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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 interacts with Robo1 and Robo2 and co-localises with both Robo1 and Robo2 in the spinal cord at E12.5.A, B:.HEK293 cells were transfected with srGAP3 and myc-tagged Robo1 or Robo2 contructs and immunoprecipitation revealed that srGAP3 interacts with Robo1 and Robo2 (upper panels, lane 1). No interaction between srGAP3 and Robo1 or Robo2 was observed when HEK293 cells were transfected with a ΔCC3 Robo1 or a ΔCC3 Robo2 construct (upper panels, lane 2) or an srGAP3 construct with a mutation in the SH3 domain (upper panels, lane 3). Control experiments where full length srGAP3, Robo1 or Robo2 were transfected alone into HEK293 cells alone showed no interaction (upper panels, lanes 4 and 5). The middle and lower panels represent the cell lysate immunoblotted with an anti-myc and anti-srGAP3 antibody respectively. C: srGAP3 and Robo1 mRNA expression in adjacent transverse sections of E11.5 spinal cords showing that srGAP3 and Robo1 are co-expressed in a subset of neurons in the dorsal spinal cord. D–F: srGAP3 and Robo1 immunohistochemistry showing co-localisation of both proteins in the longitudinal axon tracts of the spinal cord at E12.5 (arrows). srGAP3 expression is also detected in the gray matter of the spinal cord (F, I). G–I: srGAP3 and Robo2 immunohistochemistry showing expression of both proteins in the lateral funiculus of the spinal cord at E12.5 (arrows). J: Absence of srGAP3 immunostaining on transverse cryosections of srGAP3 KO spinal cords compared to WT demonstrating specificity of the srGAP3 staining in the spinal cord. Note that the brightness of the panel showing the KO spinal cord was increased in order to see the spinal cord.
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pone-0019887-g001: srGAP3 interacts with Robo1 and Robo2 and co-localises with both Robo1 and Robo2 in the spinal cord at E12.5.A, B:.HEK293 cells were transfected with srGAP3 and myc-tagged Robo1 or Robo2 contructs and immunoprecipitation revealed that srGAP3 interacts with Robo1 and Robo2 (upper panels, lane 1). No interaction between srGAP3 and Robo1 or Robo2 was observed when HEK293 cells were transfected with a ΔCC3 Robo1 or a ΔCC3 Robo2 construct (upper panels, lane 2) or an srGAP3 construct with a mutation in the SH3 domain (upper panels, lane 3). Control experiments where full length srGAP3, Robo1 or Robo2 were transfected alone into HEK293 cells alone showed no interaction (upper panels, lanes 4 and 5). The middle and lower panels represent the cell lysate immunoblotted with an anti-myc and anti-srGAP3 antibody respectively. C: srGAP3 and Robo1 mRNA expression in adjacent transverse sections of E11.5 spinal cords showing that srGAP3 and Robo1 are co-expressed in a subset of neurons in the dorsal spinal cord. D–F: srGAP3 and Robo1 immunohistochemistry showing co-localisation of both proteins in the longitudinal axon tracts of the spinal cord at E12.5 (arrows). srGAP3 expression is also detected in the gray matter of the spinal cord (F, I). G–I: srGAP3 and Robo2 immunohistochemistry showing expression of both proteins in the lateral funiculus of the spinal cord at E12.5 (arrows). J: Absence of srGAP3 immunostaining on transverse cryosections of srGAP3 KO spinal cords compared to WT demonstrating specificity of the srGAP3 staining in the spinal cord. Note that the brightness of the panel showing the KO spinal cord was increased in order to see the spinal cord.

Mentions: Co-immunoprecipitation experiments using HEK293 cells indicated that the SH3 domain of srGAP1 interacts with the CC3 domain of Robo1 [6]. Here, we used the same approach to show that srGAP3 can interact with Robo1 and Robo2 (Figure 1A, B). When we co-transfected a truncated Robo1 or Robo2 construct missing the CC3 domain with full length srGAP3, or full length Robo1 or Robo2 with an srGAP3 construct carrying a mutation in the SH3 domain, we did not observe an interaction (Figure 1A, B). This confirms that the SH3 domain of srGAP3 interacts with the CC3 region of Robo1 and Robo2.


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 interacts with Robo1 and Robo2 and co-localises with both Robo1 and Robo2 in the spinal cord at E12.5.A, B:.HEK293 cells were transfected with srGAP3 and myc-tagged Robo1 or Robo2 contructs and immunoprecipitation revealed that srGAP3 interacts with Robo1 and Robo2 (upper panels, lane 1). No interaction between srGAP3 and Robo1 or Robo2 was observed when HEK293 cells were transfected with a ΔCC3 Robo1 or a ΔCC3 Robo2 construct (upper panels, lane 2) or an srGAP3 construct with a mutation in the SH3 domain (upper panels, lane 3). Control experiments where full length srGAP3, Robo1 or Robo2 were transfected alone into HEK293 cells alone showed no interaction (upper panels, lanes 4 and 5). The middle and lower panels represent the cell lysate immunoblotted with an anti-myc and anti-srGAP3 antibody respectively. C: srGAP3 and Robo1 mRNA expression in adjacent transverse sections of E11.5 spinal cords showing that srGAP3 and Robo1 are co-expressed in a subset of neurons in the dorsal spinal cord. D–F: srGAP3 and Robo1 immunohistochemistry showing co-localisation of both proteins in the longitudinal axon tracts of the spinal cord at E12.5 (arrows). srGAP3 expression is also detected in the gray matter of the spinal cord (F, I). G–I: srGAP3 and Robo2 immunohistochemistry showing expression of both proteins in the lateral funiculus of the spinal cord at E12.5 (arrows). J: Absence of srGAP3 immunostaining on transverse cryosections of srGAP3 KO spinal cords compared to WT demonstrating specificity of the srGAP3 staining in the spinal cord. Note that the brightness of the panel showing the KO spinal cord was increased in order to see the spinal cord.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104994&req=5

pone-0019887-g001: srGAP3 interacts with Robo1 and Robo2 and co-localises with both Robo1 and Robo2 in the spinal cord at E12.5.A, B:.HEK293 cells were transfected with srGAP3 and myc-tagged Robo1 or Robo2 contructs and immunoprecipitation revealed that srGAP3 interacts with Robo1 and Robo2 (upper panels, lane 1). No interaction between srGAP3 and Robo1 or Robo2 was observed when HEK293 cells were transfected with a ΔCC3 Robo1 or a ΔCC3 Robo2 construct (upper panels, lane 2) or an srGAP3 construct with a mutation in the SH3 domain (upper panels, lane 3). Control experiments where full length srGAP3, Robo1 or Robo2 were transfected alone into HEK293 cells alone showed no interaction (upper panels, lanes 4 and 5). The middle and lower panels represent the cell lysate immunoblotted with an anti-myc and anti-srGAP3 antibody respectively. C: srGAP3 and Robo1 mRNA expression in adjacent transverse sections of E11.5 spinal cords showing that srGAP3 and Robo1 are co-expressed in a subset of neurons in the dorsal spinal cord. D–F: srGAP3 and Robo1 immunohistochemistry showing co-localisation of both proteins in the longitudinal axon tracts of the spinal cord at E12.5 (arrows). srGAP3 expression is also detected in the gray matter of the spinal cord (F, I). G–I: srGAP3 and Robo2 immunohistochemistry showing expression of both proteins in the lateral funiculus of the spinal cord at E12.5 (arrows). J: Absence of srGAP3 immunostaining on transverse cryosections of srGAP3 KO spinal cords compared to WT demonstrating specificity of the srGAP3 staining in the spinal cord. Note that the brightness of the panel showing the KO spinal cord was increased in order to see the spinal cord.
Mentions: Co-immunoprecipitation experiments using HEK293 cells indicated that the SH3 domain of srGAP1 interacts with the CC3 domain of Robo1 [6]. Here, we used the same approach to show that srGAP3 can interact with Robo1 and Robo2 (Figure 1A, B). When we co-transfected a truncated Robo1 or Robo2 construct missing the CC3 domain with full length srGAP3, or full length Robo1 or Robo2 with an srGAP3 construct carrying a mutation in the SH3 domain, we did not observe an interaction (Figure 1A, B). This confirms that the SH3 domain of srGAP3 interacts with the CC3 region of Robo1 and Robo2.

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