Contributions to Zoology, 71 (1/3) 37-45 (2002)
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Contributions to Zoology, 71 (1/3) 37-45 (2002) SPB Academic Publishing bv, The Hague Newly-discovered muscle in the larva of Patella coerulea (Mollusca, the of larval Gastropoda) suggests presence a extensor Peter Damen and Wim+J.A.G. Dictus Department ofExperimental Zoology, Utrecht University, Padualaan 8, 3584 CH Utrecht, Netherlands, [email protected] Key words: Patella, Gastropoda, Mollusca, development, larva, muscle, antagonist, cell-lineage Abstract and Dictus and Damen (1997). The formation of the 3D-macromere, which acts as an organizer and The developmentofthe muscular system ofthe gastropod mollusc is responsible for the correct dorsoventral pattern- Patella has been thoroughly studied. As a result, two larval of the has been described ing embryo, by e.g. van the main and larval had been retractors, accessory retractor, den Biggelaar and Guerrier (1979), Arnolds et al. described in ofPatella. the larva These muscles were supposed (1983), Verdonk and van den Biggelaar (1983), to be responsible for the retraction of the larval body into the Martindale et al. and Damen and Dictus shell.Previously no larval extensors, which would be responsible (1985), The of function for the extension of the larval body out ofthe shell, have been (1996). analysis gene during early described. Using cell-lineage tracer injection and phalloidin development has been described by van der Kooij staining ofmuscles, anewly-discovered muscle is herein identi- et al. (1996, 1998), Damen et al. (1994), Damen fied in the larva of Patella coerulea. A functional model is and Loon Klerkx van (1996), et al. (2001), Lartillot presented in which two muscles areresponsible for the extension et al. (2002), Lespinet et al. (2002), Nederbragt et of the larva, the newly-discovered muscle together with the al. and and but named larval (2002a, b, c), Wanninger Haszprunar previously recognized inaccurately accessory The formation ofthe musculature retractor. In our model,the latter muscle does not seem to function (2001). has been as a retractor but rather as an extensor. described by Smith (1935), Crofts (1955), and Wan- ninger et al. (1999). In the first half of the previous century, Smith Contents (1935) and Crofts (1955) studied muscle morpho- in genesis Patella with the use of a traditional Introduction 27 technique, i.e. microscopical observation of serial Materials and Methods 39 sections. Much later, Wanninger etal. (1999) studied Results 40 the formation of the musculature in the larva of Discussion 41 Patella using a much more recently introduced Acknowledgements 44 stained larvae of Patella with fluor- References 44 technique. They escently-labeled phalloidin, which binds to F-actin, in order to visualize the musculature using confocal Introduction laser scanning microscopy (CLSM). With this tech- nique, muscle development can be analyzed at a For many decades, the gastropod mollusc Patella much higher resolution and even enables a three- has been used as a model system to study various dimensionalanalysis of muscle localization. Using aspects of early development. For instance, the cell- CLSM, Wanninger et al. (1999) showed that the lineage has been described by Patten (1886), Wil- formation of the principal larval and adult muscles son den in (1904), Smith (1935), van Biggelaar (1977), Patella takes place between about 28 and 52 h Downloaded from Brill.com10/07/2021 11:07:18AM via free access - 38 P. Damen & W.J.A.G. Dictus Newly-discovered muscle in the larva ofPatella Fig. I. Post-torsional, 50 h old relaxed larva in which the muscles that are visible at this stage are shown. Right-lateral view. (A) Stereo image. Phalloidin labeling. (B) Schematic drawing ofA. In this schematic drawing and the schematic drawings that follow the muscles are drawn with different colors. For clarity, one part of the muscles is drawn with saturated colors and the other part of the muscles with the the larval and the velum muscle which correspondingpastel colors, except accessory retractor, pedal plexus ring, are drawn in saturated colors, air = larval Ism = left adult shell muscle = mantle accessory retractor (yellow); (pastel brown); m.c. cavity; mlr = main larval retractor = = = = adult shell muscle (red); oper. operculum; pp pedal plexus (green); prot. prototroch; rsm right = velum muscle Scale bar: 50 All should be with (saturated brown); vmr ring (pink). pm. stereo images examined red-green stereo glasses in order to see the three dimensional localization of the structures shown. first after cleavage. The muscles they described are Patella coerulea were injected with a fluorescent main their the larval retractor, the accessory larval re- marker to study cell-lineage. Injection of one tractor, the velum muscle ring, the pedal plexus of the blastomeres, the 2b-micromere, resulted in and the anlagen of the left and right adult shell labeling of an unknown bundle of fibers in 48 to muscles Detailed of both 52 h old larvae. After of (Fig. 1). comparison ap- post-torsional injection proaches demonstrates that the traditional approach the 3a- and 3b-micromeres small parts of this bundle Smith and labeled of (1935) Crofts (1955) is quite prone of fibers were as well. Since this unknown and result in miss- bundle of fibers to misinterpretations even may strongly resembled a muscle, 48 52 larvae with ing certain details of muscle formation. to h old post-torsional were stained The 48 to 52 h old post-torsional larva of Pa- fluorescently-labeled phalloidin. In a number of tella is a well-developed larva with a head, a foot, these larvae, besides the muscles described above, a shell and a prototroch (Fig. 1). It is capable of the unknown bundle of fibers was labeled as well, retraction into its larval shell. The main larval re- demonstrating that the unknown fibers are indeed tractor and the accessory larval retractor are sup- muscle fibers. posed to be responsible for this process, although Based on its location and orientation, together this has not been stated explicitly for Patella (Wan- with the fact that no explanation for the extension ninger et al., 1999). Besides retraction, the animal of the larva out of its shell is given in the litera- must be able to extend out of its larval shell. De- ture, it is hypothesized that the newly-discovered spite that the larva is obviously able to do so, nei- bundle of muscle fibers is involved in the exten- the ther muscles nor other mechanisms responsible for sion of the larval body out of shell. A hypothetical this action have been described in the literature. model is presented in which the newly-discovered As “retrac- part of a larger study (Damen and Dictus, muscle, together with the accessory larval submitted) various early cleavage-stage blastomeres, tor”, are responsible for the extension of the larval viz., 2b, 3a, 3b, 3c, 3d, 3A, 3B, 3C, 4d and 4D, of body out of the shell. As a result, these two muscles Downloaded from Brill.com10/07/2021 11:07:18AM via free access Contributions to Zoology, 71 (1/3) -2002 39 form the of main larval determined and in antagonists the retractor. embryos which the 2b-micromere was injected identified. The 3a- and 3b-micromeres data were were at Reanalysis of literature suggests that a struc- injected the 32-cell stage. Since at about 60 min after fifth cleavage ture similar to the previously undescribed muscle, still at the embryos are 32-cell stage, and the quadrants can be and thus a potential larval extensor, is present in at denominated at this stage, 3a- and 3b-micromeres could be least one other and that the gastropod previously identified before injection. undescribed muscle is homologous to the circular muscle fibers in the Mullers larva of the platyhel- Selection andfixation minth Hoploplana. 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 that operculum or are not able to retract into their shell, were Materials and methods discarded. The muscles ofnormal larvae of 48 to 52 h after first cleavage were relaxed by adding drops of 0.75 M MgCl to the 2 Obtaining embryos 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 Adult specimens of the limpet Patella coerulea (Mollusca, (3 times for 10 min). Gastropoda) were obtained from the Mediterraneancoast near and Trieste (Italy) near Banyuls-sur-Mer were (France). Embryos Phalloidin staining and mounting obtained as described before (van den Biggelaar, 1977; Damen and Dictus, 1996; Dictus and Damen, 1997). Before injection In order to visualize the musculature, most ofthe fixed larvae of cell-lineage tracer, embryos were treated with acidified were stained with fluorescently-labeledphalloidin (Alexa Fluor MPFSW (pH 3.9) for 2-3 min to remove the surrounding jelly 488 phalloidin, 300 U, A-12379, Molecular Probes Europe, layer. Embryos that were not injected but only culturedto stain Leiden, Netherlands), thatbinds to F-actin (modified after Wan- the musculature with phalloidin were also dejellied. To reduce et ninger al, 1999). Laryae were permeabilized for 1 h in 0.1 M microbial infection, embryos were usually transferred to anti- phosphate buffer (pH 7.4) to which 0.2% (v/v) Triton X-100 MPFSW G and 50 biotic-containing (60 mg penicillin mg strepto- was added to allow penetration of phalloidin. A stock solution liter about 6 7 h after mycin per MPFSW) to first cleavage. of phalloidin was prepared by dissolving onevial ofAlexa Fluor Some embryos were cultured in the absence ofantibiotics. During 488 phalloidin (300 U) in 1.5 ml methanol. This stock solution culturing and all experimental procedures embryos and larvae at -20°C.