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Fig. 1. Whole mount Xenopus spinal cord fluorescently labeled and viewed laterally and ventrally by confocal z-series reconstructions. (A) Schematic representation of the spinal cord viewed laterally with approximate distances between dorsal and ventral fascicles and the midline indicated in microns. (BâD) Maximum projections of confocal z-series image stacks of a 28 hpf (NF stage 25) whole mount spinal cord viewed laterally (Bâleft side, Dâright side) and ventrally (C). Rostral is to the left in all images and dorsal is up in (B) and (D). All neurons are detected with antibodies to β-tubulin (blue), while more mature CIs only are detected with antibodies against neurofilament (red). F-actin is detected using fluorescent phalloidin (green), where it is concentrated in terminal growth cones of all neurons (boxed region in C). The dorsal longitudinal fascicle (DLF), composed primarily of RohonâBeard sensory neuron axons, and the ventral longitudinal fascicle (VLF), composed primarily of axons of motoneurons (MNs) and interneurons are indicated (white arrowheads in B and D). CIs extend ventrally and at this relatively late stage of development, some CIs appear as pairs, with one CI following closely behind its pioneer (red arrowheads in B, C and boxed region in B). The ventral view of the spinal cord allows visualization of the midline with CI axons crossing from both the right and left halves of the spinal cord. Dashed lines perpendicular to the longitudinal axis in (C) indicate cross-sectional views shown in (E). (E) Three cross-sections taken at successive rostral to caudal positions along the ventrally oriented spinal cord (at positions marked by dashed lines in C) show the cord narrows posteriorly. These images also show the peripheral nerve roots (arrowheads). (F) High magnification image of boxed region in (C) shows large flattened growth cones near the midline, which are reminiscent of paused growth cones in vitro. (GâI) High magnification image of boxed region in (B) shows the close association of a pioneer CI together with a follower commissural axon. Note the pioneer axon expresses both neurofilament (G, arrow) and β-tubulin (H, arrow), whereas the follower only expresses β-tubulin (H, arrowhead). Actin in merged imaged (I, green) shows the growth cone of the follower CI (I, arrowhead). Scale bar, 50 μm in (BâE), 15 μm in (F) and 18 μm in (GâI).
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Fig. 2. Pioneering CIs extend toward and across the midline as a highly ordered array. (AâD) Spinal cord development visualized in embryos fixed at 1-h intervals, immunolabeled and viewed ventrally as described previously. The AâP axes of individual spinal cords were aligned based on the position of the first commissural axon outgrowth, which occurred near the boundary with the hindbrain (not shown). Initial commissural axon outgrowth occurs around 22 hpf (NF Stage 20â21) and is delayed slightly behind MN axon extension such that CIs always encounter the forming ventral fascicle. Multiple commissural axons extend simultaneously across the ventral fascicle in close, but non-overlapping apposition. However, overlapping phalloidin labeling suggests filopodial contact between adjacent growth cones (arrowheads in A, B and boxed insets of phalloidin labeling only at 2à magnification). Unlike CIs, RohonâBeard sensory neurons and MNs fasciculate closely within the DLF and VLF, respectively (arrows in A). CI growth cones from opposites sides of the spinal cord eventually contact one another near the midline (C, arrows), but never fasciculate tightly upon one another. Occasionally stray axons project diagonal to the more common DâV directed growth and cross multiple axons (arrowhead in C). Neurofilament labeling becomes strong in many crossed CIs in the later two stages. Ultimately, crossed CIs begin to turn longitudinally and often do so before reaching the contralateral ventral fascicle (arrows in D). Although most CIs axons extend directly toward the ventral midline, in some cases, these axons take highly tortuous paths to reach the midline (arrowheads in D). It is not clear if these tortuous axons result from serpentine process outgrowth or though the direct local displacement of pre-existing axons. Scale bar, 50 μm (25 um in insets).
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Fig. 3. Crossed CI axons turn longitudinally at various points before or after contact with the contralateral ventral fascicle. (AâB) Lateral view of a triple-labeled embryonic spinal cord showing the first crossing CIs (arrowheads mark the ventral fascicle). More mature commissural axons are labeled with antibodies to neurofilament (A), while F-actin, which marks growth cones, is labeled with fluorescent phalloidin (B). (C) Merge of β-tubulin labeling (blue), F-actin labeling in (green) and neurofilament labeling of CIs (red). Some CIs approach the contralateral ventral fascicle at acute angles (arrows in C) indicating that turning began within the floor plate. In addition, a growth cone near the ventral fascicle has F-actin localized to one side (arrow in B), suggesting turning has begun. However, other crossed commissural axons extend beyond the ventral fascicle before turning (yellow arrow in C). Scale bar, 40 μm.
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Fig. 4. CI axon outgrowth slows at the midline, but accelerates after crossing. The position of CI growth cones relative to the midline was measured from 3D image stacks of ventral spinal cords at four developmental time points. The x axis is the A–P position along the length of the spinal cord, while the y axis represents the D–V position relative to the midline. The midline (dashed line) and left and right ventral fascicles (solid lines) are indicated. At the earliest time point, many CI axons have initiated over hundreds of microns of spinal cord, however anterior axons are longer. Growth toward the midline is rapid as an hour later axons over this same length have clustered near the midline. By 24 hpf, some axons at the anterior end have crossed the midline, but many remain near the midline, suggesting neurite extension is stalled. More axons eventually cross to the contralateral side of the spinal cord as development proceeds. The downward slope of the best-fit lines indicate that CIs develop first anteriorly. While the increasing slope of best curves at later developmental times suggest that growth cones accelerate after midline crossing.
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Fig. 5. Growth cones become morphologically more elaborate and their f-actin content increases after midline crossing. (AâC) Maximum projections of confocal z-series image stacks of a 28 hpf whole mount spinal cord viewed laterally (Aâleft side, Câright side) and ventrally (B). Rostral is to the left in all images, while dorsal is up in (A) and down in (C). GFP is expressed by a subset of dorsal neurons including some CIs (green) from targeted blastomere injection of GFP mRNA. This spinal cord was immunolabeled with antibodies against neurofilament (blue) to detect all mature CIs and fluorescent phalloidin (red) to label F-actin. The cell bodies of many GFP-expressing CIs with crossed or crossing axons are evident on the left side (A), while several growth cones of these CIs are near the midline in (B). Growth cones near the midline are rounded and have few filopodia (arrows and boxed regions in B; high magnification in D). In contrast, growth cones that have crossed completely onto the contralateral side of the spinal cord (arrows and boxed regions in C; high magnification in E) are morphologically complex with more filopodia and occasional back branches (arrowheads in C). (D, Dâ²) High magnification views of GFP labeling (D) and fluorescent phalloidin staining (Dâ²) of growth cones near the midline (boxed regions in B). (E, Eâ²) High magnification views of GFP labeling (E) and fluorescent phalloidin staining (Eâ²) of growth cones crossed to the contralateral side (boxed regions in C). (F) Quantification of complexity and F-actin content of pre-crossing (ipsilateral), during crossing (midline) and post-crossing (contralateral) CI growth cones. Growth cone complexity was measured from thresholded GFP fluorescent images (see Materials and methods), while F-actin intensity was measured from the same regions of interest of the phalloidin images. Both the complexity and F-actin content of growth cones are significantly increased during and after crossing. *P < 0.05 and **P < 0.005 for each comparison, except for ipsilateral growth cone complexity versus midline growth cones, which is not significantly different. Scale bar, 40 μm in (AâC), 15 μm in (D, E).
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Fig. 6. CI axon extension slows at the ventral fascicle during midline directed outgrowth on the ipsilateral side of the spinal cord. (A–F) A GFP-expressing CI was imaged with 4D confocal microscopy during initiation and extension of its axon toward the ventral midline. Maximum projections of confocal z-series image stacks are displayed at 30–35 min intervals. The terminal growth cone (arrow) is observed extending toward the ventral midline (below), but retracts upon contact with the ventral fascicle (arrowheads) around frames (C–E). After neurite extension resumes, the proximal region of this axon becomes locally bent (red arrow), reminiscent of the highly curved axons observed in fixed preparations (see Fig. 2D). (G) Quantification of the distance to the midline of this CI growth cone over time. The red bar indicates the position of the ventral fascicle. Initially this axon extends rapidly (steep slope), but near the ventral fascicle the rate of axon extension slows and transiently retracts. Extension within the floor plate appears very slow (asterisk), but the curvature of the neural tube may skew neurite length measurements within this region. Scale bar, 15 μm.
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Fig. 7. CI axons exhibit contact-mediated repulsive turning during ventral extension on the ipsilateral side of the spinal cord. (A) xây Cartesian coordinate plots of the position of ipsilateral CI growth cones extending toward the ventral midline. The locations of five example growth cones with normalized longitudinal (x coordinate) starting positions are shown. The spinal cordânotochord boundary is the bottom this plot. Growth cones undergo seemingly random and often sharp direction changes during ventral growth sampled at 5-min intervals. The yellow plot line illustrates the path taken by the example growth cone shown in (B, yellow arrowhead). (BâG) GFP-expressing CI imaged with 4D confocal microscopy during extension of its axon toward the ventral midline. Maximum projections of confocal z-series image stacks are displayed at 10â20 min intervals. Note multiple filopodial contacts with neighboring CI axon (C, E, F). These contacts do not result in fasciculation, rather are often followed by turning away from the contact sight suggesting avoidance (D, G). However, filopodial contacts appear adhesive since they can locally displace the contacted axon (arrow in F). Scale, 20 μm.
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Fig. 8. CIs exhibit distinct pathfinding behaviors after crossing to the contralateral side of the spinal cord. (AâI) Time series of GFP expressing CI axons extending into the contralateral spinal cord. Dashed line in (A) indicates the spinal cordânotochord border. Dorsal is up and rostral is to the left in all images. CI growth cones (arrowheads in A) are observed as they first emerge from the opposite side of the spinal cord at 0 min. Many growth cones turn anteriorly or posteriorly shortly after crossing and extend longitudinally in the ventral spinal cord (arrows in D). However, some axons extend further dorsally before turning longitudinally (arrowheads in D). Dorsally projecting axons turn longitudinally at varying positions and often exhibit transient branches (arrow in B and H) often appear at turning points. However, longitudinally extending CI axons often make DâV positional adjustments by rapid ventral shifts in position (matched lettered arrowheads in GâI). As axons continue to cross to opposite side of the spinal cord they begin fasciculating upon one another (arrows in E, G and I). A dorsal barrier to growth also becomes apparent over time marked by turning axons with stabilized filopodial contacts (arrow in F). Scale, 50 μm.
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Supplemental Fig. 1. β-tubulin positive pioneer CIs express GFP after early blastomere injection. Maximum projection of confocal z-series image stack of an early neural tube stage whole mount spinal cord viewed laterally. Anterior is to the left and dorsal is up. Dorsal cells including some CIs express GFP after injection of GFP mRNA into a single blastomere at the 8-cell stage. This embryo was fixed and immunolabeled for β-tubulin (blue) and F-actin (green). Three GFP-positive pioneering CIs that express β-tubulin are apparent anteriorly. Presumptive CIs that have not as yet extended axons are present all along the dorsal margin of the spinal cord. Scale bar, 50 μm.
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