Dynamics of degeneration and regeneration in developing zebrafish peripheral axons reveals a requirement for extrinsic cell types

Abstract

Background: Understanding the cellular mechanisms regulating axon degeneration and regeneration is crucial for developing treatments for nerve injury and neurodegenerative disease. In neurons, axon degeneration is distinct from cell body death and often precedes or is associated with the onset of disease symptoms. In the peripheral nervous system of both vertebrates and invertebrates, after degeneration of detached fragments, axons can often regenerate to restore function. Many studies of axonal degeneration and regeneration have used in vitro approaches, but the influence of extrinsic cell types on these processes can only be fully addressed in live animals. Because of its simplicity and superficial location, the larval zebrafish posterior lateral line (pLL) nerve is an ideal model system for live studies of axon degeneration and regeneration.

Results: We used laser axotomy and time-lapse imaging of pLL axons to characterize the roles of leukocytes, Schwann cells and target sensory hair cells in axon degeneration and regeneration in vivo. Immune cells were essential for efficient removal of axonal debris after axotomy. Schwann cells were required for proper fasciculation and pathfinding of regenerating axons to their target cells. Intact target hair cells were not themselves required for regeneration, but chemical ablation of neuromasts caused axons to transiently deviate from their normal paths.

Conclusions: Macrophages, Schwann cells, and target sensory organs are required for distinct aspects of pLL axon degeneration or regeneration in the zebrafish larva. Our work introduces a powerful vertebrate model for analyzing axonal degeneration and regeneration in the living animal and elucidating the role of extrinsic cell types in these processes.

Figure 1

Acute axonal degeneration (AAD) and Wallerian degeneration are observed in the zebrafish posterior lateral line (pLL) nerve after laser axotomy. A three dpf larva, injected with HuC::GFP to image and axotomize a single pLL neuron. (a) Intact neuron with a typical bipolar morphology. White arrowhead shows central projection, yellow arrowhead shows peripheral projection, and square shows cell body. (b) Zero hours postaxotomy (hpa), arrowhead points to position of laser axotomy. (c) Thirty minutes after axotomy. AAD was complete: arrowheads indicate extent of AAD. (d) Three hours after axotomy, the distal axon began to fragment (arrowheads). (e) Four hours after axotomy, some fragments remained. (f) Five hours postaxotomy, clearance of debris was almost complete. Scale bar, 100 μm.

Figure 2

Leukocytes and Schwann cells contribute to axon degeneration in the axotomized posterior lateral line nerve. Length of lag phase (time from axotomy to fragmentation) and clearance phase (time from axotomy to axonal fragment removal) in minutes in control animals, animals lacking Schwann cells (AG1478 inhibitor and leo1 mutants), animals lacking leukocytes (spi1 MO) and animals lacking both cell types (spi1 MO + AG1478). The absence of leukocytes, but not Schwann cells, significantly prolonged the clearance phase (n = 10); however, combined depletion of leukocytes and Schwann cells restored the normal time course of debris clearance. *Significant difference, two-way ANOVA, P <0.05.

Figure 3

Leukocytes participate in axon fragment removal. Axonal fragments were quantified at three different time points with the Analyze Particle plugin of the ImageJ software (see Materials and Methods). At 135 minutes postaxotomy, larvae lacking Schwann cells (leo1 mutants and larvae treated with the Schwann cell inhibitor AG1478) had significantly fewer axon fragments (P <0.05) than controls or spi1 morphant larvae depleted of leukocytes. At 195 and 405 minutes spi1 morphants had more axon fragments than controls or larvae lacking Schwann cells (P <0.05). n = 10 for each treatment; all experiments were carried out in neuroD::GFP transgenic fish.

Figure 4

Leukocyte migration to degenerating axons is enhanced in the absence of glia. (a) Leukocytes (yellow arrowheads) homed to the degenerating pLL nerve in a lysC::GFP/neuroD::GFP double transgenic larva. (b) The number of leukocytes directly in contact with degenerating axons was significantly increased in the absence of glia (AG1478). Quantitative analysis was performed between 2.5 and 4.5 hours postaxotomy. Student’s t test, P = 0.00167.

Figure 5

Hair cells remain viable during Wallerian degeneration. Top panel: A single neuromast of a three dpf double transgenic neuroD::GFP/brn3c::GFP larva expressing GFP in axons and hair cells was imaged for the first 10 hours after axotomy. At six hours postaxotomy, the nerve had degenerated and one hair cell had died (red arrowhead). Lower panel: total number of hair cells in the first neuromast at three and six hours postaxotomy (hpa); pLL degeneration did not significantly alter hair cell numbers. *Two-way ANOVA, P >0.05. Scale bar 50 μm.

Figure 6

Posterior lateral line (pLL) axon degeneration is affected by the absence of Schwann cells or hair cells. Double transgenic larvae (brn3c::GFP/neuroD::GFP) were treated to remove Schwann cells (AG1478) or hair cells (laser ablation) prior to axotomy, and were examined after 24 hours to evaluate regeneration of the pLL nerve. (a) Control fish; red arrowheads indicate neuromasts; the nerve grew along its original path. (b) When Schwann cells were absent, axons regenerated after axotomy but many of them failed to grow along the normal trajectory (yellow arrowheads). (c) After physical ablation of hair cells in lateral line neuromasts (circle indicates position of ablated neuromast), axon regeneration was normal. (d) Axon regeneration was normal after inhibition of hair cell differentiation with the ath1a morpholino. (e) Ablation of neuromasts with CuSO₄ treatment produced erratic growth of regenerating axons (arrow), but the normal trajectory of neurites was restored when neuromast hair cells (arrowhead) regenerated. (f) The number of axons wandering outside of the normal pLL trajectory was quantified and compared across the different conditions as indicated. *Significant differences between treatments (one-way nonparametric ANOVA, P <0.05).

Figure 7

Posterior lateral line axons regenerate after laser axotomy. Representative images of the trunk of a three dpf brn3c::GFP/neuroD::GFP double transgenic larva imaged at the specified times (hours postaxotomy, hpa) near the lesion site. White arrow shows the leading axon tip. Scale bar, 100 μm.