Fluorescent cell-lineage tracer injectionnext section
Early cleavage-stage blastomeres, viz., 2b, 3a, 3b, 3c, 3d, 3A, 3B, 3C, 4d and 4D, were injected as part of a large study to trace the cell-lineage of the musculature in Patella coerulea (Damen and Dictus, submitted). Figure 2 shows drawings of early cleavage-stage embryos in which the localization of some injected blastomeres is indicated. Injection into one of these blastomere, the 2b-micromere (yellow in Fig. 2A), resulted in a complex pattern of labeling in 48 to 52 h old post-torsional larvae (Fig. 3). Analysis with CLSM and the subsequent analysis of z-series revealed two large, ciliated prototroch cells and a ring of fluorescent cells that bordered the prototroch at the post-trochal side (Fig. 3A). Medially, a strand of cells was labeled. This strand ran from a ventral position, between the two labeled main prototroch cells, in a dorsal direction towards the dorsal side of the prototroch and curved in a posterior direction towards a position near the visceral mass (Fig. 3). Adjacent to this strand, other cells were labeled at the level of the prototroch. In addition, some labeling was seen on the inside of the right-lateral side of the shell. Most prominently labeled was a post-trochally located transverse band of fibers that was connected to the lateral shell walls. This band of fibers was U-shaped with the base of the U at the ventral side of the larva. This transverse-oriented band of fibers strongly resembled muscle fibers. After injection of cell-lineage tracer into the 3a- and 3b-micromeres (yellow in Fig. 2B), small areas in the right, respectively the left part of the U-shaped transverse band of fibers were labeled as well (results not shown).
Fig. 3. Stereo images and schematic drawings of a 48 h old post-torsional larva that is retracted in its shell and in which the 2b-progeny are labeled. (A) Stereo image showing the fluorescence of the cell-lineage tracer. Animal view. Amongst other structures, a post-trochally located transverse band of fibers (new) is labeled that is connected to the lateral shell walls (*). (B) Schematic drawing of A. (C) Stereo image of the same larva showing the fluorescence of the cell-lineage tracer. Left-lateral view. (D) Schematic drawing of C. * = attachment of the transverse band of fibers to the shell; º = large, ciliated prototroch cells; x = labeling on the inside of the right-lateral side of the shell; ♯ = ring of cells bordering the prototroch at the post-trochal side; + = medially located strand of cells; ant. = anterior; post. = posterior. For rest of legend see figure 1. Scale bar: 50 µm.
In order to verify whether the transverse-oriented structure formed from the progeny of the 2b-micromere is indeed a muscle, 48 to 52 h old larvae were stained with phalloidin and analyzed. The fluorescence of the main larval retractor, accessory larval retractor, pedal plexus, velum muscle ring, and the anlagen of the left and right adult shell muscles was clearly visible. In addition, an unknown muscle was detected (Fig. 4). The morphology of this muscle was exactly like that of the other muscles, although the phalloidin staining of this unknown muscle was weaker. This newly-discovered muscle is located between the two lateral shell walls and is U-shaped. The base of the “U” is located near the hinge of the operculum. The fluorescence of this muscle co-localized with the fluorescence of the transverse-oriented band of fibers observed after tracing the 2b-progeny with TMR-dextran.
Fig. 4. Stereo images and schematic drawings of 48 to 52 h old larvae stained with fluorescently-labeled phalloidin. ( A) Stereo image. Ventral view of a 56 h old post-torsional larva. Phalloidin staining. The unknown muscle is clearly visible (new). In the right side of the larva, the four fibers of the accessory larval retractor (alr) are visible. (B) Schematic drawing of A. (C) Stereo image. Animal view of a 48 h old post-torsional larva. Phalloidin staining. The unknown muscle (new) is located perpendicular to and ventrally of the main larval retractor. The four fibers of the accessory larval retractor, located in the right side of the larva, are indicated as well. (D) Schematic drawing of C. For legend see figure 1. Scale bar: 50 µm.