Materials and methods
Obtaining embryosnext section
Adult specimens of the limpet Patella coerulea (Mollusca, Gastropoda) were obtained from the Mediterranean coast near Trieste (Italy) and near Banyuls-sur-Mer (France). Embryos were obtained as described before (van den Biggelaar, 1977; Damen and Dictus, 1996; Dictus and Damen, 1997). Before injection of cell-lineage tracer, embryos were treated with acidified MPFSW (pH 3.9) for 2-3 min to remove the surrounding jelly layer. Embryos that were not injected but only cultured to stain the musculature with phalloidin were also dejellied. To reduce microbial infection, embryos were usually transferred to antibiotic-containing MPFSW (60 mg penicillin G and 50 mg streptomycin per liter MPFSW) about 6 to 7 h after first cleavage. Some embryos were cultured in the absence of antibiotics. During culturing and all experimental procedures embryos and larvae were kept at 17 to 18ºC.
Cell-lineage, quadrant identification and cell-lineage tracer injection
The early development and cell-lineage of Patella have been described before (see e.g., Wilson, 1904; Smith, 1935; van den Biggelaar 1977; Dictus and Damen, 1997). The nomenclature employed is that of Wilson (1892) and Conklin (1897). For clarity, figure 2 shows some stages of the development of Patella.
The cell-lineage tracer tetramethylrhodamine dextran (MW 10,000 Da, D-1868, Molecular Probes Europe, Leiden, Netherlands; TMR-dextran) was injected using high pressure-injection as described previously (Damen and Dictus, 1994; Dictus and Damen, 1997). TMR-dextran was dissolved at a concentration of 10% (w/v) in aqua dest or in injection buffer (10 mM Hepes, 150 mM KCl, 1 mM CaCl2, 10 mM EGTA, pH 7.0).
Blastomeres, viz., 2b, 3a, 3b, 3c, 3d, 3A, 3B, 3C, 4d and 4D, were injected at specific stages of development. For instance, the 2b-micromere was injected at the 16-cell stage. Embryos of Patella are radially symmetrical up to about 60 min after the fifth cleavage (32-cell stage). Therefore, quadrant identity (A-, B-, C-, or D-quadrant) cannot be determined until after this moment (van den Biggelaar 1977; van den Biggelaar and Guerrier, 1979; Damen and Dictus, 1996). By analyzing injected larvae retrospectively, the identity of injected blastomeres was determined and embryos in which the 2b-micromere was injected were identified. The 3a- and 3b-micromeres were injected at the 32-cell stage. Since at about 60 min after fifth cleavage embryos are still at the 32-cell stage, and the quadrants can be denominated at this stage, 3a- and 3b-micromeres could be identified before injection.
Fig. 2. Schematic drawings of several stages in the development of Patella, modified after van den Biggelaar (1977). (A) Lateral view of a 16-cell stage embryo. The first quartet micromeres (1a-1d) have divided and the second quartet micromeres are formed (2a-2d). (B) Lateral view of a 32-cell stage embryo. In this view the first (1a-1d progeny), second (2a-2d progeny) and third (3a-3d) quartet micromeres as well as the third generation macromeres (3A-3D) are visible.
Selection and fixation
Abnormal larvae at 48 to 52 h after first cleavage, i.e., larvae that do not possess eyes, a prototroch, a shell, a foot and an operculum or that are not able to retract into their shell, were discarded. The muscles of normal larvae of 48 to 52 h after first cleavage were relaxed by adding drops of 0.75 M MgCl2 to the MPFSW. Subsequently, the larvae were fixed in 4% formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h and rinsed in buffer (3 times for 10 min).
Phalloidin staining and mounting
In order to visualize the musculature, most of the fixed larvae were stained with fluorescently-labeled phalloidin (Alexa Fluor 488 phalloidin, 300 U, A-12379, Molecular Probes Europe, Leiden, Netherlands), that binds to F-actin (modified after Wanninger et al., 1999). Larvae were permeabilized for 1 h in 0.1 M phosphate buffer (pH 7.4) to which 0.2% (v/v) Triton X-100 was added to allow penetration of phalloidin. A stock solution of phalloidin was prepared by dissolving one vial of Alexa Fluor 488 phalloidin (300 U) in 1.5 ml methanol. This stock solution was kept at -20°C. Some 5 or 10 µl of the stock solution were put in a small glass petri dish and the methanol was allowed to evaporate. Larvae were taken in 200 µl of the 0.1 M phosphate buffer (pH 7.4) to which 0.2% Triton X-100 was added and put on the dry phalloidin. The petri dish was gently shaken to dissolve the dried phalloidin after which the larvae were incubated for 1 h. Subsequently, the larvae were washed in 0.1 M phosphate buffer (3 times for 10 min) and dehydrated in a graded series of ethanol (70%, 80%, 90%, 96%, 100%, 100%, 100%; each step 5 min). After transferring the larvae to a 1:1 (v/v) mixture of ethanol and Murray’s (1:2 v/v benzylalcohol and benzylbenzoate), they were mounted in convex polished slides in Murray’s to which 0.1% of the antifading agent n-propyl gallate (Sigma, St. Louis, Mo, USA) was added.
Confocal laser scanning microscopy (CLSM)
All larvae were observed in a Leica upright confocal laser scanning microscope [Leica DM-RBE (microscope), Leica TCSNT (confocal laser), Leica, Heidelberg, Germany]. One, or in most cases two or even three z-series were recorded at different angles. All z-series consisted of 72 images and were transformed into stereo images that were analyzed with a pair of red-green stereo glasses.