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First published online 23 March 2005
doi: 10.1242/dev.01802

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Applications of mRNA injections for analyzing cell lineage and asymmetric cell divisions during segmentation in the leech Helobdella robusta

¹ Graduate Group in Biophysics, LSA 385, University of California, Berkeley, CA 94720-3200, USA
² Department of Molecular and Cell Biology, LSA 385, University of California, Berkeley, CA 94720-3200, USA

View larger version (26K): [in a new window]   Fig. 1. Events in the N lineage leading to gangliogenesis in H. robusta. Cleavage yields an embryo with a posterior growth zone of five bilateral pairs of segmentation stem cells (teloblasts); only the four pairs of ectoteloblasts are shown here. Ignoring gastrulation, segments arise in anteroposterior progression along the ventral midline by the stereotyped divisions of columns of segmental founder cells (blast cell bandlets). In the N (and Q lineages), clones of two successive blast cells, designated ns (red) and nf (blue) contribute distinct sets of progeny to each segment. Because blast cells of each type execute a stereotyped pattern of cell division relative to the time of their birth from the parent teloblast, events in the generic nf and ns lineages can be designated as occurring at a specific `clonal age' (cl.ag.), as indicated on the time line on the left. Primary n blast cells divide unequally, producing larger anterior (nf.a, dark blue; ns.a red) and smaller posterior (nf.p, light blue; ns.p, pink) daughter cells (40 and 44 hours cl.ag., respectively). Transverse fissures (arrows) separate nf.p clones (light blue) from ns.a clones (red) dividing the bandlets into discrete ganglionic primordia (85-95 hours cl.ag.). Anterior is upwards in all figures unless otherwise noted.

View larger version (115K): [in a new window]   Fig. 2. nXFPs as lineage tracers in Helobdella. (A) Photomontage made from stacks of confocal images (processed by Imaris, Bitplane) showing a ventral view (midline to left) of a stage 10 embryo, 140 hours after the left N teloblast had been injected with nGFP mRNA. nGFP labeled nuclei occupy the anterior and posterior lobes of N-derived ganglionic neurons and the three peripheral neurons lying just outside the posterior (nf-derived) lobe (arrows). (B) Higher magnification view of the boxed region in A. (C) Epifluorescence images of the germinal plate of a late stage 8 embryo (ventral midline towards the left) about 100 hours after the left N and P teloblasts were injected with nGFP mRNA. The N and P lineages can be distinguished by a difference in fluorescence intensity, reflecting differences in the amount of mRNA injected into the parent teloblasts. The temporal gradient of development is evident in the progress of migrating neurons (arrowheads) which originate from a single cell at the medial edge of the p blast cell clone (arrow). (D) Side view of an early stage 10 embryo, 150 hours after the left N teloblast had been injected with nGFP mRNA shows an anterior (arrowhead) to posterior gradient of GFP intensity. (E) Pseudocolored image (confocal stack) showing a ventral view of a live mid stage 8 embryo, 83 hours after one N teloblast had been injected at stage 7 with nCFP mRNA and the other with nYFP mRNA 3 hours later (nCFP, purple; nYFP, green). Scale bar: 20 µm.

View larger version (70K): [in a new window]   Fig. 3. AS MO knockdown of injected mRNA expression. (A,B) N teloblasts were injected at stage 6 with nGFP mRNA, 3 hours later with a mixture of fluorescent dextran (RDA) and antisense morpholino oligomer (AS MO), and then imaged after another 46 and 98 hours. The left side of each panel shows the combined RDA (red) and nGFP (green), and the right side shows nGFP only, revealing the decrease in nGFP fluorescence in clones produced after the AS MO injection. In these experiments, two or three blast cells were produced between the first and second injections. (C) Enlarged view of the embryo shown in A; fluorescence intensity was measured along the line shown crossing blast cell nuclei produced before and after the AS MO injection and plotted on the right. Scale bar: 10 µm.

View larger version (88K): [in a new window]   Fig. 4. Distribution and perdurance of injected mRNA. N teloblasts in stage 7 embryos were injected with nGFP mRNA. The embryos were fixed at times ranging from 1-120 hours post-injection, then processed in parallel for in situ hybridization. Arrowheads indicate the position of the first labeled clone, where visible. (A-D) Animal pole views of embryos fixed 1-72 hours after injection. The teloblast and relatively young blast cells (arrowhead in B) stained intensely, but a significant decline in staining was apparent in blast cell clones that had undergone their first mitosis (arrow in C,D). (E) Prospective posterior view of an embryo fixed 96 hours after injection. Although the teloblast still stained very intensely, the first labeled clone (not visible in this view) was barely visible. (F) By 120 hours after injection (side view), staining in the teloblast was clearly reduced, but still clear. By contrast, nGFP mRNA was no longer detectable in the first labeled clone under these conditions (arrowhead indicates the estimated position of the first labeled clone; compare with Fig. 2A,D). Scale bar: 100 µm.

View larger version (52K): [in a new window]   Fig. 5. Application of nGFP mRNA injections to analysis of the N lineage. (A) Time series showing the anterior portion of an n bandlet; one ns blast cell was uniquely labeled by tandem injections of the parent teloblast, first with nGFP mRNA and then, after one blast cell was born, with RDA. Beginning 58 hours after the first injection (I, 58-70), the uniquely labeled clone was imaged hourly for 12 hours. During this time, the clone increased from two cells to seven; a similar pattern of nuclei was observed in a sibling embryo imaged only at 70 hours cl.ag. (II, 70). (B) Reconstruction of an embryo injected as in A at 86 hours cl.ag. In the first (ns) labeled clone, superficial nuclei are pseudocolored lavender and deep nuclei (2, 4, 8, 11, 12, 14) are red (8 and 12 are obscured by overlying nuclei). In the next (nf) clone, nuclei are pseudocolored green. Those in more posterior clones appear white. The numbers over the nuclei of the ns and nf clones correspond to cells in the lineages trees in Fig. 6A and B, respectively. (C) Three-dimensional reconstruction of a similar embryo with the ns clone at 90 hours cl.ag. The ns clone (lavender) now has 18 nuclei; the posterior nf clone (green) has 18 interphase nuclei and one in mitosis (broken outline); the exact identities of these cells remain to be determined. In this panel, the cytoplasmic RDA fluorescence is shown in red surrounding nuclei in the nf and more posterior (white) clones, so that the elongating fissure between prospective ganglionic primordia is visible (arrow); midline towards left in B and C. Scale bar: 10 µm.

View larger version (14K): [in a new window]   Fig. 6. Diagram of the early ns (A) and nf (B) blast cell lineages (non-linear time scale). Cell nomenclature is adapted from C. elegans as described previously (Huang et al., 2002). anterior, a; posterior, p; medial, m; lateral, l; superficial, s; deep, d. The cl.ag. given for each division represents the average of observations drawn from three to five specimens in each case. Numbers correspond to the cells in Fig. 5B.

View larger version (58K): [in a new window]   Fig. 7. Asymmetric blast cell mitoses: nuclear volume ratios of nf and ns progeny. (A) Pseudocolored 3-D reconstruction of an nGFP labeled n bandlet, showing two two-cell ns blast cell clones (red) and two two-cell nf blast cell clones (green). (B) Graph showing the ratio of the volumes (mean±s.d.) of cells like those shown in A.

View larger version (87K): [in a new window]   Fig. 8. Early progeny of the N teloblast. (A) Lineage diagram showing divisions of the N teloblast. After its birth (stage 6a, 22 hours AZD), the N teloblast divides every 90 minutes, generating a column of progeny that mostly follow the ns and nf (segmental founder cell) fates in exact alternation. But the fourth cell (n') contributes exclusively to the provisional epithelium, and the fates of cells born prior to n' were not known with certainty. (B) Pseudocolored 3D reconstruction of an nGFP-labeled n bandlet, 48 hours after the N teloblast was injected. The clone of the anteriormost cell contains four nuclei of equal size (yellow), unlike either nf or ns, so this is designated n°. The three cells posterior to this have each divided once, in manner characteristic of the nf (green) or ns (red) clones. The nucleus in the sixth position (broken outline) has entered mitosis before the primary blast cell just ahead of it, also indicative of the nf (green) and ns (red) fates. By this time, the n' micromere clone contains six cells (blue), superficial to the bandlet. (C) Equivalent view of an older embryo, in which the N teloblast was re-injected with RDA after the birth of n° (all nuclei are shown as yellow). By cl.ag. 96 hours, the n° clone comprises 70-80 nuclei, distinguished by their smaller size and less intense fluorescence. Nuclei of the RDA labeled nf and ns blast cells are larger, brighter and surrounded by RDA fluorescence (red). The n° clone is now flanked by cells derived from the anteriormost nf clone (arrows). Fissures (arrowheads) have formed between the posterior edges of the nf clones and the anterior edges of the adjacent ns clones. (D) Ventral view of the subesophageal ganglion and presegmental tissue of an embryo (146 hours AZD) in which the left N teloblast was injected with RDA shortly before the birth of its first ns blast cell, i.e. after it had already produced cell n° and the first nf blast cell. The embryo was counterstained with Hoechst 33258 (green). Broken outlines indicate the edges of the four neuromeres (R1-R4) in the subesophageal ganglion. Neuromere R1 contains the same complement of labeled neurons as neuromeres R2-R4, even though its anterior edge is unlabeled. (E) Pseudocolored 3D representation of the RDA-labeled cells in an embryo equivalent to that shown in D. (F-H) Pseudocolored images (processed as in C) of an embryo in which the N teloblast was injected with RDA after the birth of n° (i.e. one cell cycle earlier than in D and E). The N teloblast had also been injected with nGFP mRNA prior to the birth of n°. As a result, the n° clone is labeled with nGFP only, and the first RDA-labeled clone is descended from the first nf cell. (F) Ventral view, comparable with E, showing only the RDA labeled cells; broken outline surrounds the first nf clone. (G) The same image as in F, with the addition of the n°-derived nuclei (yellow). (H) Side view (ventral to left) of the image shown in G. Scale bar: 10 µm.

View larger version (132K): [in a new window]   Fig. 9. Asymmetric mitoses of primary nf and ns blast cells: spindle dynamics. Time-lapse fluorescence video micrographs of primary blast cells (cl.ag. 40-45 hours) descended from an N teloblast injected with tau::GFP mRNA. The elapsed time (minutes) is indicated in the top right-hand corner of each panel. (A) Dividing ns cell. (B) Dividing nf cell. (C) A fortuitous specimen in which an ns and a younger nf cell (separated by a two-cell nf clone and an undivided ns cell) were dividing simultaneously. Broken outlines indicate the cell borders; arrows indicate the spindle midbody. Scale bar: 10 µm.

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