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Micromere lineages in the glossiphoniid leech Helobdella

Françoise Z. Huang¹, Dongmin Kang^1,*, Felipe-Andres Ramirez-Weber^1,, Shirley T. Bissen² and David A. Weisblat^1,

¹ Department of Molecular and Cell Biology, 385 LSA, University of California, Berkeley, CA 94720-3200, USA
² Department of Biology, University of Missouri, St Louis, MO 63121-4499, USA
^* Present address: Department of Biology, Stanford University, Stanford, CA 94305-5020, USA
Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA

View larger version (26K): [in a new window]   Fig. 1. Time line showing relevant stages of Helobdella development. The numbers on the time line are given in hours after zygote deposition (hours AZD). Micromeres arise by stereotyped cleavages; shaded cells at 7:40, 12:55, 15:55, 19:45 and at 36 hours AZD represent the micromeres born since the previous time point (for details, see Fig. 2). According to the numerical staging system of Fernández (Fernández, 1980), the micromeres are born during stages 4a to 6b, but the strictly temporal staging system is required for most of the experiments described here. For some fate-mapping experiments, embryos in later stages are designated less precisely using the numerical staging system.

View larger version (44K): [in a new window]   Fig. 2. Partial cell lineage diagram for Helobdella, emphasizing micromere lineages. The scale on the left indicates the time since zygote deposition (hours AZD). Macromeres, proteloblasts and teloblasts are indicated by capital letters; blast cells (segmental founder cells derived from teloblasts) are indicated by lower case letters. Micromeres and their progeny are indicated by '; circled lowercase letters denote the original 25 micromeres. Progeny of the micromeres are designated by a system modified from that used in C. elegans (see Materials and Methods for details). Within the lineages leading to the micromeres and their progeny, unequal divisions are indicated by a thickened horizontal line leading to the larger daughter cell. Cell divisions for which the timing is indeterminate are indicated by diagonal lines. NOPQR divisions mirror those of NOPQL and are omitted from this diagram. a, animal; d, deep; L and R, left and right; pb, polar body; s, superficial; v, vegetal.

View larger version (62K): [in a new window]   Fig. 3. Teloblastic cell divisions in the primary quartet lineages. (A) Fluorescence micrograph of an embryo at 32 hours AZD, in which micromeres c' and d' had been injected with RDA and FDA, respectively, at 8 hours AZD. The first few divisions of each primary quartet clone are unequal and oriented primarily along the animal-vegetal axis. The larger daughter cell lies animal to the smaller cell and has a much shorter cell cycle. Thus, a chain of cells is produced from each primary quartet micromere. (B) A higher magnification view of a slightly younger embryo in which b' had been injected with diI. (C) Disposition and identity of the cells shown in B. Double arrow indicates the progeny of the most recent cell division. Scale bars: 20 µm.

View larger version (83K): [in a new window]   Fig. 4. Definitive micromere progeny; lateral views at stage 10 (160-170 hours AZD). Stacked confocal images (A-H) or epifluorescence views showing embryos in which various micromeres had been labeled with lineage tracer as indicated (anterior to left except in E,F,H,J). Most embryos were counterstained with Sytox Green (for confocal microscopy) or Hoechst 33258. (A) Anterior end of an embryo in which micromere d' was injected with RDA shows labeled progeny in supraesophageal ganglion (black arrow), prostomial epidermis (white arrow), and epithelial cells of the provisional integument (arrowhead). (B) An entire embryo in which micromere a' had been injected with RDA. This view illustrates that, in addition to the anterior cells (as in A), the primary quartet micromeres contribute progeny to the epithelium of the provisional integument, which by stage 10 lie compressed along the dorsal midline (arrows). (C,D) Anterior ends of embryos at early and late stage 10, respectively, in which micromere a'' had been injected with RDA. At early stage 10, the a'' clone comprises an undifferentiated set of cells within the left half of the proboscis (C); by late stage 10 (D), these cells project narrow processes, suggesting that they are neurons (arrow) or connective tissue (arrowhead), or both. (E) Right side of the anterior end of an embryo in which micromere c'' had been injected with RDA (compare with C). (F) Right side of the anterior end of an embryo in which micromere b'' had been injected with RDA and micromere c'' was ablated by over-injection (compare with E). (G) Anterior end of an embryo in which micromere a''' had been injected with RDA. Progeny include putative neural or connective tissue cells (arrowheads), or both, associated with the supraesophageal ganglion and a parallel array of elongated cells (arrow), perhaps retractor muscles, within the dorsal proboscis sheath. (H) Right side of the anterior end of an embryo in which micromere dnopq' had been injected with RDA. Progeny include epidermal cells of the proboscis sheath, what appear to be glial cells in the subesophageal (slanted arrow) and supraesophageal (arrowhead) ganglia, and some neurons or connective tissue in the proboscis (horizontal arrow). Micromere dnopq'' generates a mirror image clone on the left side of the embryo (not shown). (I) Side view, focusing on the posterior sucker, of an embryo in which micromere opq'' had been injected with RDA. Progeny include epidermal cells in the provisional integument (horizontal arrow), which are in the process of being sloughed off in this late stage 10 embryo and in the skin of the posterior sucker (vertical arrow). Inset shows a ventral view of the posterior sucker, where the opq''-derived epidermal cells seem to persist. (J) Right side of the anterior end of an embryo in which micromere dm'' had been injected with RDA. Progeny include what appear to be epidermal cells on the outer surface of the proboscis sheath (arrow); from this clone, cell debris (arrowheads) is usually seen between the yolk cell and the germinal plate. (K) Side view of an embryo in which nopq'L had been injected with RDA and nopq''L had been injected with FDA. Both clones gives rise to epidermal cells of the provisional integument (arrowheads) plus a few neurons in the anterior portion of the subesophageal ganglion (vertical arrow) and, more anteriorly, epidermal cells in the anterior sucker or mouth, or both (horizontal arrow). Within this latter group, the nopq'-derived cells invariably lie anterior to the nopq''-derived cells. The right nopq' and nopq'' clones (not shown) are bilaterally symmetric to those of the left nopq' and nopq'' clones, respectively. (L) Side view of an embryo in which the opq' clone was uniquely labeled with RDA by injecting blastomere OPQ with RDA and OPQ' with FDA (see Materials and Methods). Progeny (arrow) comprise cells in the putative anteroventral adhesive organ. Inset shows boxed area at higher magnification. Scale bar: 50 µm in A,C,D-H; 100 µm in B,I-L; 50 µm in inset to I; 30 µm in inset to L.

View larger version (82K): [in a new window]   Fig. 5. Definitive progeny of micromeres dm' and c''' at stage 10 (160 hours AZD). (A) Digital montage, combining the in focus portions of 59 optical sections (10x, 0.45 NA objective; 0.8 µm steps; 2D ‘no neighbors’ deconvolution (Metamorph, UIC) of each section prior to montaging) comprising a side view through an embryo in which micromere dm' had been injected with RDA and micromere c''' with FDA (dorsal is up; anterior to the left; see Materials and Methods for details). (B) The same embryo and imaging procedures as in A, but viewed through a 20x, 0.75 NA objective for higher resolution (74 optical sections; 0.7 µm steps), and without the Hoechst fluorescence images, to bring out the details of the labeled cells. The dm' and c''' clones contribute prominent, interdigitated sets of circumferential fibers, presumably muscles, to the proboscis. Additional labeled cells lie within the proboscis (arrows in C), some of which appear to be part of a most curious and hitherto undescribed network of nonsegmental, interconnected fibers that reaches throughout the body wall of the embryo. (The following abbreviated description of this network was drawn from observations of more than 15 embryos in which both c''' and dm' were labeled, three in which c''' alone was labeled and six in which dm' alone was labeled.) The network consists of five main fibers on each side of the animal. Two roughly parallel fibers on each side (vertical arrows) run the length of the animal near the surface of the body wall; a dorsolateral fiber runs near the edge of the germinal plate and a ventromedial fiber lies 1/4 of the distance from the ventral midline to the dorsal midline. These two fibers extend to the posterior end, where one or both ramify just dorsal to the seven fused ganglia that innervate the tail sucker. Within the anterior midbody, three more main fibers on each side loop between the dorsolateral and ventromedial fibers, at the approximate levels of midbody segments 2, 5 and 8 (horizontal arrows). Additional fibers are present in the anterior midbody segments, but could not be reconstructed in their entirety. This accounts for apparent discontinuities in some fibers; note that many breaks in the fibers in the low resolution image (A) are shown to be continuous in the higher resolution image (B). Note in particular that individual, apparently continuous fibers comprise juxtaposed segments of distinct red (dm'-derived) and green (c'''-derived) cells. (C) Lateral view of the proboscis of the same preparation (anterior towards the left, dorsal is upwards) using the same imaging procedures as in A,B, but viewed through a 40x, NA 0.75 objective (29 optical sections; 0.5 µm steps). Arrows indicate additional cells within the proboscis and proboscis sheath. (D) Combined fluorescence and brightfield (DIC optics) images of a transverse plastic section (10 µm) through the proboscis at about the level of the vertical arrow in C. Circumferential fibers are visible within the proboscis (p). Segments of the fiber network (arrows) are visible in the sheath (s). (E) Digital montage of fluorescence micrographs made from three obliquely horizontal plastic sections (10 µm each) through the dorsal region of the posterior sucker in a roughly horizontal plane (anterior is upwards). The dm'-derived region of the fiber network ramifies in a bilaterally symmetric fashion (better visible on the left, owing to the oblique plane of section). The branches tend to lie between the seven segmental ganglia that have fused to form the caudal ganglion (see also A). Some cell bodies are visible (arrows). (F) Digital montage of the developing posterior sucker of the embryo shown in A,B (20x, 0.75 NA objective; 58 sections, 0.7 µm steps) showing a side view of the ramifying fibers in the tail sucker, just dorsal to the seven fused ganglia (C1-C7). To maintain the same orientation as in A,B, prospective dorsal is downwards in this panel, owing to the curvature of the embryo. Scale bar: 100 µm in A,B; 50 µm in C-F.

View larger version (52K): [in a new window]   Fig. 6. Rise and fall of the micromere b''' clone in normal development. Combined brightfield and fluorescence live images of one typical embryo (22 total) in which micromere b'' had been injected with RDA (0 hours clonal age, 12 hours AZD, not shown). (A) Animal view at 24 hours clonal age (36 hours AZD; see Fig. 1); the labeled clone comprises two cells (arrowheads) within the micromere cap (dotted contour). (B) Dorsal view at 63 hours clonal age (75 hours AZD, see Fig. 1). By this point, the clone (arrowhead) comprises six to ten cells within the anterior portion of the germinal plate (dotted contour shows germinal plate and part of right germ band), but cell death is probably already under way, as evidenced by isolated cellular debris (arrow). (C) Lateral view at 111 hours clonal age (123 hours AZD); by this stage the remnants of the b''' clone (arrow) are confined to a fluorescent ‘bag’ trapped between the germinal plate and the yolk. (D,E) Each panel shows two closely timed (0.5 second apart) lateral views at 129 hours (D) or 147 hours (E) clonal age (141 and 159 hours AZD, respectively). Once the embryo has initiated peristalsis, the fluorescent remnants of the b''' clone (arrows) can be seen to drift back and forth in response to muscle contractions. The b'' clone follows a very similar time course (not shown). Scale bar: 100 µm.

View larger version (26K): [in a new window]   Fig. 7. Micromere fate differences between H. robusta (Sacramento) and Helobdella sp. (Galt). Fluorescence micrographs of stage 10 embryos in which micromeres b'' and c'' had been injected with RDA and FDA, respectively. (A-C) H. robusta (Sacramento). (A) In normal development, c'' contributes a group of cells to the proboscis (see also Fig. 4), and the b'' clone has died. (B) When c'' is ablated (by photolesioning at clonal age 36 hours; see Fig. 8), the b'' clone survives and generates a set of cells resembling the normal c'' progeny. (C) In a single embryo in which the c'' clone was photolesioned at clonal age 48 hours, some progeny of both b'' and c'' survived and intermingled both anteroposteriorly and dorsoventrally. (D) In contrast to the foregoing, the normal development of Helobdella sp. (Galt) entails the contribution of definitive progeny by both micromeres b'' and c''. Note that in this species, the b'' and c'' progeny are largely confined to the ventral and dorsal portions of the proboscis, respectively. Scale bar: 50 µm.

View larger version (11K): [in a new window]   Fig. 8. Time dependence of b'' rescue. Except for the earliest time point (at which micromere c'' was killed directly by over-injection), the c'' clone was photolesioned at selected intervals after injecting c'' with FDA and b'' with RDA (see Materials and Methods for details). The star indicates one of the nine embryos tested at clonal age 48 hours that exhibited in a partial rescue (see Fig. 7C).