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First published online 3 November 2004
doi: 10.1242/dev.01452

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Evolutionary diversification of specification mechanisms within the O/P equivalence group of the leech genus Helobdella

Dian-Han Kuo^*, and Marty Shankland

Graduate Program in Zoology, Section of Molecular Cell and Developmental Biology and Institute of Cellular and Molecular Biology, The University of Texas at Austin, TX 78712, USA

View larger version (77K): [in a new window]   Fig. 1. Cell lineage arrangement in the germinal band of a stage 7/8 leech embryo. Each of the five teloblasts produces a column or `bandlet' of primary blast cells, shown here in cross-section. The four ectodermal bandlets (white) lie in parallel above a single mesodermal (m) bandlet (cyan), and are overlaid by a provisional integument composed of micromere-derived epithelial cells (yellow). The n and q bandlets are located respectively on the future ventral and dorsal sides of the germinal band. The o and p bandlets are initially equipotent, and are committed to distinct O and P developmental pathways during normal development on the basis of positional cues encountered in the germinal band (Shankland and Weisblat, 1984; Huang and Weisblat, 1996).

View larger version (84K): [in a new window]   Fig. 2. Normal cleavage pattern of the O/P lineage. (A) Image of the o (red) and p (green) bandlets in the germinal band of a stage 8 embryo. Ventral is towards the left, and anterior towards the top. Nuclei are stained with Hoechst 33258, and appear blue. (B) Hoechst staining shows the shape and location of the nucleus of each individual cell. One o primary blast cell is undergoing mitosis (red arrowhead), and the more anterior (i.e. older) o blast cell clones consist of the larger o.a daughter cell and the smaller o.p daughter cell (arrows). Further anterior, an o.a cell has divided into two cells, o.aa and o.ap. The most posterior cells in the p bandlet are primary blast cells. Anterior to the p primary blast cells, the older p blast cell clones consist of paired daughter cells p.a and p.p. A p.a cell undergoing mitosis is labeled with a yellow arrowhead. Scale bar: 10 µm.

View larger version (101K): [in a new window]   Fig. 3. Differentiated pattern elements arising from the O and P lineages in stage 9 embryos. (A) The descendants of the O lineage (red) are generally located more ventrally than are the descendants of the P lineage (green). The arrow indicates the ventral midline. For labeled tissues, ventral is towards the left and anterior towards the top. (B,C) Pattern elements derived from the O lineage. (D,E) Pattern elements derived from the P lineage. The more superficial pattern elements of the body wall are shown in B and D. Pattern elements in the ganglia of the ventral nerve cord are shown in C and E, in which Hoechst 33258 counterstaining (blue) was used to visualize the segmental ganglia. CR, crescent neuron cluster; AD, anterodorsal neuron cluster; PV, posteroventral neuron cluster; WE, wedge-shaped neuron cluster; c.f.3, cell floret 3; n.t., nephridial tubule. pz6, pz10, LD1 and LD2 are identified peripheral neurons. Scale bar: 30 µm in A; 15 µm in B,E.

View larger version (102K): [in a new window]   Fig. 4. Effects of the bilateral ablation of the Q lineage on the cleavage patterns of o/p blast cell clones in stage 8 embryos. (A,B) Cleavage pattern of the presumptive o bandlet (A) and the presumptive p bandlet (B) of H. robusta (Austin) shown by Hoechst 33258 nuclear staining. Presumptive o blast cell clones express the normal O-type cleavage pattern. The o.p nuclei are marked with red arrows. Most presumptive p blast cell clones express a normal P-type cleavage pattern, and white lines mark the p.a and p.p daughter cell pairs. A few presumptive p blast cell clones express an O-type cleavage pattern (yellow arrows). (C,D) Ablation of bilateral Q lineages causes both o/p bandlets to express the O-type cleavage pattern in H. robusta (Sacramento) (C) and Helobdella sp. (Galt) (D). The small nuclei of o.p cells in the presumptive o bandlet are labeled with red arrows, and the nuclei of ectopic o.p cells in the presumptive p bandlet are labeled with yellow arrows. See Fig. 2 for orientation. Scale bar: 20 µm.

View larger version (59K): [in a new window]   Fig. 5. Following ablation of the Q lineages in H. robusta (Austin), the cleavage pattern of presumptive p blast cell clones is correlated to the contact area between the o/p bandlets. (A,B) The presumptive o and p bandlets shown here were labeled with injected rhodamine cell lineage tracer and Hoechst 33258 nuclear counterstaining. The two fluorescent signals are superimposed in A, and nuclear staining alone is shown in B. At the region where the presumptive p bandlets are in better contact with the presumptive o bandlet (between the white arrowheads in A), a presumptive p blast cell expresses the P-type cleavage pattern (white lines). In regions in which the contact area of the presumptive o and p bandlets is reduced, the presumptive p blast cell clones express an O-type cleavage pattern. See Fig. 4 for labeling of cells, and Fig. 2 for orientation. (C,D) Optical cross-sections of the germinal band shows the relative position of the m bandlet (green) and the o/p bandlets (red) in the area where the presumptive p bandlet expresses a P-type cleavage pattern (C) and in the area where the presumptive p bandlet expresses an O-type cleavage pattern (D). The contacting surface between the two o/p bandlets is marked with white arrows. Scale bar: 40 µm in A,B; 80 µm in C,D.

View larger version (95K): [in a new window]   Fig. 6. The differentiated pattern elements arising from the O/P lineages following bilateral ablation of the Q lineage differ in H. robusta (Austin) (A-D) and H. robusta (Sacramento) (E-J). (A,E) The presumptive O and P lineages give rise to their normally distinct sets of pattern elements in H. robusta (Austin) (A), but give rise to nearly identical sets of O pattern elements in H. robusta (Sacramento) (E). In both laboratory populations, the presumptive O lineage (red) and the presumptive P lineage (green) are fluorescently labeled. (B,F) Superimposed images of presumptive o bandlet descendants (red), presumptive p bandlet descendants (green), and nuclear staining (blue) in the ventral nerve cord of H. robusta (Austin) (B) and H. robusta, Sacramento (F). (C,G) The presumptive O lineage gives rise to O pattern elements in both laboratory populations. (D,H) The presumptive P lineage gives rise to normal P pattern elements in H. robusta (Austin) (D), but to O pattern elements in H. robusta (Sacramento) (H). (I,J) Following Q lineage ablation in H. robusta (Sacramento), O pattern elements that are represented by single cells in normal embryos are present as duplicate cells. Rhodamine- and fluorescein-labeling of these duplicate pattern elements reflects their derivation from the two different ipsilateral O/P teloblasts. See Fig. 3 for labeling of pattern elements and orientation. Scale bar: 200 µm in A-E; 50 µm in B-D,F-H; 33 µm I,J.

View larger version (90K): [in a new window]   Fig. 7. Mesoderm is required in the Q lineage-independent O/P specification pathway in H. robusta (Austin). (A) Ipsilateral o/p bandlets in stage 8 embryo in which both the M and Q lineages were ablated. Both bandlets exhibit the O-type cleavage pattern, with the small o.p nuclei marked by arrows. The presumptive p bandlet has transfated to the O-type cleavage pattern, and is labeled o'. (B) Ipsilateral o/p bandlets in stage 8 embryo in which only the M lineage was ablated. The presumptive o bandlet expresses the O-type cleavage pattern, while the presumptive p bandlet expresses the P-type cleavage pattern. See Fig. 2 for orientation. Scale bar: 30 µm.

View larger version (115K): [in a new window]   Fig. 8. Development of a `lone' O/P lineage following ablation of the Q lineages in H. robusta (Austin). (A) In early stage 8, the blast cell clones in the `lone' o/p bandlet express the O-type cleavage pattern. The small o.p nuclei are marked with arrows. Orientation of this panel is the same as Fig. 2. (B,C) Differentiated pattern elements derived from a `lone' O/P lineage in stage 9 embryos. In some embryos the `lone' O/P lineage gives rise to O pattern elements in every segment (B); in other embryos it gives rise to a combination of O pattern elements in more anterior segments and P pattern elements in more posterior segments (C). See Fig. 3 for orientation. Scale bar: 30 µm.

View larger version (15K): [in a new window]   Fig. 9. Evolution of O/P specification mechanisms in clitellate annelids deduced from phylogenetic analysis. The relationship of the three Helobdella laboratory populations used here is based on cytochrome oxidase I sequence analysis by A. Bely (personal communication); the oligochaete Tubifex hattai is an outgroup. Branch lengths do not reflect the genetic distance between taxa. Evolutionary changes of developmental characters are marked by short bars striking across the tree branches, and are assigned on the basis of parsimony. The role of the Q lineage as a P fate inducer evolved either as an apomorphy (1a) on the branch leading to the Helobdella leech species shown here, or is a plesiomorphic trait lost (1b) on the branch leading to Tubifex. The role of mesoderm as a redundant P fate inducer appears to have evolved as an apomorphy (2) on the branch leading to H. robusta (Austin) following its separation from H. robusta (Sacramento).

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