MICROMERE FORMATION AT 3RD CLEAVAGE IS DECISIVE FOR TROCHOBLAST SPECIFICATION IN THE EMBRYOGENESIS OF PATELLA-VULGATA

Trochoblasts of the mollusc Patella vulgata differentiate early in dev elopment into ciliated cells due to a cell intrinsic developmental cap acity. The third cleavage appears to be decisive for their specificati on. Permanent inhibition of cleavage after the third cleavage, as indu ced by a continuous incubation with the drug Cytochalasin B, had no ef fect on trochoblast-specific gene expression later in development. How ever, permanent inhibition of cleavage before third cleavage abolished trochoblast differentiation including trochoblast-specific gene expre ssion. Inhibition of just the third cleavage itself was sufficient to repress trochoblast-specific gene expression. In contrast, inhibiting the second or fourth cleavage did not effect trochoblast-specific gene expression. Correct formation of micromeres at the third cleavage is required to obtain trochoblast-specific gene expression as was shown i n experiments in which the formation of micromeres during third cleava ge was suppressed by pressure. In addition, centrifugation before or d uring the third cleavage disturbs trochoblast-specific gene expression , whereas centrifugation after the third cleavage does not affect troc hoblast-specific gene expression. Thus, during the third cleavage a de cisive step in the determination of developmental fate of trochoblasts takes place, likely resulting in a segregation of activating and inhi bitory determinants. Trochoblast-specific markers in Patella were only expressed in embryos that were division arrested after third cleavage . This suggests that a segregation of differentiation potentials has t o take place before trochoblast differentiation markers are expressed. The restriction of differentiation potentials in the cleaving stage e mbryo is thus required to enable trochoblast-specific gene expression. (C) 1996 Academic Press, Inc.