Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail

Abstract

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.

Keywords: Lizard; Neuromuscular junction; Regeneration; Reptile; de novo.

Figure 1

Peripheral axons regrow early in regeneration and are subsequently remodeled. Transverse sections of original and regenerated Anolis carolinensis tails distal to the site of autotomy following immunostaining with β-III tubulin (TUJ1, red) and nuclei (DAPI, blue). (A–C) Representative confocal images showing the anatomical organization of nerves in the original tail in a region 2–5 mm distal to the site of autotomy (n=5). The spinal cord (sc) is enclosed by the vertebrae (v) and prominent scales (s) are visible. (D–F) Peripheral nerves (pn) and the ependyma (ep) readily regrow into the regenerated tail at 15 DPA (n=3). (G–I) At 30 DPA peripheral nerves continue to regenerate and cartilage (ct) has begun to differentiate and form around the ependymal tube 2–5 mm past the breakpoint (n=5). (J–L) Peripheral nerves localize deep to newly synthesized muscle at 120 DPA in the region 2–5 mm distal to the autotomy zone (n=4). (M) Quantification of axonal density in non-injured, 30 DPA, and 120 DPA regenerated tails. A statistically significant increase in TUJ1⁺ pixel density relative to non-injured tails was detected at 30 DPA during early regeneration and outgrowth (n=3). At 120 DPA, axonal density was decreased relative to 30 DPA, but increased relative to the original tail. Data are presented as mean sd; statistical analysis performed by one-way ANOVA followed by Tukey’s post-hoc test. **p<0.001, *p<0.05. The non-parametric, Kruskal-Wallis rank test followed by Dunn’s post-hoc test further confirmed the statistical significance of these changes (Dunn’s post-hoc p=0.049). Insets show a single muscle and its peripheral innervation. Scale bars A, D, G, J=200 µm; C, F, I, L=100 µm.

Figure 2

Concurrent myelination of regenerating axons occurs along the rostro-caudal axis. Immunofluorescent micrographs of myelinated peripheral nerves stained with myelin basic protein (MBP, green) and β-III tubulin (TUJ1, red). Myelinated peripheral nerves are more abundant in (A–H) proximal sections when compared to (I–P) distal sections (n=3). Scale bar: 200 µm.

Figure 3

Morphology and formation of the neuromuscular junction in the regenerating tail. Representative morphology of BTX-labeled neuromuscular junctions in high resolution confocal images of (A) original, (B) 15 DPA, (C) 30 DPA, (D) 70 DPA, (E) 90 DPA, (F) 120 DPA, (G) and 250 DPA tails (n=at least 3 tails for each time point). Axons were labeled via β-III tubulin staining (TUJ1, red) and acetylcholine receptors by Alexa-fluor 488-conjugated α-bungarotoxin (BTX, green). Scale bar: 200 µm.

Figure 4

Spatiotemporal changes in regenerated NMJ number and distribution. Representative confocal images of motor endplate distribution within a single muscle group labeled with TUJ1 (red), α-bungarotoxin (BTX, green), and DAPI (blue) in (A) original, (B) 120 DPA, (C) and 250 DPA tails. (D) Quantification of NMJ density between each time point following regeneration of the lizard tail. At 120 DPA (n=3), NMJ density is statistically increased relative to original tails (n=5) and 250 DPA (n=3) regenerated tails. Though lower than 120 DPA, NMJ density at 250 DPA is still greater than original tails. Data are represented as mean sd; statistical analysis performed by one-way ANOVA analysis test followed by Tukey’s post-hoc test. **p <0.001, *p < 0.05, n.s. = not significant. The non-parametric, Kruskal-Wallis rank test followed by Dunn’s post-hoc test further confirmed the statistical significance of the changes in first regenerated tails (Dunn’s post-hoc p<0.05). R1: first regenerated tail, R2: second regenerated tail. Scale bars: 50 µm.

Figure 5

Central axons radiate from the terminal neuroependyma into the periphery. Transverse sections within 1 mm of the regenerated tail tip at 30 DPA (n=3). (A–B) The ependyma (ep) projects towards the distal tip. (C–H) Regenerating axons radiate from the terminal neuroependyma and radiate outwards towards peripheral tissue. Yellow arrows indicate axons exiting the ependyma. Scale bars: 50 µm.

Figure 6

Foramina in the cartilaginous tube serve as passageways for axons from the neuroependyma to the periphery. (A–C) Transverse sections through 30 DPA tails (n=3) and (D–F) sagittal sections through 90 DPA tails (n=3) stained for axons (TUJ1, red) and nuclei (DAPI, blue). Insets show axons traversing foramina found in the hyaline cartilage tube into peripheral tissues. Yellow arrows indicate TUJ1 labeled axons, yellow dashed lines label boundary of the cartilage tube. (G) Schematic representation of proposed neuroanatomical conduits of regenerating axons. Scale bars: 200 µm