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Axon-Schwann cell interactions during peripheral nerve regeneration in zebrafish larvae.

Ceci ML, Mardones-Krsulovic C, Sánchez M, Valdivia LE, Allende ML - Neural Dev (2014)

Bottom Line: Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve.We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL.The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.

View Article: PubMed Central - HTML - PubMed

Affiliation: FONDAP Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. allende@uchile.cl.

ABSTRACT

Background: Peripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells.

Results: Using pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored.

Conclusion: The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.

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Axonal and Schwann cell behaviors at the point of neurectomy. Three days post fertilization transgenic Tg(neurod:TagRFP;foxd3:GFP) zebrafish larvae were neurectomized and imaged from 5 hpn to 11 hpn every 2 min following complete nerve transection (axons in red and Schwann cells in green). In all panels, the arrows show the behavior of axons and Schwann cells proximal to the gap whereas the arrowhead shows the behavior of Schwann cells distal to the gap. (A, B) A few hours after neurectomy, distal (denervated) Schwann cells extend their processes within the gap and show an exploratory behavior, whereas proximal Schwann cells are less motile. (C) At 6.5 hpn, the regrowing axons have contacted distal Schwann cells and have formed a bridge across the gap (asterisk). (D, E) After 7 hpn, the axons complete the crossing of the gap; often, the first axon to navigate the gap stops growing and another axon takes the lead. (F) After 11 hpn, the regrowing nerve has grown past the gap enabling the reconnection between proximal and distal Schwann cells in 100% of neurectomized larvae.
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Figure 3: Axonal and Schwann cell behaviors at the point of neurectomy. Three days post fertilization transgenic Tg(neurod:TagRFP;foxd3:GFP) zebrafish larvae were neurectomized and imaged from 5 hpn to 11 hpn every 2 min following complete nerve transection (axons in red and Schwann cells in green). In all panels, the arrows show the behavior of axons and Schwann cells proximal to the gap whereas the arrowhead shows the behavior of Schwann cells distal to the gap. (A, B) A few hours after neurectomy, distal (denervated) Schwann cells extend their processes within the gap and show an exploratory behavior, whereas proximal Schwann cells are less motile. (C) At 6.5 hpn, the regrowing axons have contacted distal Schwann cells and have formed a bridge across the gap (asterisk). (D, E) After 7 hpn, the axons complete the crossing of the gap; often, the first axon to navigate the gap stops growing and another axon takes the lead. (F) After 11 hpn, the regrowing nerve has grown past the gap enabling the reconnection between proximal and distal Schwann cells in 100% of neurectomized larvae.

Mentions: Given that a failure in axonal regeneration usually arises as a result of sprouting of damaged axons that must break through the injury site without guidance cues, we took advantage of our experimental setup of neurectomy, which not only sections the nerve but also leads to local death of Schwann cells within a diameter of 80 to 100 μm, to examine this process. We analyzed the behavior and interaction between glial cells and damaged axons at the injury site during the first hours of axon regeneration in vivo. We neurectomized 3-day-old Tg(neurod:TagRFP;foxd3:GFP) double transgenic larvae that exhibit differentially labeled pLL axons and neural crest derived Schwann cells[28,54]. We carried out time-lapse recording beginning 5 h after nerve transection, a time in which removal of axonal debris has concluded and nerve elongation begins (see Additional file3). We noticed a difference in the behavior of Schwann cells located proximal to the gap (still in contact with the axonal stump) in comparison to the denervated Schwann cells located distal to the injury site. While distal Schwann cells showed increased motility and exploratory behavior with process extensions (Figure 3A and B arrowhead), the proximal population extended processes only following pioneering axon extension through the gap (Figure 3A and B, arrow). The exploratory behavior of the regrowing axons and the protrusion and movements of denervated Schwann cells, contributed to form a bridge between both elements in such way that the axon regrowth was guided across the gap permitting the reconnection between Schwann cells located on both sides (asterisk in Figure 3C).


Axon-Schwann cell interactions during peripheral nerve regeneration in zebrafish larvae.

Ceci ML, Mardones-Krsulovic C, Sánchez M, Valdivia LE, Allende ML - Neural Dev (2014)

Axonal and Schwann cell behaviors at the point of neurectomy. Three days post fertilization transgenic Tg(neurod:TagRFP;foxd3:GFP) zebrafish larvae were neurectomized and imaged from 5 hpn to 11 hpn every 2 min following complete nerve transection (axons in red and Schwann cells in green). In all panels, the arrows show the behavior of axons and Schwann cells proximal to the gap whereas the arrowhead shows the behavior of Schwann cells distal to the gap. (A, B) A few hours after neurectomy, distal (denervated) Schwann cells extend their processes within the gap and show an exploratory behavior, whereas proximal Schwann cells are less motile. (C) At 6.5 hpn, the regrowing axons have contacted distal Schwann cells and have formed a bridge across the gap (asterisk). (D, E) After 7 hpn, the axons complete the crossing of the gap; often, the first axon to navigate the gap stops growing and another axon takes the lead. (F) After 11 hpn, the regrowing nerve has grown past the gap enabling the reconnection between proximal and distal Schwann cells in 100% of neurectomized larvae.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4214607&req=5

Figure 3: Axonal and Schwann cell behaviors at the point of neurectomy. Three days post fertilization transgenic Tg(neurod:TagRFP;foxd3:GFP) zebrafish larvae were neurectomized and imaged from 5 hpn to 11 hpn every 2 min following complete nerve transection (axons in red and Schwann cells in green). In all panels, the arrows show the behavior of axons and Schwann cells proximal to the gap whereas the arrowhead shows the behavior of Schwann cells distal to the gap. (A, B) A few hours after neurectomy, distal (denervated) Schwann cells extend their processes within the gap and show an exploratory behavior, whereas proximal Schwann cells are less motile. (C) At 6.5 hpn, the regrowing axons have contacted distal Schwann cells and have formed a bridge across the gap (asterisk). (D, E) After 7 hpn, the axons complete the crossing of the gap; often, the first axon to navigate the gap stops growing and another axon takes the lead. (F) After 11 hpn, the regrowing nerve has grown past the gap enabling the reconnection between proximal and distal Schwann cells in 100% of neurectomized larvae.
Mentions: Given that a failure in axonal regeneration usually arises as a result of sprouting of damaged axons that must break through the injury site without guidance cues, we took advantage of our experimental setup of neurectomy, which not only sections the nerve but also leads to local death of Schwann cells within a diameter of 80 to 100 μm, to examine this process. We analyzed the behavior and interaction between glial cells and damaged axons at the injury site during the first hours of axon regeneration in vivo. We neurectomized 3-day-old Tg(neurod:TagRFP;foxd3:GFP) double transgenic larvae that exhibit differentially labeled pLL axons and neural crest derived Schwann cells[28,54]. We carried out time-lapse recording beginning 5 h after nerve transection, a time in which removal of axonal debris has concluded and nerve elongation begins (see Additional file3). We noticed a difference in the behavior of Schwann cells located proximal to the gap (still in contact with the axonal stump) in comparison to the denervated Schwann cells located distal to the injury site. While distal Schwann cells showed increased motility and exploratory behavior with process extensions (Figure 3A and B arrowhead), the proximal population extended processes only following pioneering axon extension through the gap (Figure 3A and B, arrow). The exploratory behavior of the regrowing axons and the protrusion and movements of denervated Schwann cells, contributed to form a bridge between both elements in such way that the axon regrowth was guided across the gap permitting the reconnection between Schwann cells located on both sides (asterisk in Figure 3C).

Bottom Line: Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve.We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL.The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.

View Article: PubMed Central - HTML - PubMed

Affiliation: FONDAP Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. allende@uchile.cl.

ABSTRACT

Background: Peripheral nerve injuries can severely affect the way that animals perceive signals from the surrounding environment. While damage to peripheral axons generally has a better outcome than injuries to central nervous system axons, it is currently unknown how neurons re-establish their target innervations to recover function after injury, and how accessory cells contribute to this task. Here we use a simple technique to create reproducible and localized injury in the posterior lateral line (pLL) nerve of zebrafish and follow the fate of both neurons and Schwann cells.

Results: Using pLL single axon labeling by transient transgene expression, as well as transplantation of glial precursor cells in zebrafish larvae, we individualize different components in this system and characterize their cellular behaviors during the regenerative process. Neurectomy is followed by loss of Schwann cell differentiation markers that is reverted after nerve regrowth. We show that reinnervation of lateral line hair cells in neuromasts during pLL nerve regeneration is a highly dynamic process with promiscuous yet non-random target recognition. Furthermore, Schwann cells are required for directional extension and fasciculation of the regenerating nerve. We provide evidence that these cells and regrowing axons are mutually dependant during early stages of nerve regeneration in the pLL. The role of ErbB signaling in this context is also explored.

Conclusion: The accessibility of the pLL nerve and the availability of transgenic lines that label this structure and their synaptic targets provides an outstanding in vivo model to study the different events associated with axonal extension, target reinnervation, and the complex cellular interactions between glial cells and injured axons during nerve regeneration.

Show MeSH
Related in: MedlinePlus