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Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord.

Bacon C, Endris V, Andermatt I, Niederkofler V, Waltereit R, Bartsch D, Stoeckli ET, Rappold G - PLoS ONE (2011)

Bottom Line: Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO.However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect.We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics, University of Heidelberg, Heidelberg, Germany. Claire.bacon@med.uni-heidelberg.de

ABSTRACT
Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.

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srGAP3 co-localizes with TAG-1 in the majority of commissural axons but TAG-1 positive axons project normally towards the floor plate in srGAP3 KO spinal cords.A–C: srGAP3 is expressed in the majority of TAG-1 positive axons (grey arrows). D–F: Enlarged images of the areas indicated by white boxes in A–C, showing the floor plate region more clearly and the co-localisation of srGAP3 and TAG-1 (grey arrows) in most axons. G–H: TAG-1 immunohistochemistry on WT and srGAP3 KO sections showed that TAG-1 positive axons project normally towards and across the floor plate. TAG-1 staining was also present in the ventral funiculus (white arrows). This was observed in three different srGAP3 KO embryos.
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pone-0019887-g002: srGAP3 co-localizes with TAG-1 in the majority of commissural axons but TAG-1 positive axons project normally towards the floor plate in srGAP3 KO spinal cords.A–C: srGAP3 is expressed in the majority of TAG-1 positive axons (grey arrows). D–F: Enlarged images of the areas indicated by white boxes in A–C, showing the floor plate region more clearly and the co-localisation of srGAP3 and TAG-1 (grey arrows) in most axons. G–H: TAG-1 immunohistochemistry on WT and srGAP3 KO sections showed that TAG-1 positive axons project normally towards and across the floor plate. TAG-1 staining was also present in the ventral funiculus (white arrows). This was observed in three different srGAP3 KO embryos.

Mentions: We have shown that srGAP3 protein co-localises with Robo1 in the ventrolateral funiculus and with Robo2 in the lateral funiculus. To examine whether srGAP3 protein also localises to pre-crossing axons we performed double immunohistochemistry of TAG-1 and srGAP3 (Figure 2A–F). SrGAP3 is expressed diffusely throughout the gray matter of the spinal cord and therefore almost all TAG-1 positive axons co-express srGAP3 (Figure 2A–F, grey arrows). In Slit triple mutant mice, TAG-1 positive axons were defasciculated at the floor plate and did not leave the floor plate into the ventral funiculus [2]. However, in the srGAP3 KO, TAG-1 positive commissural axons projected normally across the floor plate and staining in the ventral funiculus was present (Figure 2G–H, white arrows). This was observed in three different srGAP3 KO embryos. TAG-1 also labels sensory axons and in Robo1/Robo2 double mutants, TAG-1 positive sensory axons overshoot the dorsal root entry zone [4]. However, we did not observe any overshooting of TAG-1 positive sensory axons in the srGAP3 KO (Figure 2H).


Evidence for a role of srGAP3 in the positioning of commissural axons within the ventrolateral funiculus of the mouse spinal cord.

Bacon C, Endris V, Andermatt I, Niederkofler V, Waltereit R, Bartsch D, Stoeckli ET, Rappold G - PLoS ONE (2011)

srGAP3 co-localizes with TAG-1 in the majority of commissural axons but TAG-1 positive axons project normally towards the floor plate in srGAP3 KO spinal cords.A–C: srGAP3 is expressed in the majority of TAG-1 positive axons (grey arrows). D–F: Enlarged images of the areas indicated by white boxes in A–C, showing the floor plate region more clearly and the co-localisation of srGAP3 and TAG-1 (grey arrows) in most axons. G–H: TAG-1 immunohistochemistry on WT and srGAP3 KO sections showed that TAG-1 positive axons project normally towards and across the floor plate. TAG-1 staining was also present in the ventral funiculus (white arrows). This was observed in three different srGAP3 KO embryos.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104994&req=5

pone-0019887-g002: srGAP3 co-localizes with TAG-1 in the majority of commissural axons but TAG-1 positive axons project normally towards the floor plate in srGAP3 KO spinal cords.A–C: srGAP3 is expressed in the majority of TAG-1 positive axons (grey arrows). D–F: Enlarged images of the areas indicated by white boxes in A–C, showing the floor plate region more clearly and the co-localisation of srGAP3 and TAG-1 (grey arrows) in most axons. G–H: TAG-1 immunohistochemistry on WT and srGAP3 KO sections showed that TAG-1 positive axons project normally towards and across the floor plate. TAG-1 staining was also present in the ventral funiculus (white arrows). This was observed in three different srGAP3 KO embryos.
Mentions: We have shown that srGAP3 protein co-localises with Robo1 in the ventrolateral funiculus and with Robo2 in the lateral funiculus. To examine whether srGAP3 protein also localises to pre-crossing axons we performed double immunohistochemistry of TAG-1 and srGAP3 (Figure 2A–F). SrGAP3 is expressed diffusely throughout the gray matter of the spinal cord and therefore almost all TAG-1 positive axons co-express srGAP3 (Figure 2A–F, grey arrows). In Slit triple mutant mice, TAG-1 positive axons were defasciculated at the floor plate and did not leave the floor plate into the ventral funiculus [2]. However, in the srGAP3 KO, TAG-1 positive commissural axons projected normally across the floor plate and staining in the ventral funiculus was present (Figure 2G–H, white arrows). This was observed in three different srGAP3 KO embryos. TAG-1 also labels sensory axons and in Robo1/Robo2 double mutants, TAG-1 positive sensory axons overshoot the dorsal root entry zone [4]. However, we did not observe any overshooting of TAG-1 positive sensory axons in the srGAP3 KO (Figure 2H).

Bottom Line: Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO.However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect.We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Molecular Genetics, University of Heidelberg, Heidelberg, Germany. Claire.bacon@med.uni-heidelberg.de

ABSTRACT
Slit-Robo signaling guides commissural axons away from the floor-plate of the spinal cord and into the longitudinal axis after crossing the midline. In this study we have evaluated the role of the Slit-Robo GTPase activating protein 3 (srGAP3) in commissural axon guidance using a knockout (KO) mouse model. Co-immunoprecipitation experiments confirmed that srGAP3 interacts with the Slit receptors Robo1 and Robo2 and immunohistochemistry studies showed that srGAP3 co-localises with Robo1 in the ventral and lateral funiculus and with Robo2 in the lateral funiculus. Stalling axons have been reported in the floor-plate of Slit and Robo mutant spinal cords but our axon tracing experiments revealed no dorsal commissural axon stalling in the floor plate of the srGAP3 KO mouse. Interestingly we observed a significant thickening of the ventral funiculus and a thinning of the lateral funiculus in the srGAP3 KO spinal cord, which has also recently been reported in the Robo2 KO. However, axons in the enlarged ventral funiculus of the srGAP3 KO are Robo1 positive but do not express Robo2, indicating that the thickening of the ventral funiculus in the srGAP3 KO is not a Robo2 mediated effect. We suggest a role for srGAP3 in the lateral positioning of post crossing axons within the ventrolateral funiculus.

Show MeSH
Related in: MedlinePlus