Mesoderm specification and myogenesis in Priapulus caudatus. (A-D) Whole-mount in situ hybridization of twist. twi is expressed in the most vegetal endomesodermal cells, and in two lateral bands of the gastrula (black arrows in A) and later in the developing musculature of the introvert (black arrow in C) and trunk. In (A-C) the asterisk indicates the blastopore/anus. Drawings depict the expression domains. The slight elongation of embryos might be an artifact of fixation. (E, F) z projections of confocal stacks of embryos at 5, 6, 7, and 8 days post-fertilization, hatching larva, and the first lorica larva, stained with phallacidin (green) and propidium iodide (magenta). (E) Muscle fibers appear as actin-positive bundles around the equator of the embryo (white arrows). (F) Subsequently, the trunk musculature and longitudinal muscles (lm) (inset) become visible. (G) Before introvert retraction, the musculature appears more developed. (H) Introvert retraction extends the circular and longitudinal musculature of the trunk, while short retractor muscles attach the introvert to the trunk. (I) The hatching larva exhibits a musculature similar to that of the late embryo, while (J) the first molt involves an increase in general complexity. (E’-J’) Schematic drawings depicting the basic muscular patterns. A, C, E-J, lateral views; B, vegetal view; and D, anterior view. All panels and drawings are oriented with the animal/anterior pole to the top. In E, ventral side to the left. The pairs of arrowheads in C and E-J’ indicate the position of the introvert-trunk boundary. Drawings are not to scale. Scale bars in E-H, 50 μm; and I and J, 100 μm. an, anus; bp, blastopore; cm, circular muscles; em, endomesoderm; im, Introvert musculature; lrm, long retractor muscles; mo, mouth; pm, primary muscles; sc, scalids; srm, short retractor muscles; tm, trunk musculature.