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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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Neuromast reinnervation after PLL nerve regeneration. Tg(neurod:TagRFP) fish that have a red labeled pLL nerve were injected at the one cell stage with the pE46:GFP DNA construct and were selected if they displayed green fluorescence in a single sensory neuron in the pLL ganglion (inset in A). The innervation pattern of the single sensory neuron was recorded both before neurectomy and after regeneration of the axon. Two different examples of reorganization during pLL nerve regeneration are shown, referred to as larva 1 (A-F) and larva 2 (G-J). Larva 1 shows a pLL ganglion neuron that innervates the terminal neuromasts before injury (B, D). Neurectomy is carried out about 200 μm away from the ganglion severing all axons of the pLL nerve (C). Twenty-four hours post neurectomy (hpn), the pLL nerve has regenerated about half way down the body of the larva (E). At 48 hpn, the nerve has completely regenerated (F) altough the green-labeled axon now innervates a different neuromast, the L4 (small inset in F) and does not innervate its original targets, which are innervated by other neurons (large inset in F). Larva 2 shows innervation of the terminal neurmasts before injury (G, H) and, after neurectomy and regeneration, the same neuromasts are reinnervated (I, J). Scale: A, B, E, F, G, I: 200 μm; C: 100 μm; inset in A, D, larger inset in F, inset in G, H; small inset in F: 20 μm.
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Figure 1: Neuromast reinnervation after PLL nerve regeneration. Tg(neurod:TagRFP) fish that have a red labeled pLL nerve were injected at the one cell stage with the pE46:GFP DNA construct and were selected if they displayed green fluorescence in a single sensory neuron in the pLL ganglion (inset in A). The innervation pattern of the single sensory neuron was recorded both before neurectomy and after regeneration of the axon. Two different examples of reorganization during pLL nerve regeneration are shown, referred to as larva 1 (A-F) and larva 2 (G-J). Larva 1 shows a pLL ganglion neuron that innervates the terminal neuromasts before injury (B, D). Neurectomy is carried out about 200 μm away from the ganglion severing all axons of the pLL nerve (C). Twenty-four hours post neurectomy (hpn), the pLL nerve has regenerated about half way down the body of the larva (E). At 48 hpn, the nerve has completely regenerated (F) altough the green-labeled axon now innervates a different neuromast, the L4 (small inset in F) and does not innervate its original targets, which are innervated by other neurons (large inset in F). Larva 2 shows innervation of the terminal neurmasts before injury (G, H) and, after neurectomy and regeneration, the same neuromasts are reinnervated (I, J). Scale: A, B, E, F, G, I: 200 μm; C: 100 μm; inset in A, D, larger inset in F, inset in G, H; small inset in F: 20 μm.

Mentions: Thus, in order to determine the fidelity of this system upon nerve injury, we first stochastically labeled single pLL neurons by injection of HuC:mem-TdTomato or pE46:GFP DNA into transgenic tg(neuroD:EGFP) or tg(neuroD:tagRFP) embryos at the one-cell stage, respectively. We screened for transient transgenic embryos expressing TdTomato or GFP in single lateral line neurons at 3dpf. Selected larvae were imaged 1 h before neurectomy (hbn) to identify the neuromast(s) innervated by the labeled neuron. Afterwards, larvae were neurectomized using an electrical pulse between the pLL ganglion and the first neuromast (L1) and the larvae were left to recover at 28°C, as decribed before[52]. We analyzed the structure of both the axon and the nerve at 24 and 48 hours post neurectomy (hpn) (Figure 1).We found that axons displayed a variable reinnervation behavior during regeneration. In Figure 1 we show two different examples that are representative of the different situations encountered. Larva 1 shows a single neuron labeled by GFP that innervated the terminal-most neuromasts (L5-L7; Figure 1A-D). After neurectomy (Figure 1C), this neuron changed its sensory target once regeneration was achieved (48 hpn) innervating a different neuromast (L4). After regeneration, the neuromasts originally innervated by this neuron are now innervated by other neurons, labeled by RFP (insets in Figure 1F). The second example (larva 2) shows a neuron displaying a large soma innervating the terminal-most neuromasts. After neurectomy and regeneration, this cell extended its peripheral axons to the same targets (Figure 1G-J).


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)

Neuromast reinnervation after PLL nerve regeneration. Tg(neurod:TagRFP) fish that have a red labeled pLL nerve were injected at the one cell stage with the pE46:GFP DNA construct and were selected if they displayed green fluorescence in a single sensory neuron in the pLL ganglion (inset in A). The innervation pattern of the single sensory neuron was recorded both before neurectomy and after regeneration of the axon. Two different examples of reorganization during pLL nerve regeneration are shown, referred to as larva 1 (A-F) and larva 2 (G-J). Larva 1 shows a pLL ganglion neuron that innervates the terminal neuromasts before injury (B, D). Neurectomy is carried out about 200 μm away from the ganglion severing all axons of the pLL nerve (C). Twenty-four hours post neurectomy (hpn), the pLL nerve has regenerated about half way down the body of the larva (E). At 48 hpn, the nerve has completely regenerated (F) altough the green-labeled axon now innervates a different neuromast, the L4 (small inset in F) and does not innervate its original targets, which are innervated by other neurons (large inset in F). Larva 2 shows innervation of the terminal neurmasts before injury (G, H) and, after neurectomy and regeneration, the same neuromasts are reinnervated (I, J). Scale: A, B, E, F, G, I: 200 μm; C: 100 μm; inset in A, D, larger inset in F, inset in G, H; small inset in F: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Neuromast reinnervation after PLL nerve regeneration. Tg(neurod:TagRFP) fish that have a red labeled pLL nerve were injected at the one cell stage with the pE46:GFP DNA construct and were selected if they displayed green fluorescence in a single sensory neuron in the pLL ganglion (inset in A). The innervation pattern of the single sensory neuron was recorded both before neurectomy and after regeneration of the axon. Two different examples of reorganization during pLL nerve regeneration are shown, referred to as larva 1 (A-F) and larva 2 (G-J). Larva 1 shows a pLL ganglion neuron that innervates the terminal neuromasts before injury (B, D). Neurectomy is carried out about 200 μm away from the ganglion severing all axons of the pLL nerve (C). Twenty-four hours post neurectomy (hpn), the pLL nerve has regenerated about half way down the body of the larva (E). At 48 hpn, the nerve has completely regenerated (F) altough the green-labeled axon now innervates a different neuromast, the L4 (small inset in F) and does not innervate its original targets, which are innervated by other neurons (large inset in F). Larva 2 shows innervation of the terminal neurmasts before injury (G, H) and, after neurectomy and regeneration, the same neuromasts are reinnervated (I, J). Scale: A, B, E, F, G, I: 200 μm; C: 100 μm; inset in A, D, larger inset in F, inset in G, H; small inset in F: 20 μm.
Mentions: Thus, in order to determine the fidelity of this system upon nerve injury, we first stochastically labeled single pLL neurons by injection of HuC:mem-TdTomato or pE46:GFP DNA into transgenic tg(neuroD:EGFP) or tg(neuroD:tagRFP) embryos at the one-cell stage, respectively. We screened for transient transgenic embryos expressing TdTomato or GFP in single lateral line neurons at 3dpf. Selected larvae were imaged 1 h before neurectomy (hbn) to identify the neuromast(s) innervated by the labeled neuron. Afterwards, larvae were neurectomized using an electrical pulse between the pLL ganglion and the first neuromast (L1) and the larvae were left to recover at 28°C, as decribed before[52]. We analyzed the structure of both the axon and the nerve at 24 and 48 hours post neurectomy (hpn) (Figure 1).We found that axons displayed a variable reinnervation behavior during regeneration. In Figure 1 we show two different examples that are representative of the different situations encountered. Larva 1 shows a single neuron labeled by GFP that innervated the terminal-most neuromasts (L5-L7; Figure 1A-D). After neurectomy (Figure 1C), this neuron changed its sensory target once regeneration was achieved (48 hpn) innervating a different neuromast (L4). After regeneration, the neuromasts originally innervated by this neuron are now innervated by other neurons, labeled by RFP (insets in Figure 1F). The second example (larva 2) shows a neuron displaying a large soma innervating the terminal-most neuromasts. After neurectomy and regeneration, this cell extended its peripheral axons to the same targets (Figure 1G-J).

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