Three Distinct Distributions of F-Actin Occur during the Divisions of Polar Surface Caps to Pole Cells in Drosophila Embryos Produce

The F-actin distribution was studied during pole cell formation in Drosophila embryos using the phalloidin derivative rhodaminyl-lysine-phallotoxin. Nuclei were also stained with 4'-6 diamidine-2-phenylindole dihydrochloride to correlate the pattern seen with the nuclear cycle. The precursors of the pole cells, the polar surface caps, were found to have an F-actin-rich cortex distinct from that of the rest of the embryo surface and an interior cytoplasm that was less intensely stained but brighter than the cytoplasm deeper in the embryo. They were found to divide once without forming true cells and then a second time when cells formed as a result of a meridional and a basal cleavage. Three distinct distributions of the cortical F-actin have been identified during these cleavages. It is concluded that the first division, which cleaves the polar caps but does not separate them from the embryo, involves very different processes from those that lead to the formation of the pole cells. A contractile-ring type of F-actin organization may not be present during the first cleavage but is suggested to occur during the second. The mechanism of Drosophila pole cell formation is of interest to both cell and developmental biologists because the pole plasm, from which the cells are formed, is a very welldocumented case of a region of an egg that contains morphogenetic determinants. These determinants have important controlling influences over the spatial organization of the cells formed and quite possibly over the determination of their fate (for reviews see references 4, 12, 19, and 23). Pole plasm forms as an area of cortical cytoplasm at the posterior tip of the growing oocyte. It is characterized by the presence of polar granules, small basophilic bodies 0.5-1.0 ~tm diam, of unknown significance but present in the germ cell lines of many groups. The pole cells, which are derived from this region only, have a roughly spherical shape quite distinct from the columnar morphology of the somatic blastoderm cells that make up the rest of the embryo. Once formed, they migrate to the gonads where they give rise to the germ cells. As a first step towards understanding the controlling influences of pole plasm, it is necessary to understand how pole ceils are formed and how this process is distinct from the formation of the blastoderm cells and the somatic surface caps that are their progenitors. The caps are unusual structures. They are not true cells but surface protuberances each 1010 overlying a nucleus (6, 24). During the syncytial blastoderm stage, they undergo four cycles of expansion and flattening followed by division and a rounding up of the daughter caps. Cell membranes then extend simultaneously between all the caps to form the single layered cellular blastoderm. Fluorescent derivatives of the cyclic peptide phalloidin, derived from the mushroom Amanita phalloides, have proved to be specific stains for F-actin microfilaments in vitro (22, 28). By using this technique, it is possible to visualize the Factin distribution associated with the surface caps during the syncytial and cellular blastoderm stages (25, 26). In this paper we describe the distribution of F-actin during the formation of the progenitors of the pole cells, the polar surface caps, their divisions, and pole cell formation. MATERIALS AND METHODS The techniques for embryo preparation, staining, mounting, and epifluorescence microscopy have been previously described (25, 26). To obtain embryos with precisely staged pole cells, we observed them until the correct stage was reached and then immediately pierced them with a microneedle. Staining of Factin was done with rhodaminyl-lysine-phallotoxin (RLP), ~ a chemically synAbbreviations used in this paper: DAPI, 4 ' -6 diamidine-2-phenylindole dihydrochloride; RLP, rhodaminyllysine-phallotoxin; st,