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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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Quantification of proliferative Schwann cells in control vs. neurectomized larvae. (A) Schematic representation of the two areas of the larvae in which the number of proliferative (BrdU incorporating) Schwann cells was determined. The proximal area spans between the sixth to the eighth somites (two somites behind the neurectomy point) and the distal area begins two somites posterior to the anus. Proliferation was measured at different time points by BrdU incorporation in tg(foxd3:GFP) larvae (see Methods). (B, C) Schwann cells are actively proliferating from 3 to 4 dpf in control larvae. By day 6 however, there is a significant reduction in the number of proliferative cells in both areas analyzed, and by day 7 there is essentially no BrdU labeled Schwann cells. In (D), data from panels B and C is graphed to compare proliferation in both areas. (E) The proliferative response of Schwann cells to denervation was evaluated at days 6 and 7 post neurectomy, when proliferation in these cells has normally ceased. tg(foxd3:GFP) transgenic larvae were neurectomized at 5 dpf and the number of GFP+/BrdU + cells was quantified in control and neurectomized larvae at 6 dpf (24 hpn) and 7 dpf (48 hpn). The number of proliferating Schwann cells was different between control and neurectomized fish at 48 hpn in the proximal area. n.s: non-significant, **P <0.01, ***P <0.001.
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Figure 4: Quantification of proliferative Schwann cells in control vs. neurectomized larvae. (A) Schematic representation of the two areas of the larvae in which the number of proliferative (BrdU incorporating) Schwann cells was determined. The proximal area spans between the sixth to the eighth somites (two somites behind the neurectomy point) and the distal area begins two somites posterior to the anus. Proliferation was measured at different time points by BrdU incorporation in tg(foxd3:GFP) larvae (see Methods). (B, C) Schwann cells are actively proliferating from 3 to 4 dpf in control larvae. By day 6 however, there is a significant reduction in the number of proliferative cells in both areas analyzed, and by day 7 there is essentially no BrdU labeled Schwann cells. In (D), data from panels B and C is graphed to compare proliferation in both areas. (E) The proliferative response of Schwann cells to denervation was evaluated at days 6 and 7 post neurectomy, when proliferation in these cells has normally ceased. tg(foxd3:GFP) transgenic larvae were neurectomized at 5 dpf and the number of GFP+/BrdU + cells was quantified in control and neurectomized larvae at 6 dpf (24 hpn) and 7 dpf (48 hpn). The number of proliferating Schwann cells was different between control and neurectomized fish at 48 hpn in the proximal area. n.s: non-significant, **P <0.01, ***P <0.001.

Mentions: To examine cell proliferation in these cells, we began by assessing Bromodeoxyuridine (BrdU) incorporation from 72 hpf to 7 dpf in tg(foxd3:GFP) untreated larvae (Figure 4). We carried out the analysis in two areas: in the trunk (proximal), near the point where we routinely carry out neurectomy, and in the tail (distal) (Figure 4A, see methods). In both areas analyzed, Schwann cells showed the same behavior. Between 72 hpf to 4 dpf, these cells proliferate at approximately the same rate, but this rate drops to zero around 6 to 7 dpf, coinciding with the start of mielynation[61] (Figure 4B and C). The proliferative rate of Schwann cells until 4dpf was always higher in the proximal region compared to the distal region (Figure 4D).Since Schwann cells are actively proliferating until 4 dpf, we decided to carry out neurectomy at 5 dpf and follow proliferation until 7 dpf, so that the normal proliferation in these cells does not interfere with our analysis. Neurectomized and control larvae were given a BrdU pulse at different time windows (24 to 27 hpn; and 48 to 51 hpn, see Methods). The incubated larvae were then fixed immediately after BrdU incubation. Whereas in control larvae there is no proliferation at 7 dpf, the Schwann cells of neurectomized larvae continued to proliferate, albeit only in the proximal region, at this stage (48 hpn) (Figure 4E).


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)

Quantification of proliferative Schwann cells in control vs. neurectomized larvae. (A) Schematic representation of the two areas of the larvae in which the number of proliferative (BrdU incorporating) Schwann cells was determined. The proximal area spans between the sixth to the eighth somites (two somites behind the neurectomy point) and the distal area begins two somites posterior to the anus. Proliferation was measured at different time points by BrdU incorporation in tg(foxd3:GFP) larvae (see Methods). (B, C) Schwann cells are actively proliferating from 3 to 4 dpf in control larvae. By day 6 however, there is a significant reduction in the number of proliferative cells in both areas analyzed, and by day 7 there is essentially no BrdU labeled Schwann cells. In (D), data from panels B and C is graphed to compare proliferation in both areas. (E) The proliferative response of Schwann cells to denervation was evaluated at days 6 and 7 post neurectomy, when proliferation in these cells has normally ceased. tg(foxd3:GFP) transgenic larvae were neurectomized at 5 dpf and the number of GFP+/BrdU + cells was quantified in control and neurectomized larvae at 6 dpf (24 hpn) and 7 dpf (48 hpn). The number of proliferating Schwann cells was different between control and neurectomized fish at 48 hpn in the proximal area. n.s: non-significant, **P <0.01, ***P <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Quantification of proliferative Schwann cells in control vs. neurectomized larvae. (A) Schematic representation of the two areas of the larvae in which the number of proliferative (BrdU incorporating) Schwann cells was determined. The proximal area spans between the sixth to the eighth somites (two somites behind the neurectomy point) and the distal area begins two somites posterior to the anus. Proliferation was measured at different time points by BrdU incorporation in tg(foxd3:GFP) larvae (see Methods). (B, C) Schwann cells are actively proliferating from 3 to 4 dpf in control larvae. By day 6 however, there is a significant reduction in the number of proliferative cells in both areas analyzed, and by day 7 there is essentially no BrdU labeled Schwann cells. In (D), data from panels B and C is graphed to compare proliferation in both areas. (E) The proliferative response of Schwann cells to denervation was evaluated at days 6 and 7 post neurectomy, when proliferation in these cells has normally ceased. tg(foxd3:GFP) transgenic larvae were neurectomized at 5 dpf and the number of GFP+/BrdU + cells was quantified in control and neurectomized larvae at 6 dpf (24 hpn) and 7 dpf (48 hpn). The number of proliferating Schwann cells was different between control and neurectomized fish at 48 hpn in the proximal area. n.s: non-significant, **P <0.01, ***P <0.001.
Mentions: To examine cell proliferation in these cells, we began by assessing Bromodeoxyuridine (BrdU) incorporation from 72 hpf to 7 dpf in tg(foxd3:GFP) untreated larvae (Figure 4). We carried out the analysis in two areas: in the trunk (proximal), near the point where we routinely carry out neurectomy, and in the tail (distal) (Figure 4A, see methods). In both areas analyzed, Schwann cells showed the same behavior. Between 72 hpf to 4 dpf, these cells proliferate at approximately the same rate, but this rate drops to zero around 6 to 7 dpf, coinciding with the start of mielynation[61] (Figure 4B and C). The proliferative rate of Schwann cells until 4dpf was always higher in the proximal region compared to the distal region (Figure 4D).Since Schwann cells are actively proliferating until 4 dpf, we decided to carry out neurectomy at 5 dpf and follow proliferation until 7 dpf, so that the normal proliferation in these cells does not interfere with our analysis. Neurectomized and control larvae were given a BrdU pulse at different time windows (24 to 27 hpn; and 48 to 51 hpn, see Methods). The incubated larvae were then fixed immediately after BrdU incubation. Whereas in control larvae there is no proliferation at 7 dpf, the Schwann cells of neurectomized larvae continued to proliferate, albeit only in the proximal region, at this stage (48 hpn) (Figure 4E).

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