoma cell motility and metastasis (1). During the EMT process the typical architecture of epithelia undergoes changes, cells loose cellular contacts and increase their migratory ability, acquiring a mesenchymal phenotype. In sea urchin embryos, early EMT is related to the physiological migration of primary mesenchyme cells (PMC) into the blastocoele where later they give rise to the embryonic skeleton. In preliminary screenings, treatment of P. lividus embryos from fertilization up to 16 hr with different concentrations of Kenpaullone (9-bromo-7,12- dihydroindolo[3,2-d][1]benzazepin-6(5H)-one), led to the development of empty blastulae, suggesting that Kp did not inhibit cleavage per se, but prevented EMT. Embryos treated at hatching blastula stage were instead more robust. Kp, a synthetic molecule, was described as an ATP-competitive kinase inhibitor of GSK3 and CDKs(2). In order to define embryonic sensitivity to Kp, linked to specific developmental stages, we added the drug at different concentrations immediately after fertilization for 10 minutes; Kp was then removed and embryos let to develop. Mesenchyme cells differentiation was strongly perturbed, as manifested by aberrant development and the lack of expression of the mesenchymal-specific markers. Moreover, the expression of two genes, WNT8 and GSK3, involved in EMT was evaluated. In Kp treated embryos from 8-cell to early blastula stages Wnt8 gene expression was down-regulated, while GSK3 levels did not change in treated and untreated embryos. At late gastrula stage, Wnt8 expression level was recovered and GSK3 was over-expressed, but the altered phenotipe did not revert. These results indicate that Kp treatments at very early cleavage stages severely perturb the PMCs specification and the EMT process in a irreversible manner.