Multiple roles for protein kinase C in gastropod embryogenesis

Protein kinase C (PKC) contributes to the correct development of organisms, but its importance to the embryogenesis of molluscs is not yet known. We report here that PKC activation is cyclic within early developing embryos of the gastropod snail Lymnaea stagnalis, and that activation with phorbol myristate acetate (PMA) results in disorganised and developmentally arrested embryos within 24 h. Moreover, chronic modulation of PKC activation by PMA or by the PKC inhibitor GF109203X in early embryos results in altered rotation and gliding behaviours and heartbeat during development. Finally, dis-regulation of PKC activity during early development significantly increased the duration to hatching. Our findings thus support novel roles for PKC in L. stagnalis embryos, in several physiological contexts, providing further insights into the importance of protein kinases for gastropod development in general

PKC and 'Lymnaea stagnalis' embryogenesis

Embryonic development and morphogenesis rely on the co-ordination of complex cell signalling mechanisms. In this study, embryos of the freshwater pond snail 'Lymnaea stagnalis' were used as a model to study Protein kinase C (PKC) signalling during molluscan embryogenesis. Using anti-phospho-specific antibodies against conventional PKC isoforms and confocal immunofluorescence microscopy, phosphorylated (activated) PKC was found in early 1-24 cell and late 24-36 cell embryos. In early embryos, PKC activation appeared transient and cyclical; in addition, PKC activation was observed in the nuclear region during cell division. The PKC inhibitor GFI09203X blocked PKC activation in developing embryos providing a tool to explore the role of PKC during embryogenesis. Early-cleaving embryos were exposed to different concentrations of either GF109203X or the PKC activator phorbol-myristate acetate (PMA). Results revealed that early cleaving embryos which were exposed to 1 !lM and 10 !lM PMA did not develop further and appeared abnormal after 24 hours; however when exposed to 1 !lM and 10,!lM GFl09203X embryo development proceeded. Chronic PMA treatment (0.01 !lM and 0.1 !lM) of early cleavage stage embryos during development resulted in 20% death prior to hatching whilst exposure of later embryonic stages produced 60% embryo lethality. Chronic GF109203X treatment showed no lethal effects on later stage embryos, although 20% death was observed with 10 /lM GF109203X treatment during development of cleavage stage embryos. Cleavage stage embryos exposed to GF l 09203X displayed delayed hatching whereas PMA did not affect duration to hatching; GFl 09203X had the opposite effect when used with later stage embryos. During these experiments rotation, heart beat and gliding were observed. Results revealed that early cleavage stage embryos exposed to PMA throughout development had significantly increased rotation and heartbeat, whereas GFl09203X treated embryos showed significantly lower rotation as well as slower heartbeat. GF109203X and PMA had considerably less effect on these parameters when short term incubations (O - 60 min) were performed, demonstrating that the effects seen following exposure of early cleavage stage embryos are likely due to developmental defects. From these findings it can be concluded that PKC plays an important role during early embryogenesis of 'L. stagnalis'