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Target-selective vertebrate motor axon regeneration depends on interaction with glial cells at a peripheral nerve plexus [1]

['Lauren J. Walker', 'Department Of Cell', 'Developmental Biology', 'Perelman School Of Medicine', 'University Of Pennsylvania', 'Philadelphia', 'Pennsylvania', 'United States Of America', 'Camilo Guevara', 'Koichi Kawakami']

Date: 2023-08 

A critical step for functional recovery from peripheral nerve injury is for regenerating axons to connect with their pre-injury targets. Reestablishing pre-injury target specificity is particularly challenging for limb-innervating axons as they encounter a plexus, a network where peripheral nerves converge, axons from different nerves intermingle, and then re-sort into target-specific bundles. Here, we examine this process at a plexus located at the base of the zebrafish pectoral fin, equivalent to tetrapod forelimbs. Using live cell imaging and sparse axon labeling, we find that regenerating motor axons from 3 nerves coalesce into the plexus. There, they intermingle and sort into distinct branches, and then navigate to their original muscle domains with high fidelity that restores functionality. We demonstrate that this regeneration process includes selective retraction of mistargeted axons, suggesting active correction mechanisms. Moreover, we find that Schwann cells are enriched and associate with axons at the plexus, and that Schwann cell ablation during regeneration causes profound axonal mistargeting. Our data provide the first real-time account of regenerating vertebrate motor axons navigating a nerve plexus and reveal a previously unappreciated role for Schwann cells to promote axon sorting at a plexus during regeneration.

Here, we use larval zebrafish to visualize the multistep process of axonal regeneration through a peripheral nerve plexus in real time and to determine the role of local glia cells in this process. We demonstrate that within 2 days of complete motor nerve transection, motor axons regenerate robustly to reestablish functional synapses. We find that individual axons faithfully sort at the plexus to reinnervate their original muscle fiber targets and that mistargeted axons are selectively retracted to correct targeting errors. Finally, we demonstrate that Schwann cells are required for regenerating axons to properly navigate through the plexus, in part by preventing axonal mistargeting to the incorrect muscle. Combined, this work reveals a previously unappreciated role for Schwann cells to ensure appropriate axon sorting at a plexus, thereby promoting target-selective axon regeneration.

(A) Schematic of a 5 dpf larval zebrafish. Inset shows motor pools from SC segments 3–6 that form pectoral fin nerves 1–4 in the body wall. Axons sort at a DP or VP and innervate the musculature of the pectoral fin topographically. Innervation domains are labeled 1–4 and shown in corresponding colors. (B) Dorsal view. Motor neurons in SC segments innervate either the abductor or adductor muscle. (C) Lateral view. Schematic of abductor innervation of the pectoral fin. Nerves were transected using a laser in the locations shown with the lightning bolts. (D, E) Images from the transected side of maximum projections of fin motor innervation labeled with Tubb:dsRed. This example is from the same animal through regeneration. At 5 h hpt, axons have fragmented. At 1 dpt, axons have started to grow into the fin. At 2 dpt, axons have regenerated. (D) Abductor muscle innervation. (E) Abductor and adductor innervation pseudo-colored in green and magenta, respectively. (F) Corresponding time projections (<700 ms) of spontaneous pectoral fin movements. The region shown is indicated by the green dotted box in G. Only the nerves of the right fin were transected. At 5 hpt, the transected fin does not move. At 1 dpt, axons have just begun to grow into the fin but the transected fin still does not move. However, by 2 dpt, axons have regenerated and the transected fin can move again. The green and orange arrows point to the maximum fin position for the uninjured and transected sides, respectively. (G) The maximum angle of the tip of the fin compared to the body wall was measured during spontaneous fin movements. (H) Quantification of the maximum angle of the uninjured and transected fins pre-transection through regeneration. Each dot represents 1 fish and 1 movement. The black bar represents the mean. One-way ANOVA. *p = 0.04, **p < 0.005, ***p < 0.0001, ns = not significant. Original data for panel H is in S1 Data . dpf, day post fertilization; dpt, day post transection; DP, dorsal plexus; hpt, hour post transection; SC, spinal cord; VP, ventral plexus.

The larval zebrafish pectoral fin, evolutionarily analogous to tetrapod forelimbs [ 14 ], has stereotyped motor innervation. At 5 days post fertilization (dpf), pectoral fins are comprised of 2 antagonistic muscles, the abductor and the adductor. The fin musculature is innervated by 4 distinct motor nerves, which we refer to here as nerves 1–4, with cell bodies in anterior spinal cord segments 3 through 6 ( Fig 1A ) [ 15 , 16 ]. Motor nerves grow through axial trunk muscle and converge at the dorsal plexus located at the dorsal anterior edge of the fin (nerves 1–3) or the ventral plexus located at the ventral anterior edge of the fin (nerve 4). At these plexuses, axons sort between the abductor or adductor muscles ( Fig 1B ) [ 16 ] and then segregate into target-specific bundles ( Fig 1A and 1C ). Motor axons generally innervate the fin musculature topographically, such that neurons from the more anterior spinal cord segment 3 (nerve 1) innervate the dorsal region of fin musculature, while neurons from spinal segments 4 and 5 (nerves 2 and 3) innervate the middle region, and neurons from spinal segment 6 (nerve 4) innervate the ventral region of the musculature ( Fig 1A ) [ 16 , 17 ]. Consequently, to connect to their correct muscle fibers, fin motor axons navigate a series of stepwise choice points. They first sort between muscles at the plexus and then, at subsequent choice points within the fin musculature, axons select a path to innervate the proper muscle domain. Thus, the complex pectoral fin motor innervation pattern combined with the genetic-tractability, optical transparency suitable for live imaging, and the ability to measure fin movement as a functional readout of regeneration, make the larval zebrafish pectoral fin an ideal vertebrate system to interrogate mechanisms of target-selective axon regeneration.

Target specificity is particularly challenging when regenerating axons navigate through a series of choice points. The brachial plexus, the complex network of peripheral nerves at the base of the tetrapod forelimb, is one such region. Limb-innervating peripheral nerve axons exit from the spinal cord within discrete nerves that converge at the brachial plexus. There, axons from several nerves intermingle and then sort into target-specific bundles prior to innervating certain muscles in the forelimb. Recent work has revealed several molecular mechanisms that instruct axon guidance decisions at choice points during regeneration [ 8 , 9 , 11 , 12 ], yet the molecular and cellular mechanisms that enable proper axon navigation through a plexus followed by multiple choice points to innervate the proper muscle targets are largely unknown. Denervated Schwann cells, which up-regulate trophic factors and form tracks, called Bands of Bungner, upon which regenerating axons grow [ 13 ], are one cell type that could function in this process.

Axons of the peripheral nervous system (PNS) have significant capacity to regenerate after injury from chemical or mechanical insults. To achieve functional recovery, regenerating motor axons face the challenge of reconnecting with their original muscle targets. In mammals, motor axon targeting errors during regeneration are common and can cause long-term deficits (reviewed in: [ 1 , 2 ]). Recently discovered pro-regenerative molecular pathways enhance axon growth during regeneration [ 3 – 7 ]; however, these manipulations frequently lack the spatial cues that direct axons to their original targets and therefore limit functional recovery. Furthermore, while we now have a broad understanding of the developmental cues that shape the nervous system, there is growing evidence that axon regeneration is not simply a recapitulation of development, but that it requires unique injury-dependent signals [ 8 – 10 ].

Results

Pectoral fin motor axons regenerate robustly To identify the mechanisms that guide regenerating peripheral nerve axons through a plexus, we first observed axons during the process of regeneration. To visualize motor nerves that innervate the pectoral fin, we used transgenic Xla.Tubb:dsRed (hereafter referred to as Tubb:dsRed) larvae. At 5 dpf, pectoral fin targeting motor axons have established an elaborate innervation field across the abductor and adductor musculature of the fin (Fig 1D and 1E). We used a laser to transect nerves 1/2 and 3 dorsal to the dorsal plexus and nerve 4 at the same approximate area within the body wall (Fig 1C). This laser transection strategy yielded complete motor denervation of the pectoral fin while leaving the fin itself uninjured. Within 2 to 3 hpt, the distal portion of transected axons began to bleb and axons fragmented by 5 hpt. Following an initial period of stasis, axons initiated growth and navigated the dorsal plexus at 12 hpt ± 2.6 h (n = 9) to sort between the abductor and adductor muscles. Axons exit the dorsal or ventral plexus as a single fascicle prior to segregating into discrete bundles. In this study, we focused predominately on the dorsal plexus and hereafter most results pertain to axon regeneration through the dorsal plexus. By 24 hpt, these discrete bundles were apparent as regenerating axons extended partially across the musculature. Axon regeneration proceeded rapidly, with axon regrowth largely completed by 48 hpt (2 days post transection (dpt)) (Fig 1D and 1E). Thus, pectoral fin motor axons regenerate robustly to reestablish the complex innervation patterning.

Pectoral fin motor axons achieve functional regeneration To determine the degree of functional regeneration, we measured fin movements prior to and following nerve transection. Larval zebrafish pectoral fins perform spontaneous movements of alternating pectoral fins that are dependent on motor axon innervation [18]. To determine if and to what extent pectoral fin innervating motor axons achieve functional recovery, we used high-speed imaging at millisecond resolution to record spontaneous pectoral fin movements. From these movies, we then determined the maximum fin movement angle (Fig 1F–H). Concurrently, we also imaged fin motor axon innervation in the same animal throughout the regeneration process and then correlated fin movement with the extent of innervation. Transecting nerves that innervate the right pectoral fin while sparing those that innervate the left pectoral fin provided an internal control. Prior to nerve transection, left and right pectoral fins moved rhythmically (mean maximum angles for left fin 62.45 ± 22.7 degrees and for right fin 63.68 ± 18.73 degrees, n = 19; p = 0.856, unpaired t test) (Fig 1H, S1 Movie). Five hours after nerve transection, motor axons that innervated the right pectoral fin had fragmented and all movements of the right, transected fin ceased, whereas the uninjured left fin continued to move. At 1 dpt, regenerating axons had partially grown back into the dorsal region of the right fin, yet the fin still failed to move. In contrast, at 2 dpt axons had fully regrown across the fin musculature and the injured fin performed spontaneous movements comparable to the uninjured fin (mean maximum angle at 2 dpt for uninjured fin 84.96 ± 23.3.1 degrees and for transected fin 75.83 ± 21.8 degrees; p = 0.233, unpaired t test) (Fig 1G and 1H). This recovery of movement demonstrates that pectoral fin motor axons regrow robustly towards their original targets, and they also reform functional synapses.

Single axon labeling reveals target specificity We next examined the specificity with which regenerating axons grow back to their original muscle domains that were established during development. For this, we sparsely labeled axons using mnx1:mKate in the context of the entire population of labeled motor neurons (mnx1:GFP) (Fig 2A). This approach allows for a direct comparison of the trajectory of one or only a few axons before transection and after regeneration. Two days after complete transection of all pectoral fin innervating motor nerves, sparsely labeled axons had correctly navigated many choice points to reestablish an innervation pattern similar to their pre-injury route (Figs 2B and 2C). Next, we examined the frequency by which regenerating axons reinnervated their original muscle (abductor versus adductor) and their original topographic domains. We found that 94.4% of sparsely labeled axons re-occupied their original muscle (n = 51/54 to the correct muscle) (Fig 2D). To determine the specificity of axon targeting within the musculature, we divided the fin into 4 domains based on axon targeting patterns (Fig 2E). We found that 87% of axons reinnervate their original domains (n = 47/54 to the correct domain) (Fig 2F). Thus, following complete nerve transection, pectoral fin motor axons reinnervate their original muscle and muscle domains with high specificity. This specificity strongly suggests the existence of robust mechanisms during regeneration that mediate axon sorting at the plexus between the abductor versus the adductor muscle and precise domain targeting of axons throughout the fin musculature. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Target-selective regeneration of motor axons. (A) Schematic of a lateral view of pectoral fin abductor muscle motor innervation. An example trajectory of a single axon is shown in magenta. DP labels dorsal plexus, motor nerves in the body wall are labeled 1–4. (B, C) A timecourse of sparsely labeled axon(s) showing the innervation pattern pre-transection, after axon degeneration, and at 2 dpt. Sparsely labeled axons are labeled in white (B) and magenta (C) and all adductor motor axons are labeled in green (C). The white arrow points to a fascicle that did not degenerate and the orange arrow points to ectopic growth during regeneration. (D) Quantification of the muscle localization of sparsely labeled axons pre-transection compared to where these labeled axons innervated after regeneration. Small numbers on pre-transection data represent n. Diagonal lines indicate the axon mistargeted during regeneration. (E) Schematic defining domains for axon domain scoring. For category 3, which are the minority, axons entered the fin at the DP but innervated the ventral region of the fin, overlapping with domain 4 (like the sparsely labeled axon in A). The domains used to quantify sparsely labeled axon targeting are a broader categorization than the topographic territories schematized in Fig 1A. (F) Quantification of fin domain localization of sparsely labeled axons pre-transection compared to where these labeled axons innervated after regeneration. Small numbers on pre-transection data represent n. (G) Example of sparsely labeled axons (magenta) that form new trajectories and reestablish previous trajectories during regeneration with both motor axons (mnx1:GFP) and muscle fibers (α-actin:GFP) labeled. (H) The original (green) and regenerated (orange) trajectories of the sparsely labeled axons in G. Here, part of the pre-injury and regenerated trajectory can be overlayed precisely (arrows). Additionally, an axon does not follow its original trajectory (double arrows) but instead is mistargeted along the base of the fin (triangle). Original data for panels D and F are in S1 Data. DP, dorsal plexus; dpt, day post transection; GFP, green fluorescent protein. https://doi.org/10.1371/journal.pbio.3002223.g002 Both the abductor and adductor muscles are comprised of approximately 50 muscle fibers arranged longitudinally across the fin [19]. To determine how precise fin motor axon targeting is during regeneration, we next asked if labeled motor axons reinnervate their original muscle fibers. For this, we used larvae in which all motor neurons and muscle fibers were labeled with green fluorescent protein (GFP) (mnx1:GFP; α-actin:GFP), and then used mnx1:mKate to sparsely label axons. The position and morphology of individual muscle fibers remain consistent through the course of the experiment, allowing them to serve as landmarks. Using this framework, after injury, sparsely labeled motor axons regenerated to their original muscle fiber targets with high but not perfect precision (Figs 2G and S1). Specifically, we observed that during the regeneration process some terminal axonal branches vacated their original, pre-injury muscle fibers and that some now occupied new muscle fibers. The example shown in Fig 2G and 2H illustrates the variability of axon targeting we observe during regeneration. Here, a section of the route from sparsely labeled regenerated axons was distinct from the pattern prior to injury indicating that during regeneration axons sometimes form divergent projections. However, the distal segment of the regenerated axon pattern can be directly overlayed on the pre-injury axon path, demonstrating that the distal end of this axon followed the same route it had prior to injury (of 9 muscles with sparse axon labeling: 3 had labeled axons that terminated on their original muscle fibers, 3 had axons that partially grew across their original muscle fibers but terminated elsewhere, 2 had labeled axons that failed to regrow, and 1 had labeled axons that innervated the original domain but not the original muscle fibers). Thus, pectoral fin motor axon regeneration is remarkably precise, such that axons preferentially re-innervate their original fin domains, with specificity as accurate as their original muscle fibers.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002223

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