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First published online 27 July 2004
doi: 10.1242/dev.01271

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Cell interactions that affect axonogenesis in the leech Theromyzon rude

¹ Department of Biology, Rutgers, The State University of New Jersey, Camden, NJ 08102, USA
² Department of Molecular and Cell Biology, 385 LSA, University of California, Berkeley, CA 94720-3200, USA

View larger version (28K): [in a new window]   Fig. 1. Segmental development in Theromyzon rude illustrating the temporal progression of the main neurogenic lineage (N) and final positions of major segmental and longitudinal nerves. The relative positions of the mesodermal (M) and ectodermal (O/P, O/P and Q) teloblasts and their progeny are indicated on the right side only. Bilaterally paired N teloblasts (NL and NR) give rise to coherent columns of cells (n bandlets). Within the germinal plate, contralateral n blast cell clones lie in apposition across the ventral midline (broken line) and subsequently give rise to the bulk of the segmental ganglia of the ventral nerve cord, along with segmentally iterated peripheral neurons (nz1, nz2 and nz3) and a few epidermal cells (not shown). The O, P and Q teloblasts on each side give rise to distinct lineages that generate progressively more lateral and dorsal ectoderm (not shown here) (Weisblat et al., 1984). Ganglionic primordia result from the formation of transverse fissures that arise when the parent blast cell clones are 50 hours old (Shain et al., 1998). Immediately following fissure formation (50-55 hours), the first axonal processes appear between the anterior and posterior lobes of the N lineage, as evidenced by acetylated -tubulin (ACT) antibody staining (see Fig. 2). The outgrowth of two ventrolateral stripes of cells from each posterior lobe (the anterior of which expresses the leech engrailed-class gene) occurs later at 65 hours clonal age. By 100 hours, the major nerve tracts have acquired their final positions in the juvenile leech (AA, anterior-anterior; MA, medial-anterior; PP, posterior-posterior; DP, dorsal-posterior; UP, ultraposterior; C, connective; F, Faivre's nerve). Approximate clonal ages for n blast cells and their derivatives are indicated at right. Anterior is upwards. Not drawn to scale.

View larger version (21K): [in a new window]   Fig. 2. Development of axonal processes in the leech nervous system. A monoclonal antibody to acetylated -tubulin (ACT; Sigma) was used to stain axons in embryos of different stages. Tracings of nerves from representative segments were fused into a single image that depicts the major events during T. rude axogenesis. The time line indicates the approximate clonal age of axon fibers with respect to the corresponding birth of blast cells within the neuroectodermal (N) lineage. The first axon processes were observed at 50 hours (arrowhead), appearing on either side of the prospective midline. These projected medially and met at the midline 1 hour later, upon which they extended anteriorly to pioneer Faivre's nerve (arrow identified by F). Paired longitudinal axons occupying positions of the future connective nerves (arrow C) appeared at 55 hours clonal age. The pioneering process of the MA and AA segmental nerves (arrows MA and AA, respectively) appeared almost simultaneously between 55-58 hours clonal age; the MA axon fiber(s) originated from a peripheral location corresponding to pz8/cf3 (Braun and Stent, 1989a), while AA fibers appear to extend both medially and laterally from unidentified cells at the ganglionic margin. Processes of the PP segmental nerve (arrow PP) appeared at 60 hours clonal age. A transient, lateral longitudinal nerve (arrows) was apparent at 65 hours clonal age, positioned slightly medial to peripheral floret cf3 along the MA nerve. An axon marking the UP nerve (arrow UP) appeared at 70 hours clonal age. At 120 hours clonal age, the medial portion of the DP nerve was apparent (arrow DP), the segmental nerves were compressed longitudinally forming a cluster of three nerve roots and the ganglion was surrounded by glial sheaths. Anterior is upwards.

View larger version (114K): [in a new window]   Fig. 3. Axonal architecture following lineage-specific ablations. Each panel shows the medial region of two (E) or three (A-D) midbody segments immunostained for ACT to reveal axon patterning (red). (A-D) Germinal plates from embryos in which one teloblast of a given pair was injected with lineage tracer (green; left side) and its contralateral homolog was ablated (right side). (A) Unilateral M lineage ablation resulted in a random array of axon fibers. (B) Unilateral N lineage ablation resulted in coalescence of the PP and MA segmental nerves at the ganglionic margin (arrows). The ipsilateral connective was often shifted towards the midline and appeared smaller in diameter (arrowheads). (C) Unilateral O lineage ablations resulted in a truncated and/or displaced PP segmental nerve (arrows) on the experimental side relative to the contralateral control at the same clonal age. (D) Unilateral P lineage ablations had little effect other than a decrease in diameter and moderate displacement of the medial end of the MA segmental nerve (arrows). The transient, lateral longitudinal nerve failed to appear (not shown). (E) A similar preparation in which O, P and Q lineages are labeled on the left side and missing from the right side, while both N lineages were labeled with a different lineage tracer (blue). Unilateral OPQ ablations resulted in abnormalities of all the segmental nerves ranging from displacement to reduction in size and/or length, but all nerves could be recognized. Anterior is upwards. Scale bar: 25 µm.

View larger version (141K): [in a new window]   Fig. 4. Coincidence of nerve and muscle fibers in T. rude. A view of five segments (95-105 hours clonal age) in a germinal plate immunostained with anti-ACT (green) and Lan3-14 (red) to identify axons and muscle fibers, respectively. The right N teloblast was labeled with RDA (blue). More than 30 bilaterally paired, longitudinal muscle fibers are apparent; between 20-25 circular muscle fibers can be identified per segment. Arrows indicate the largest gap between identifiable circular muscle fibers and correspond to the position of the iterated MA nerve tract. Other nerve tracts (AA, PP and UP) consistently align between characteristic sets of muscle fibers. Anterior is upwards. Scale bar: 25 µm.

View larger version (105K): [in a new window]   Fig. 5. Early development of the PP nerve. Each panel shows one or more hemiganglia on the right side of a germinal plate in which the N lineage was labeled with RDA. (A) A series of three hemiganglia (65-70 hours clonal age), from a preparation that was also immunostained for ACT (green), reveals the close association of the developing PP nerve (arrows) with the laterally migrating nz1 and nz2 axons (arrowheads). (B) A single hemiganglion at the same age from a preparation in which the O lineage was labeled with FDA lineage tracer (green). There is a close association between processes originating from the PD neuron (arrow) and the peripheral oz2 and/or oz3 neurons (lower arrowhead). O-neurons PD, oz2 and oz3 are coincident with the PP nerve and the latter two (oz2 and oz3) are closely associated with N-derived neurons nz1 and nz2 (overlap appears yellow). Anterior is upwards. Scale bar: 25 µm.

View larger version (82K): [in a new window]   Fig. 6. Effects of specific N lineage ablations on PP nerve patterning. Each panel shows a right hemiganglion (80 hours clonal age) from a preparation in which the N lineage was labeled with FDA (green) and axons were labeled by ACT immunostaining (red). The PP nerve is indicated by an arrow in each panel. (A) An unablated control preparation shows the normal, posterior origin of the PP nerve and its association with peripheral nz neurons (arrowhead). (B) Ablation of an ns primary blast cell resulted in a significant reduction in size of the anterior region of the ganglion (broken outline approximates the normal contour), but the PP nerve root maintained its normal position. (C) Ablation of an nf primary blast cell resulted in a significant reduction in size of the posterior region of the ganglion (broken line shows the normal contour). The putative PP nerve branched from the MA nerve at the margin of the ganglion. Anterior is upwards. Scale bar: 25 µm.

View larger version (72K): [in a new window]   Fig. 7. N-derived progeny are required for the normal intraganglionic migration of the O-derived PD neuron. Each panel shows one (D) or two (A-C) segmental ganglia from germinal plates in which both the left and right O lineages were labeled with lineage tracer (green). PD (arrows) and oz2/oz3 (arrowheads) are indicated when they can be distinguished from other O-derived cells. In A and B, the N lineage was labeled with lineage tracer (red) on the left side and is absent on the right side, owing to teloblast ablation. (A) By 80-85 hours clonal age, PD and oz2/oz3 were readily distinguished on the control (left) side. In the absence of the N lineage (right side), oz2/oz3 migrated to their normal peripheral positions, but PD apparently remained within a central cluster of O-derived cells. (B) Earlier in development (50-55 hours clonal age), PD, oz2 and oz3 assumed their normal positions, independent of the N lineage (compare positions of O lineage cells on left and right sides). PD has not yet separated from the central cluster of O-derived cells (compare with A). (C,D) ACT immunostaining (red) of axon projections reveals that PP nerve fibers still connect PD and oz2/oz3 in N-depleted hemiganglia (right side). As PD has failed to migrate normally, the PP nerve now branches from a central position in N-depleted hemiganglia (compare right side to left side). Ganglia are between 80-85 hours clonal age. Anterior is upwards. Scale bars: 25 µm in all panels.

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