Fig. 7. Cells expressing Hro-hh in proboscis arise from specific micromere lineages. (A) Transverse section through the proboscis of an adult Helobdella; the triangular lumen (*) is surrounded by an inner ring (i) comprising mainly the thick ends and nuclei of radial muscles, a middle ring (m) comprising circumferential muscles and an outer ring (o) comprising longitudinal muscles and salivary gland ductules. (B) Magnified view of the boxed area in A highlighting two radial muscles (green), a circumferential muscle fiber (m, pink), plus several longitudinal muscle fibers (pink) and ductules (circles). (Labels i and o have been omitted from B for clarity.) (C) Combined bright-field and fluorescence micrographs showing anterior portion of an embryo in which micromeres a', b' and d' had been injected with RDA (red), FDA (green) and both (yellow), respectively, at stage 4 (8 hours AZD); the embryo was processed for in situ hybridization at early stage 10 (135 hours AZD), and then sectioned in obliquely transverse orientation through the long axis of the embryo (dorsal is up in all sections). (D) A section through the anterior proboscis (left paired arrows in C). Micromeres a', b' and d' contribute cells to the left dorsal, right dorsal and left ventral quadrants of the outer ring (o) of the proboscis, respectively (black arrows). Other experiments demonstrated that the unlabeled right ventral quadrant contains progeny of micromere c' (data not shown), consistent with the established symmetry of the clones of these four cells (Nardelli-Haefliger and Shankland, 1993; Smith and Weisblat, 1994; Huang et al., 2002). Hro-hh transcripts (dark grey) are localized to the inner ring (i) of presumptive radial muscles surrounding the lumen (white *); some of these cells are co-labeled with FDA (green, partially masked by the in situ signal), indicating their descent from micromere b'. Progeny of a' are also present in the proboscis sheath (white arrow). Note also the presence of a middle ring (m, black arrowhead) containing neither lineage tracer nor Hro-hh transcripts. (E) A section through the mid-portion of the proboscis (middle arrows in C) shows Hro-hh transcripts in presumptive longitudinal muscles at the inner edge of the outer ring (white arrowheads); some of these cells appear to co-label with lineage tracer. At this level, all three of the labeled micromeres contribute to the proboscis sheath (white arrow). (F) A section near the posterior end of the proboscis (right arrows in C) includes part of the left side of the supraesophageal ganglion (sg), containing progeny of a' and d'; at this level, the outer portions of the section pass through definitive epidermis ventrally and a temporary embryonic covering [provisional integument (Weisblat et al., 1984)] dorsally. Progeny of all three micromeres are seen in the epithelium of the provisional integument (black arrow) and in the inner (i) and outer (o) rings of the proboscis, including cells that express Hro-hh. (G,H) Obliquely transverse sections (at roughly the level and orientation indicated by the paired arrowheads in C) through an embryo in which micromere dm' (G) or a'' (H) had been injected with RDA (red) at stage 4 (10 hours AZD). (G) Progeny of dm' occupy the middle ring (black arrowhead) of the proboscis, between the inner and outer rings of cells expressing Hro-hh. [The seemingly double-labeled cell (white arrow) is an artifact resulting from the thickness and obliquity of the section.] Other dm' progeny occupy the outer ring (black arrow) and external surface (white arrowhead) of the proboscis. (H) Progeny of micromere a'' contribute to the proboscis sheath (white arrow), and to both the outer (o) and inner (i, white arrowhead) rings of the proboscis. Scale bar: (A,C-H) 50 µm, (B) 30 µm.

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