F-actin localization during trochoblast differentiation embryos

0 Present address: Hubrecht Laboratory, Netherlands Institute for Developmental Biology , Uppsalalaan 8, 3584 CT Utrecht , The Netherlands 1 Department of Experimental Zoology, University of Utrecht , Padualaan 8, 3584 CH Utrecht , The Netherlands F-actin localization during trochoblast differentiation in Patella vulgata - We have studied the development of the ciliated, locomotory organ of Patella vulgata trochophore larvae. This organ, the prototroch, arises from different clones of trochoblasts. In each of these trochoblasts, a band of filamentous (F-) actin is formed at the time that ciliogenesis starts. This band, which we visualized with TRITC-phalloidin, is positioned at the base of therow of cilia that crosses each trochoblast. Isolated trochoblasts, as well as isolated quartets of animal micromeres (from which the trochoblasts are derived), similarly form rows of cilia and F-actin bands at the proper time in development. In whole embryos, the trochoblasts shift their position following ciliogenesis, and finally form a ring of differentiated prototroch cells with a continuous band of F-actin encircling the entire larva. At the dorsal side, a double row of prototroch cells and thus a double band of F-actin is present. In contrast, multiple regions with a double F-actin band are found in trochophores in Embryos of the gastropod mollusc Patella vulgata develop a ciliated organ, called the prototroch, which is responsible for the locomotion of the trochophore larva. The prototroch has a polyclonal origin: it arises from a well-ordered ring of two types of intercalated clones of trochoblasts, i.e. the primary and the accessory trochoblasts. The primary trochoblasts, formed at the 16-cell stage, and the accessory trochoblasts, formed at the 32-cell stage, divide only two more times, after which they start to differentiate: they become cell-cycle arrested, reduce their communication via gap junctions and develop cilia (Wilson, 1904; Van den Biggelaar, 1977; Janssen-Dommerholt et al. 1983; Serras et al. 1990). Later in development, these trochoblasts shift their position becoming organized into a ring of ciliated prototroch cells that encircles the entire embryo. In this paper, we have studied the organization of F-actin during development of the prototroch in Patella which the establishment of a dorsoventral axis is inhibited experimentally. Confocal laser scanning microscopy shows that the F-actin band extends from the apical surface deep into the cytoplasm of the prototroch cells. At the ultrastructural level, a single striated rootlet connected to the basal body of each cilium can be seen to extend deep into the cytoplasm toward the nucleus, and a band of actin-like filaments is found to interconnect neighboring basal apparatus. Treatment of trochophores with cytochalasin B disrupts the organization of the F-actin band as visualized with TRITC-phalloidin, affects the angle of the effective stroke of ciliary beat and reduces their swimming capacity. This suggests that the F-actm band is essential for the normal locomotory behavior of the Patella trochophore larva. vulgata embryos. We found that differentiation of the trochoblasts is accompanied by the formation of a distinct F-actin band, which underlies the row of cilia that develops on the surface of each trochoblast. The fate of this band during subsequent formation of the prototroch as well as its possible significance for locomotion of the trochophore larva were studied. In addition, the identity of the F-actin-containing structure was investigated at the ultrastructural level. Materials and methods Embryos Patella vulgata was collected at Roscoff (Bretagne, France) and kept in aquaria at 15 C. Oocytes and sperm were obtained by dissection. Maturation of the oocytes was induced by treatment with Millipore-fUtered sea water pH 8.9, for 7min. The mature eggs were inseminated by adding a diluted solution of sperm collected from two or three males. Synchronously cleaving embryos were selected and kept in small Petri dishes. All experiments were carried out at19C. F. Serras and J. E. Embryos used for cell isolation experiments, monensintreatment and scanning electron microscopy were dejellied at the required stage by a 1-2 min treatment with filtered sea water pH3.8. The developmental stage of the embryos is indicated by the number of cells up to the 88-cell stage. At later stages, when the cell pattern becomes very complex and the number of cells cannot be determined accurately, time after first cleavage is indicated instead. In all experiments, groups of 20-30 synchronously cleaving embryos were fixed at the desired stage and further processed for fluorescence labeling or electron microscopy. Labeling with TRITC-phalloidin and Hoechst 33258 Phalloidin is a toxin that stains F-actin in fixed material (Wulf et al. 1979; Barak et al. 1980). The pattern of filamentous (F-) actin was studied by fixing embryos in 3.7 % formaldehyde in buffer (10mM Pipes pH6.8, IOOITIM KC1, 5mM magnesium acetate, 5mM EGTA pH6.8) for lOmin, after which they were rinsed in buffer, transferred to 0.5 % Nonidet P-40 in buffer for 5min, rinsed in buffer, incubated in Cl/jgrnl"1 TRITC-phalloidin (Sigma, St Louis, MO) in buffer for 1 h and rinsed in buffer. Control groups were incubated in 0.1/igml"1 TRITC-phalloidin in buffer to which lO/igml"1 unlabeled phalloidin was added. Only slight background fluorescence was observed in such controls. The TRITC-phalloidin-labeled embryos were subsequently stained with 10/igml"1 of the fluorescent nucleic acid-specific dye Hoechst 33258 (Riedel de Haen AG, Hannover, FRG) in buffer. After 10 min, the embryos were rinsed extensively and mounted on a microscope slide in buffer. They were examined with a Zeiss Axiovert 35 M microscope using Differential Interference Contrast (DIC) optics as well as epifluorescence illumination (TRTTC: exc. 510-560 nm, em. 590 nm; Hoechst: exc. 365 nm, em. 420 nm). Photographs were taken on Kodak Ektachrome 400 ASA and Tmax 100 ASA film. Confocal laser scanning microscopy (CLSM) Specimens were also examined with a Bio-Rad Lasersharp MRC-500 confocal laser scanning microscope (Bio-Rad Lasersharp Ltd, Oxfordshire, UK) to determine more precisely the position of the F-actin band relative to the apical surface of the prototroch cells. Series of optical sections at steps of 1.1/xm each were made using a Zeiss Axioplan microscope equipped with a 63x/NA 1.4 oil immersion objective. Isolation of animal caps and trochoblasts Pipettes with a tip diameter of about 10-20 /an were mounted on a micromanipulator attached to a stereo microscope. The back of the pipet was connected to a Teflon tube, through which suction was applied. By means of the micromanipulator, the tip of the pipet was brought to the surface of the embryo, and single cells were removed from the embryo. A first series of operations was performed at the 8-cell stage. By removing the four vegetal macromeres (...truncated)