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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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Axons within the enlarged ventral funiculus of the srGAP3 KO are Robo1 but not Robo2 positive.A: Robo1 immunohistochemistry on transverse section of E12.5 WT spinal cord, showing the normal size of the Robo1 positive ventral funiculus (white arrows). B: The enlarged ventral funiculus in the srGAP3 KO spinal cord is visible with Robo1 immunohistochemistry (white arrows) C–D: Immunohistochemistry of Robo2 in WT (C) and srGAP3 KO (D) cords showing lack of Robo2 expression in the axons of the ventral funiculus in both WT and srGAP3 KO spinal cords (white arrows). E: The Robo1 positive ventral funiculus is significantly thicker in srGAP3 KO spinal cords (mean value 0.0262) compared to WT (mean value 0.0223). F: The Robo1 positive lateral funiculus was significantly thinner in srGAP3 KO (mean value 0.062) compared to WT (mean value 0.0705) spinal cords (P = 0.0296). Quantifications were performed on cervical sections only. I: Summary of the total number of embryos and cervical sections quantified.
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pone-0019887-g005: Axons within the enlarged ventral funiculus of the srGAP3 KO are Robo1 but not Robo2 positive.A: Robo1 immunohistochemistry on transverse section of E12.5 WT spinal cord, showing the normal size of the Robo1 positive ventral funiculus (white arrows). B: The enlarged ventral funiculus in the srGAP3 KO spinal cord is visible with Robo1 immunohistochemistry (white arrows) C–D: Immunohistochemistry of Robo2 in WT (C) and srGAP3 KO (D) cords showing lack of Robo2 expression in the axons of the ventral funiculus in both WT and srGAP3 KO spinal cords (white arrows). E: The Robo1 positive ventral funiculus is significantly thicker in srGAP3 KO spinal cords (mean value 0.0262) compared to WT (mean value 0.0223). F: The Robo1 positive lateral funiculus was significantly thinner in srGAP3 KO (mean value 0.062) compared to WT (mean value 0.0705) spinal cords (P = 0.0296). Quantifications were performed on cervical sections only. I: Summary of the total number of embryos and cervical sections quantified.

Mentions: The size of the ventrolateral funiculus is altered in Robo1 and Robo2 mutants [2], [4]. To investigate the possibility that the thickening of the ventral funiculus in the srGAP3 KO is a Robo mediated effect, we visualised Robo1 and Robo2 positive axons in WT and srGAP3 KO spinal cords using immunohistochemistry. Robo1 positive axons were found throughout the ventrolateral funiculus in both srGAP3 KO and WT sections. Interestingly the enlarged ventral funiculus in the srGAP3 KO was only visible with Robo1 staining (Figure 5A–B; white arrows). No Robo2-positive axons were found in the ventral funiculus (Figure 5C–D; white arrows), as might have been expected based on published results [4]. We quantified the Robo1-positive area of the ventral and lateral funiculi as previously described in cervical spinal cord sections and were able to confirm that the increase of the ventral funiculus and reduction of the lateral funiculus is statistically significant (Figure 5E–F). Additionally, the mean Robo1 staining intensity did not differ between the KO and the WT spinal cord sections.


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)

Axons within the enlarged ventral funiculus of the srGAP3 KO are Robo1 but not Robo2 positive.A: Robo1 immunohistochemistry on transverse section of E12.5 WT spinal cord, showing the normal size of the Robo1 positive ventral funiculus (white arrows). B: The enlarged ventral funiculus in the srGAP3 KO spinal cord is visible with Robo1 immunohistochemistry (white arrows) C–D: Immunohistochemistry of Robo2 in WT (C) and srGAP3 KO (D) cords showing lack of Robo2 expression in the axons of the ventral funiculus in both WT and srGAP3 KO spinal cords (white arrows). E: The Robo1 positive ventral funiculus is significantly thicker in srGAP3 KO spinal cords (mean value 0.0262) compared to WT (mean value 0.0223). F: The Robo1 positive lateral funiculus was significantly thinner in srGAP3 KO (mean value 0.062) compared to WT (mean value 0.0705) spinal cords (P = 0.0296). Quantifications were performed on cervical sections only. I: Summary of the total number of embryos and cervical sections quantified.
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Related In: Results  -  Collection

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pone-0019887-g005: Axons within the enlarged ventral funiculus of the srGAP3 KO are Robo1 but not Robo2 positive.A: Robo1 immunohistochemistry on transverse section of E12.5 WT spinal cord, showing the normal size of the Robo1 positive ventral funiculus (white arrows). B: The enlarged ventral funiculus in the srGAP3 KO spinal cord is visible with Robo1 immunohistochemistry (white arrows) C–D: Immunohistochemistry of Robo2 in WT (C) and srGAP3 KO (D) cords showing lack of Robo2 expression in the axons of the ventral funiculus in both WT and srGAP3 KO spinal cords (white arrows). E: The Robo1 positive ventral funiculus is significantly thicker in srGAP3 KO spinal cords (mean value 0.0262) compared to WT (mean value 0.0223). F: The Robo1 positive lateral funiculus was significantly thinner in srGAP3 KO (mean value 0.062) compared to WT (mean value 0.0705) spinal cords (P = 0.0296). Quantifications were performed on cervical sections only. I: Summary of the total number of embryos and cervical sections quantified.
Mentions: The size of the ventrolateral funiculus is altered in Robo1 and Robo2 mutants [2], [4]. To investigate the possibility that the thickening of the ventral funiculus in the srGAP3 KO is a Robo mediated effect, we visualised Robo1 and Robo2 positive axons in WT and srGAP3 KO spinal cords using immunohistochemistry. Robo1 positive axons were found throughout the ventrolateral funiculus in both srGAP3 KO and WT sections. Interestingly the enlarged ventral funiculus in the srGAP3 KO was only visible with Robo1 staining (Figure 5A–B; white arrows). No Robo2-positive axons were found in the ventral funiculus (Figure 5C–D; white arrows), as might have been expected based on published results [4]. We quantified the Robo1-positive area of the ventral and lateral funiculi as previously described in cervical spinal cord sections and were able to confirm that the increase of the ventral funiculus and reduction of the lateral funiculus is statistically significant (Figure 5E–F). Additionally, the mean Robo1 staining intensity did not differ between the KO and the WT spinal cord sections.

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