Developmentally regulated impediments to skin reinnervation by injured peripheral sensory axon terminals

Abstract

The structural plasticity of neurites in the central nervous system (CNS) diminishes dramatically after initial development, but the peripheral nervous system (PNS) retains substantial plasticity into adulthood. Nevertheless, functional reinnervation by injured peripheral sensory neurons is often incomplete [1-6]. To investigate the developmental control of skin reinnervation, we imaged the regeneration of trigeminal sensory axon terminals in live zebrafish larvae following laser axotomy. When axons were injured during early stages of outgrowth, regenerating and uninjured axons grew into denervated skin and competed with one another for territory. At later stages, after the establishment of peripheral arbor territories, the ability of uninjured neighbors to sprout diminished severely, and although injured axons reinitiated growth, they were repelled by denervated skin. Regenerating axons were repelled specifically by their former territories, suggesting that local inhibitory factors persist in these regions. Antagonizing the function of several members of the Nogo receptor (NgR)/RhoA pathway improved the capacity of injured axons to grow into denervated skin. Thus, as in the CNS, impediments to reinnervation in the PNS arise after initial establishment of axon arbor structure.

Figure 1. Laser axotomy and imaging of peripheral axon regeneration

(A) Experimental design. Dorsal view of zebrafish embryo head; eyes are grey, trigeminal axons are green. Arrowhead indicates site of axotomy in all panels. Axon regeneration was monitored by time lapse for ≥ 12 hours and reinnervation of the denervated territory (yellow shading) by new axon growth (red) was calculated. (B) Dorsal and lateral views of a trigeminal axon reconstructed in 3-D. The axon arborizes within a mostly 2-D plane. (C–G) Time series of confocal image stacks. Same axon as in 1B. Time stamps are in minutes relative to axotomy at 30 hpf. Olive shading highlights the denervated region. Scale bar = 50 μm. See Movie S1.

Figure 2. The capacity for reinnervation diminishes during development

Axotomy at 30hpf (A–C), 54 hpf (D–F), or 78 hpf (G–I). Left two columns are confocal projections. Scale bars = 50 μm. Yellow arrowheads indicate site of axotomy, and olive overlay marks the territory that was denervated after axotomy. Blue dashed boxes indicate the area represented in rightmost panels (C, F, I), which show 3-D reconstructions of the same axon before axotomy (green) and 12 hours after axotomy (red), aligned at the shared branchpoint most proximal to the axotomy site. (J) Quantification of the average area reinnervated by injured axons, calculated as surface area of regenerated axon in denervated territory/surface area of denervated territory. (K) Quantification of average new axon growth that entered the denervated territory, calculated as length of new growth in denervated region/total length of new growth. Error bars ± S.E.M. See Table S1 and Movies S2–S4.

Figure 3. Developmental regulation of territory reinnervation strategy

(A–C) Growth potential of uninjured axons is developmentally regulated. (A and B) Confocal projections. Dorsal view of zebrafish head, anterior up. Olive indicates denervated half of head. Scale bars = 50 μm. Ablation of the left trigeminal ganglion at 30 hpf (A) or 78 hpf (B). (C) Quantification of axon growth rate in unablated (olive) vs. ablated (red) animals. Values are the average growth in μm per hour of individual branch tips. Error bars ± S.E.M. See Table S3 and Movies S5–S6. (D–G) Examples of control axons (wildtype cells transplanted into wildtype host) axotomized at 30 hpf (D) or 78 hpf (F), compared to isolated regenerating axons (wildtype cells transplanted into ngn-1 morphant host) axotomized at 30 hpf (E) or 78 hpf (G). Tracing overlays as in Figure 2. Arrowhead is site of axotomy, olive marks denervated territory, and scale bars = 50 μm. (H) Quantification of the area reinnervated by the injured axon, calculated as in Figure 2J. Error bars ± S.E.M. See Table S1 and Movies S7–S10. (I) Model of the developmental regulation of skin reinnervation by the terminal arbors of peripheral sensory axons. Black bar indicates site of axotomy. Injured axons are red, uninjured axons are green.

Figure 4. Inhibition of skin reinnervation by injured axons is mediated by the NgR/RhoA pathway

(A–C) Tracing overlays as in Figure 2. Arrowhead is site of axotomy, olive marks denervated territory, and scale bars = 50 μm. 78 hpf axotomy of a trigeminal neuron expressing GFP and RFP (A), DN NgR (B), or DN RhoA (C). (D) Quantification of fraction of axons that entered the denervated territory. Data to the left of the double bar were from axons expressing GFP and dominant negative or full length versions of the genes indicated. Data to the right of the double bar were from Tg(sensory:GFP) embryos injected with indicated morpholinos. Red indicates fraction of axons that grew into denervated territory, blue indicates axons that avoided denervated territory (See supplemental methods). See Table S2 and Movies S11–S13.