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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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Loss of Schwann cell differentiation markers after denervation. Tg(foxD3:GFP) larvae were left untreated or neurectomized and then fixed at different times and processed for immunostaining with anti-Myelin Basic Protein (MBP; labeled in red). (A) The expression of MBP was evaluated in a proximal and distal area with respect to the point of neurectomy (black circle). (B-I) In control larvae. MBP is expressed at proximal and distal levels at 3 dpf and increases as the larva ages. (J-M) Expression of MBP becomes absent after neurectomy in denervated Schwann cells. Five-day-old tg(NeuroD:GFP) larvae were neurectomized and were fixed and processed for immunostaining with anti-MBP 1 (J, K) or 2 days (L, M) after neurectomy. Nerve degeneration (green label in inset in K) is followed by fragmentation of MBP expression in Schwann cells distal to the injury point (white dotted circle) at 24 hpn (J, K, inset in K shows both channels). At 48 hpn (L, M), MBP expression has disappeared distal to the injury site. (N-Q) Neurectomy at earlier stages, when MBP becomes expressed (at 3 pdf), showed the same results. In this case, Tg(foxD3:GFP) larvae were used to follow Schwann cells; note that despite fragmentation and loss of the MBP label, Schwann cells still remain viable as revealed by GFP expression. Scale bars: J, K, inset in N: 100 μm; B-I, L-Q, inset in E, inset in O: 50 μm.
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Figure 5: Loss of Schwann cell differentiation markers after denervation. Tg(foxD3:GFP) larvae were left untreated or neurectomized and then fixed at different times and processed for immunostaining with anti-Myelin Basic Protein (MBP; labeled in red). (A) The expression of MBP was evaluated in a proximal and distal area with respect to the point of neurectomy (black circle). (B-I) In control larvae. MBP is expressed at proximal and distal levels at 3 dpf and increases as the larva ages. (J-M) Expression of MBP becomes absent after neurectomy in denervated Schwann cells. Five-day-old tg(NeuroD:GFP) larvae were neurectomized and were fixed and processed for immunostaining with anti-MBP 1 (J, K) or 2 days (L, M) after neurectomy. Nerve degeneration (green label in inset in K) is followed by fragmentation of MBP expression in Schwann cells distal to the injury point (white dotted circle) at 24 hpn (J, K, inset in K shows both channels). At 48 hpn (L, M), MBP expression has disappeared distal to the injury site. (N-Q) Neurectomy at earlier stages, when MBP becomes expressed (at 3 pdf), showed the same results. In this case, Tg(foxD3:GFP) larvae were used to follow Schwann cells; note that despite fragmentation and loss of the MBP label, Schwann cells still remain viable as revealed by GFP expression. Scale bars: J, K, inset in N: 100 μm; B-I, L-Q, inset in E, inset in O: 50 μm.

Mentions: Since the differentiation status of Schwann cells has been seen to change after neurectomy in mammals, we analyzed the expression of Myelin Basic Protein (MBP) in control and neurectomized zebrafish larvae. MBP is a terminal differentiation marker of myelinating glia in the central and peripheral nervous systems[61]. At 3 dpf, it was possible to observe incipient expression of MBP in the lateral line at proximal and distal levels, suggesting that Schwann cells associated with this nerve have begun myelination at this stage (Figure 5B, C). The expression of this marker increases progressively over time from 4 dpf to 8 dpf in untreated larvae (Figure 5D-I). Larvae that were neurectomized at either 3 dpf or 5 dpf, showed fragmentation of the MBP label beyond the injury site by 24 hpn (Figure 5J. K, N, and O) and a total loss of this marker from the injury site to caudal regions after 48 hpn (Figure 5L, M, P, and Q). Interestingly, this rapid loss of MBP in Schwann cells of neurectomized fish happens despite the fact that Schwann cells continue to express GFP driven by the foxd3 promoter (see Figure 3) and display motile behavior that allows reconnection of the gap created at neurectomy (inset in Figure 5N). In larvae that were neurectomized at 3 dpf, 2 days after neurectomy (48 hpn), an anteroposterior wave of MBP reappearance was observed (Additional file4A) that reached the tip of the tail after 5 dpn, even though the label was considerably lower in neurectomized larvae when compared to controls (Additional file4, compare B vs. C).


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)

Loss of Schwann cell differentiation markers after denervation. Tg(foxD3:GFP) larvae were left untreated or neurectomized and then fixed at different times and processed for immunostaining with anti-Myelin Basic Protein (MBP; labeled in red). (A) The expression of MBP was evaluated in a proximal and distal area with respect to the point of neurectomy (black circle). (B-I) In control larvae. MBP is expressed at proximal and distal levels at 3 dpf and increases as the larva ages. (J-M) Expression of MBP becomes absent after neurectomy in denervated Schwann cells. Five-day-old tg(NeuroD:GFP) larvae were neurectomized and were fixed and processed for immunostaining with anti-MBP 1 (J, K) or 2 days (L, M) after neurectomy. Nerve degeneration (green label in inset in K) is followed by fragmentation of MBP expression in Schwann cells distal to the injury point (white dotted circle) at 24 hpn (J, K, inset in K shows both channels). At 48 hpn (L, M), MBP expression has disappeared distal to the injury site. (N-Q) Neurectomy at earlier stages, when MBP becomes expressed (at 3 pdf), showed the same results. In this case, Tg(foxD3:GFP) larvae were used to follow Schwann cells; note that despite fragmentation and loss of the MBP label, Schwann cells still remain viable as revealed by GFP expression. Scale bars: J, K, inset in N: 100 μm; B-I, L-Q, inset in E, inset in O: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214607&req=5

Figure 5: Loss of Schwann cell differentiation markers after denervation. Tg(foxD3:GFP) larvae were left untreated or neurectomized and then fixed at different times and processed for immunostaining with anti-Myelin Basic Protein (MBP; labeled in red). (A) The expression of MBP was evaluated in a proximal and distal area with respect to the point of neurectomy (black circle). (B-I) In control larvae. MBP is expressed at proximal and distal levels at 3 dpf and increases as the larva ages. (J-M) Expression of MBP becomes absent after neurectomy in denervated Schwann cells. Five-day-old tg(NeuroD:GFP) larvae were neurectomized and were fixed and processed for immunostaining with anti-MBP 1 (J, K) or 2 days (L, M) after neurectomy. Nerve degeneration (green label in inset in K) is followed by fragmentation of MBP expression in Schwann cells distal to the injury point (white dotted circle) at 24 hpn (J, K, inset in K shows both channels). At 48 hpn (L, M), MBP expression has disappeared distal to the injury site. (N-Q) Neurectomy at earlier stages, when MBP becomes expressed (at 3 pdf), showed the same results. In this case, Tg(foxD3:GFP) larvae were used to follow Schwann cells; note that despite fragmentation and loss of the MBP label, Schwann cells still remain viable as revealed by GFP expression. Scale bars: J, K, inset in N: 100 μm; B-I, L-Q, inset in E, inset in O: 50 μm.
Mentions: Since the differentiation status of Schwann cells has been seen to change after neurectomy in mammals, we analyzed the expression of Myelin Basic Protein (MBP) in control and neurectomized zebrafish larvae. MBP is a terminal differentiation marker of myelinating glia in the central and peripheral nervous systems[61]. At 3 dpf, it was possible to observe incipient expression of MBP in the lateral line at proximal and distal levels, suggesting that Schwann cells associated with this nerve have begun myelination at this stage (Figure 5B, C). The expression of this marker increases progressively over time from 4 dpf to 8 dpf in untreated larvae (Figure 5D-I). Larvae that were neurectomized at either 3 dpf or 5 dpf, showed fragmentation of the MBP label beyond the injury site by 24 hpn (Figure 5J. K, N, and O) and a total loss of this marker from the injury site to caudal regions after 48 hpn (Figure 5L, M, P, and Q). Interestingly, this rapid loss of MBP in Schwann cells of neurectomized fish happens despite the fact that Schwann cells continue to express GFP driven by the foxd3 promoter (see Figure 3) and display motile behavior that allows reconnection of the gap created at neurectomy (inset in Figure 5N). In larvae that were neurectomized at 3 dpf, 2 days after neurectomy (48 hpn), an anteroposterior wave of MBP reappearance was observed (Additional file4A) that reached the tip of the tail after 5 dpn, even though the label was considerably lower in neurectomized larvae when compared to controls (Additional file4, compare B vs. C).

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