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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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Fate of Schwann cells after neurectomy. (A) Schematic representation of the transplantation scheme used to obtain mosaic labeling of Schwann cells. Briefly, 10 to 20 donor cells of Tg(Ubi:zebrabow:cherry) blastula stage embryos were aspirated and transplanted into Tg(8.0cldnb:lynEGFP) host embryos of the same stage. The transplanted embryos were screened for the presence of red fluorescent donor cells in the pLL at 48 hpf. (B) Distribution and morphology of transplanted Schwann cells in a mock-neurectomized larva (control). The rectangle indicates the region examined in the same fish at different timepoints. (C-E) Schwann cells were examined from 1 hpn (C) to 5 dpn (E); the cells show the same distribution and general morphology through time; however, at 5 dpn (which corresponds to 8 dpf) the cell extensions form a tubular structure typical of myelinating Schwann cells (arrow). (F) A transplanted larva that was neurectomized; the rectangle indicates the region detailed in G-I. (G-I) Organization and morphology of Schwann cells over time, from 1 hpn (G) to 5 dpn (I). At 24 hpn (H) the cells exhibited vacuoles and lost their typical bipolar morphology. After 5 dpn (8 dpf) Schwann cell numbers appear reduced and the cells show thinner processes and fail to form the extensions seen in control fish (compare E to I arrows). The rectangle in H shows the region expanded in J-L. (J-L) One day after neurectomy, Schwann cells appear to engulf debris from damaged axons (arrows in L). Scale bars: B, F: 200 μm; C-E, G-I: 25 μm; J-L: 20 μm; insets in J-L: 10 μm.
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Figure 6: Fate of Schwann cells after neurectomy. (A) Schematic representation of the transplantation scheme used to obtain mosaic labeling of Schwann cells. Briefly, 10 to 20 donor cells of Tg(Ubi:zebrabow:cherry) blastula stage embryos were aspirated and transplanted into Tg(8.0cldnb:lynEGFP) host embryos of the same stage. The transplanted embryos were screened for the presence of red fluorescent donor cells in the pLL at 48 hpf. (B) Distribution and morphology of transplanted Schwann cells in a mock-neurectomized larva (control). The rectangle indicates the region examined in the same fish at different timepoints. (C-E) Schwann cells were examined from 1 hpn (C) to 5 dpn (E); the cells show the same distribution and general morphology through time; however, at 5 dpn (which corresponds to 8 dpf) the cell extensions form a tubular structure typical of myelinating Schwann cells (arrow). (F) A transplanted larva that was neurectomized; the rectangle indicates the region detailed in G-I. (G-I) Organization and morphology of Schwann cells over time, from 1 hpn (G) to 5 dpn (I). At 24 hpn (H) the cells exhibited vacuoles and lost their typical bipolar morphology. After 5 dpn (8 dpf) Schwann cell numbers appear reduced and the cells show thinner processes and fail to form the extensions seen in control fish (compare E to I arrows). The rectangle in H shows the region expanded in J-L. (J-L) One day after neurectomy, Schwann cells appear to engulf debris from damaged axons (arrows in L). Scale bars: B, F: 200 μm; C-E, G-I: 25 μm; J-L: 20 μm; insets in J-L: 10 μm.

Mentions: A consistent observation in fish neurectomized at both 5 dpf and 3 dpf was that GFP levels in tg(foxd3:GFP) larvae diminished caudal to the neurectomy point even though the axons had regenerated to the tip of the tail after 48 h (Additional file5, compare 5B vs. 5C, and 5E vs. 5 F). This lower level of GFP label in the line of Schwann cells that accompany the pLL nerve persisted for several days after neurectomy (data not shown). The changes detected in Schwann cells after neurectomy (extension of the proliferative phase, loss of MBP, and diminishing GFP expression), prompted us to look for additional signs that could be indicative of a reversion of the differentiated state such as changes in cell shape or cell size. GFP expression in tg(foxd3:GFP) larvae did not allow us to observe individual cells, so we generated mosaic animals by transplanting cells from Tg(ubi:zebrabow:cherry) embryos, which stably and permanently express red fluorescent protein, into Tg(8.0cldnb:lynEGFP) embryos as hosts, in which all the cells derived of the primordium and the nerve of the lateral line express a membrane bound GFP[62]. In transplanted fish at 48 hpf we screened for the presence of red-labeled donor cells in the myoseptum, where Schwann cells are expected to be (Figure 6A). These patches of genetically labeled glial cells allowed us to analyze their number, distribution, and morphological changes through time after denervation, with a higher level of resolution. We imaged transplanted Schwann cells from 1 hpn to 5 dpn at low magnification (20×) and we also imaged selected regions with a 40× objective. In control larvae, the distribution and the number of Schwann cells remained essentially unchanged from 3 dpf to 8 dpf, both at proximal and distal positions, although a small increase in the number of cells over time can be seen, especially in the anteriormost portion of the glial chain of cells (Figure 6B-E). From 4 dpf to 8 dpf, cell processes extended, suggesting the formation of the tubular structure that is typical of myelinating Schwann cells (Figure 6E, arrow). In neurectomized larvae, the morphology and number of Schwann cells changes compared to control fish. At 1 hpn the cells showed the same bipolar morphology as controls did (Figure 6G) but, after 24 hpn, the cells appeared vacuolated (Figure 6H). Figures 6J-L show higher magnification images of Schwann cells (in red, Figure 6J) and the degenerating nerve (in green, Figure 6K) at this stage after neurectomy. Combination of both images allows the detection of GFP labeled membranes closely associated with Schwann cells, suggesting that axonal debris may be engulfed by Schwann cells in vivo (Figure 6L). Later, at 5 dpn, (8 dpf), neurectomized larvae displayed abnormal Schwann cells, both in shape and in number, compared to controls (compare Figure 6E to6I). The Schwann cells display a bipolar morphology, more typical of the more undifferentiated state. Furthermore, at this stage, Schwann cells have not yet formed the tubular structures seen in control larvae (compare Figure 6I with6E, arrow).


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)

Fate of Schwann cells after neurectomy. (A) Schematic representation of the transplantation scheme used to obtain mosaic labeling of Schwann cells. Briefly, 10 to 20 donor cells of Tg(Ubi:zebrabow:cherry) blastula stage embryos were aspirated and transplanted into Tg(8.0cldnb:lynEGFP) host embryos of the same stage. The transplanted embryos were screened for the presence of red fluorescent donor cells in the pLL at 48 hpf. (B) Distribution and morphology of transplanted Schwann cells in a mock-neurectomized larva (control). The rectangle indicates the region examined in the same fish at different timepoints. (C-E) Schwann cells were examined from 1 hpn (C) to 5 dpn (E); the cells show the same distribution and general morphology through time; however, at 5 dpn (which corresponds to 8 dpf) the cell extensions form a tubular structure typical of myelinating Schwann cells (arrow). (F) A transplanted larva that was neurectomized; the rectangle indicates the region detailed in G-I. (G-I) Organization and morphology of Schwann cells over time, from 1 hpn (G) to 5 dpn (I). At 24 hpn (H) the cells exhibited vacuoles and lost their typical bipolar morphology. After 5 dpn (8 dpf) Schwann cell numbers appear reduced and the cells show thinner processes and fail to form the extensions seen in control fish (compare E to I arrows). The rectangle in H shows the region expanded in J-L. (J-L) One day after neurectomy, Schwann cells appear to engulf debris from damaged axons (arrows in L). Scale bars: B, F: 200 μm; C-E, G-I: 25 μm; J-L: 20 μm; insets in J-L: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214607&req=5

Figure 6: Fate of Schwann cells after neurectomy. (A) Schematic representation of the transplantation scheme used to obtain mosaic labeling of Schwann cells. Briefly, 10 to 20 donor cells of Tg(Ubi:zebrabow:cherry) blastula stage embryos were aspirated and transplanted into Tg(8.0cldnb:lynEGFP) host embryos of the same stage. The transplanted embryos were screened for the presence of red fluorescent donor cells in the pLL at 48 hpf. (B) Distribution and morphology of transplanted Schwann cells in a mock-neurectomized larva (control). The rectangle indicates the region examined in the same fish at different timepoints. (C-E) Schwann cells were examined from 1 hpn (C) to 5 dpn (E); the cells show the same distribution and general morphology through time; however, at 5 dpn (which corresponds to 8 dpf) the cell extensions form a tubular structure typical of myelinating Schwann cells (arrow). (F) A transplanted larva that was neurectomized; the rectangle indicates the region detailed in G-I. (G-I) Organization and morphology of Schwann cells over time, from 1 hpn (G) to 5 dpn (I). At 24 hpn (H) the cells exhibited vacuoles and lost their typical bipolar morphology. After 5 dpn (8 dpf) Schwann cell numbers appear reduced and the cells show thinner processes and fail to form the extensions seen in control fish (compare E to I arrows). The rectangle in H shows the region expanded in J-L. (J-L) One day after neurectomy, Schwann cells appear to engulf debris from damaged axons (arrows in L). Scale bars: B, F: 200 μm; C-E, G-I: 25 μm; J-L: 20 μm; insets in J-L: 10 μm.
Mentions: A consistent observation in fish neurectomized at both 5 dpf and 3 dpf was that GFP levels in tg(foxd3:GFP) larvae diminished caudal to the neurectomy point even though the axons had regenerated to the tip of the tail after 48 h (Additional file5, compare 5B vs. 5C, and 5E vs. 5 F). This lower level of GFP label in the line of Schwann cells that accompany the pLL nerve persisted for several days after neurectomy (data not shown). The changes detected in Schwann cells after neurectomy (extension of the proliferative phase, loss of MBP, and diminishing GFP expression), prompted us to look for additional signs that could be indicative of a reversion of the differentiated state such as changes in cell shape or cell size. GFP expression in tg(foxd3:GFP) larvae did not allow us to observe individual cells, so we generated mosaic animals by transplanting cells from Tg(ubi:zebrabow:cherry) embryos, which stably and permanently express red fluorescent protein, into Tg(8.0cldnb:lynEGFP) embryos as hosts, in which all the cells derived of the primordium and the nerve of the lateral line express a membrane bound GFP[62]. In transplanted fish at 48 hpf we screened for the presence of red-labeled donor cells in the myoseptum, where Schwann cells are expected to be (Figure 6A). These patches of genetically labeled glial cells allowed us to analyze their number, distribution, and morphological changes through time after denervation, with a higher level of resolution. We imaged transplanted Schwann cells from 1 hpn to 5 dpn at low magnification (20×) and we also imaged selected regions with a 40× objective. In control larvae, the distribution and the number of Schwann cells remained essentially unchanged from 3 dpf to 8 dpf, both at proximal and distal positions, although a small increase in the number of cells over time can be seen, especially in the anteriormost portion of the glial chain of cells (Figure 6B-E). From 4 dpf to 8 dpf, cell processes extended, suggesting the formation of the tubular structure that is typical of myelinating Schwann cells (Figure 6E, arrow). In neurectomized larvae, the morphology and number of Schwann cells changes compared to control fish. At 1 hpn the cells showed the same bipolar morphology as controls did (Figure 6G) but, after 24 hpn, the cells appeared vacuolated (Figure 6H). Figures 6J-L show higher magnification images of Schwann cells (in red, Figure 6J) and the degenerating nerve (in green, Figure 6K) at this stage after neurectomy. Combination of both images allows the detection of GFP labeled membranes closely associated with Schwann cells, suggesting that axonal debris may be engulfed by Schwann cells in vivo (Figure 6L). Later, at 5 dpn, (8 dpf), neurectomized larvae displayed abnormal Schwann cells, both in shape and in number, compared to controls (compare Figure 6E to6I). The Schwann cells display a bipolar morphology, more typical of the more undifferentiated state. Furthermore, at this stage, Schwann cells have not yet formed the tubular structures seen in control larvae (compare Figure 6I with6E, arrow).

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