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Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

Welte C, Engel S, Stuermer CA - Neural Dev (2015)

Bottom Line: MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. Cornelia.welte@uni-konstanz.de.

ABSTRACT

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

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Expression pattern of Rtn4b in the zebrafish retina and optic nerve. Cross sections of the zebrafish retina normal (A) and 10 days after ONS (B,C) were exposed to ABs against Rtn4b (A,B) and MBP (C). Weak Rtn4b staining is seen across all retinal layers including RGCs (white arrow) in the normal retina (A). RGCs robustly upregulate Rtn4b 10 days after ONS (B). The RGC axons in the retina on top of the RGCs (bracket) are also weakly labeled but are more intensely stained by the AB against MBP (C). Scale bar, 50 μm. Cross sections through the normal zebrafish optic nerve (D,E,F) show very weak labeling with Rtn4b AB (D). (E) Rtn4a AB stains across the normal nerve similar to MBP. Labeling with the MBP AB is strong in the normal nerve (F) as well as at 10 days after ONS (M). Scale bar, 10 μm. Rtn4b AB in nerves at 10 days after ONS (G,I) labels portions of RGC axons identified as axonal (rather than glial) by the AB against neurolin (H). (I), overlay. Scale bar, 20 μm. (J) Rtn4a AB in the10-days ONS nerve stains, in particular, the boundaries of fascicles and subdivisions of the fascicles. (K,L) The neurolin-positive regenerating RGC axons are not labeled with Rtn4a to any significant extent, in contrast to Rtn4b labeling (I). (M) MBP staining is intense in the 10-day ONS nerve. (N,O) Neurolin-positive regenerating RGC axons are located amidst the myelin. Boxed areas in (I,L,O) are enlarged in (P,Q,R). Arrows point to neurolin-positive axons which are also Rtn4b AB-positive (P), but seem not significantly co-localized with Rtn4a (Q), and lie amidst the MBP labeling of myelin (R). Scale bar, 10 μm.
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Fig1: Expression pattern of Rtn4b in the zebrafish retina and optic nerve. Cross sections of the zebrafish retina normal (A) and 10 days after ONS (B,C) were exposed to ABs against Rtn4b (A,B) and MBP (C). Weak Rtn4b staining is seen across all retinal layers including RGCs (white arrow) in the normal retina (A). RGCs robustly upregulate Rtn4b 10 days after ONS (B). The RGC axons in the retina on top of the RGCs (bracket) are also weakly labeled but are more intensely stained by the AB against MBP (C). Scale bar, 50 μm. Cross sections through the normal zebrafish optic nerve (D,E,F) show very weak labeling with Rtn4b AB (D). (E) Rtn4a AB stains across the normal nerve similar to MBP. Labeling with the MBP AB is strong in the normal nerve (F) as well as at 10 days after ONS (M). Scale bar, 10 μm. Rtn4b AB in nerves at 10 days after ONS (G,I) labels portions of RGC axons identified as axonal (rather than glial) by the AB against neurolin (H). (I), overlay. Scale bar, 20 μm. (J) Rtn4a AB in the10-days ONS nerve stains, in particular, the boundaries of fascicles and subdivisions of the fascicles. (K,L) The neurolin-positive regenerating RGC axons are not labeled with Rtn4a to any significant extent, in contrast to Rtn4b labeling (I). (M) MBP staining is intense in the 10-day ONS nerve. (N,O) Neurolin-positive regenerating RGC axons are located amidst the myelin. Boxed areas in (I,L,O) are enlarged in (P,Q,R). Arrows point to neurolin-positive axons which are also Rtn4b AB-positive (P), but seem not significantly co-localized with Rtn4a (Q), and lie amidst the MBP labeling of myelin (R). Scale bar, 10 μm.

Mentions: The affinity purified antiserum against zebrafish Rtn4b [16] labeled all retinal layers but was brighter over RGC somata compared to other retinal neurons (Figure 1A). The RGC axon layer which was intensely labeled by the anti-MBP antibody (AB) (fish RGC axons are myelinated in their intraretinal path) was only weakly stained by the Rtn4b AB (Figure 1A, B, C). Ten days after ONS, RGC somata had significantly increased expression of Rtn4b indicating that ONS leads to Rtn4b upregulation in neurons (Figure 1B). In the normal optic nerve, Rtn4b labeling was weak (Figure 1D) whereas anti-MBP AB strongly labeled the myelin (Figure 1F, M) in the normal nerve and after ONS. The staining with Rtn4a AB was similar to MBP, but the AB labeled in addition the boundaries of axon fascicles and further subdivisions of the fascicles (Figure 1E). Rtn4a therefore appears to reside in astrocytic structures as suggested earlier [18] and myelin. In the nerve 10 days after ONS, Rtn4b labeling was associated with glial cell processes around fascicles and more strikingly with regenerating RGC axons which were identified by anti-neurolin AB [19] (Figure 1G, H, I, P). Accordingly, axons and growth cones in culture were also labeled (Figure 2E). Rtn4a AB also stains RGC growth cones in vitro [18] but in sections through the nerve strongly stained the fascicle boundaries and subdivisions rather than neurolin-positive regenerating axons (Figure 1J, K, L, Q). In the nerve 10 days after ONS, myelin detected by MBP AB was intense and the neurolin-positive regenerating axons were located amidst the myelin staining (Figure 1M, N, O, R). Together, this staining shows that regenerating RGC axons in the nerve and in vitro are Rtn4b-positive and cross through MBP-labeled myelin. Rtn4a is in myelin and astrocytic fascicle boundaries and subdivisions but not to the same extent in neurolin-positive axons as Rtn4b. Rtn4b appears less prominent in CNS myelin in the retina and optic nerve but is significantly upregulated in RGCs and RGC axons after ONS.Figure 1


Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

Welte C, Engel S, Stuermer CA - Neural Dev (2015)

Expression pattern of Rtn4b in the zebrafish retina and optic nerve. Cross sections of the zebrafish retina normal (A) and 10 days after ONS (B,C) were exposed to ABs against Rtn4b (A,B) and MBP (C). Weak Rtn4b staining is seen across all retinal layers including RGCs (white arrow) in the normal retina (A). RGCs robustly upregulate Rtn4b 10 days after ONS (B). The RGC axons in the retina on top of the RGCs (bracket) are also weakly labeled but are more intensely stained by the AB against MBP (C). Scale bar, 50 μm. Cross sections through the normal zebrafish optic nerve (D,E,F) show very weak labeling with Rtn4b AB (D). (E) Rtn4a AB stains across the normal nerve similar to MBP. Labeling with the MBP AB is strong in the normal nerve (F) as well as at 10 days after ONS (M). Scale bar, 10 μm. Rtn4b AB in nerves at 10 days after ONS (G,I) labels portions of RGC axons identified as axonal (rather than glial) by the AB against neurolin (H). (I), overlay. Scale bar, 20 μm. (J) Rtn4a AB in the10-days ONS nerve stains, in particular, the boundaries of fascicles and subdivisions of the fascicles. (K,L) The neurolin-positive regenerating RGC axons are not labeled with Rtn4a to any significant extent, in contrast to Rtn4b labeling (I). (M) MBP staining is intense in the 10-day ONS nerve. (N,O) Neurolin-positive regenerating RGC axons are located amidst the myelin. Boxed areas in (I,L,O) are enlarged in (P,Q,R). Arrows point to neurolin-positive axons which are also Rtn4b AB-positive (P), but seem not significantly co-localized with Rtn4a (Q), and lie amidst the MBP labeling of myelin (R). Scale bar, 10 μm.
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Related In: Results  -  Collection

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Fig1: Expression pattern of Rtn4b in the zebrafish retina and optic nerve. Cross sections of the zebrafish retina normal (A) and 10 days after ONS (B,C) were exposed to ABs against Rtn4b (A,B) and MBP (C). Weak Rtn4b staining is seen across all retinal layers including RGCs (white arrow) in the normal retina (A). RGCs robustly upregulate Rtn4b 10 days after ONS (B). The RGC axons in the retina on top of the RGCs (bracket) are also weakly labeled but are more intensely stained by the AB against MBP (C). Scale bar, 50 μm. Cross sections through the normal zebrafish optic nerve (D,E,F) show very weak labeling with Rtn4b AB (D). (E) Rtn4a AB stains across the normal nerve similar to MBP. Labeling with the MBP AB is strong in the normal nerve (F) as well as at 10 days after ONS (M). Scale bar, 10 μm. Rtn4b AB in nerves at 10 days after ONS (G,I) labels portions of RGC axons identified as axonal (rather than glial) by the AB against neurolin (H). (I), overlay. Scale bar, 20 μm. (J) Rtn4a AB in the10-days ONS nerve stains, in particular, the boundaries of fascicles and subdivisions of the fascicles. (K,L) The neurolin-positive regenerating RGC axons are not labeled with Rtn4a to any significant extent, in contrast to Rtn4b labeling (I). (M) MBP staining is intense in the 10-day ONS nerve. (N,O) Neurolin-positive regenerating RGC axons are located amidst the myelin. Boxed areas in (I,L,O) are enlarged in (P,Q,R). Arrows point to neurolin-positive axons which are also Rtn4b AB-positive (P), but seem not significantly co-localized with Rtn4a (Q), and lie amidst the MBP labeling of myelin (R). Scale bar, 10 μm.
Mentions: The affinity purified antiserum against zebrafish Rtn4b [16] labeled all retinal layers but was brighter over RGC somata compared to other retinal neurons (Figure 1A). The RGC axon layer which was intensely labeled by the anti-MBP antibody (AB) (fish RGC axons are myelinated in their intraretinal path) was only weakly stained by the Rtn4b AB (Figure 1A, B, C). Ten days after ONS, RGC somata had significantly increased expression of Rtn4b indicating that ONS leads to Rtn4b upregulation in neurons (Figure 1B). In the normal optic nerve, Rtn4b labeling was weak (Figure 1D) whereas anti-MBP AB strongly labeled the myelin (Figure 1F, M) in the normal nerve and after ONS. The staining with Rtn4a AB was similar to MBP, but the AB labeled in addition the boundaries of axon fascicles and further subdivisions of the fascicles (Figure 1E). Rtn4a therefore appears to reside in astrocytic structures as suggested earlier [18] and myelin. In the nerve 10 days after ONS, Rtn4b labeling was associated with glial cell processes around fascicles and more strikingly with regenerating RGC axons which were identified by anti-neurolin AB [19] (Figure 1G, H, I, P). Accordingly, axons and growth cones in culture were also labeled (Figure 2E). Rtn4a AB also stains RGC growth cones in vitro [18] but in sections through the nerve strongly stained the fascicle boundaries and subdivisions rather than neurolin-positive regenerating axons (Figure 1J, K, L, Q). In the nerve 10 days after ONS, myelin detected by MBP AB was intense and the neurolin-positive regenerating axons were located amidst the myelin staining (Figure 1M, N, O, R). Together, this staining shows that regenerating RGC axons in the nerve and in vitro are Rtn4b-positive and cross through MBP-labeled myelin. Rtn4a is in myelin and astrocytic fascicle boundaries and subdivisions but not to the same extent in neurolin-positive axons as Rtn4b. Rtn4b appears less prominent in CNS myelin in the retina and optic nerve but is significantly upregulated in RGCs and RGC axons after ONS.Figure 1

Bottom Line: MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. Cornelia.welte@uni-konstanz.de.

ABSTRACT

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

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Related in: MedlinePlus