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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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The ventral funiculus of srGAP3 KO spinal cords is significantly thicker compared to WT.A–B: L1 staining on transverse sections of srGAP3 WT (A) and srGAP3 KO (B) spinal cords revealed an enlargement of the ventral funiculus in srGAP3 KO cords compared to WT (white arrows). C: The area of the L1-positive ventral funiculus was quantified in WT and srGAP3 KO cord sections at cervical, thoracic and caudal levels and the ventral funiculus of srGAP3 KO spinal cords was found to be significantly thicker at all levels using the Independent Samples T-test (P<0.0001). D: The L1 positive lateral funiculus area was also quantified in the same sections and was found to be significantly thinner in srGAP3 KO cervical sections (P = 0.0204) compared to WT and srGAP3 KO thoracic sections (P = 0.0335) compared to WT. The lateral funiculus was also found to be slightly thinner in caudal sections of srGAP3 KO spinal cords compared to WT, but this difference was not significant (P = 0.339). E: The number of embryos and sections analysed is provided in the table.
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pone-0019887-g004: The ventral funiculus of srGAP3 KO spinal cords is significantly thicker compared to WT.A–B: L1 staining on transverse sections of srGAP3 WT (A) and srGAP3 KO (B) spinal cords revealed an enlargement of the ventral funiculus in srGAP3 KO cords compared to WT (white arrows). C: The area of the L1-positive ventral funiculus was quantified in WT and srGAP3 KO cord sections at cervical, thoracic and caudal levels and the ventral funiculus of srGAP3 KO spinal cords was found to be significantly thicker at all levels using the Independent Samples T-test (P<0.0001). D: The L1 positive lateral funiculus area was also quantified in the same sections and was found to be significantly thinner in srGAP3 KO cervical sections (P = 0.0204) compared to WT and srGAP3 KO thoracic sections (P = 0.0335) compared to WT. The lateral funiculus was also found to be slightly thinner in caudal sections of srGAP3 KO spinal cords compared to WT, but this difference was not significant (P = 0.339). E: The number of embryos and sections analysed is provided in the table.

Mentions: We used L1 staining to visualise the longitudinal axon tracts in E12.5 transverse cryosections (Figure 4A–B). We observed and were able to quantify a thickening of the ventral funiculus next to the floor plate in srGAP3 KO spinal cords, which was absent in WT sections at the same rostro-caudal level (Figure 4A–B, white arrows). We quantified the area of the ventral funiculus (Figure 4C) and the increase in the ventral funiculus was found to be 16% in cervical sections (P  =  <0.0001), 11% in thoracic sections (P  =  <0.0001) and 10% in caudal sections (P  =  <0.0001). Additionally, we quantified the area of the lateral funiculus (Figure 4D) and found a significant thinning of the lateral funiculus in cervical (8% decrease, P = 0.0204) and thoracic (8% decrease, P = 0.0335) sections of srGAP3 KO spinal cords. The lateral funiculus was also thinner in caudal KO sections compared to caudal WT sections but this was not statistically significant, probably because the lateral funiculus is still in early stages of development at the caudal level (4% thinner, P = 0.339). The total normalised ventrolateral funiculus area was not significantly different between srGAP3 KO and WT spinal cords (Cervical level: WT (n = 38)  = 0.0942, KO (n = 72)  = 0.0937, P = 0.8; Thoracic level: WT (n = 41)  = 0.0859, KO (n = 58)  = 0.0843, P = 0.52; Caudal level: WT (n = 30)  = 0.0784, KO (n = 61)  = 0.0799, P = 0.6). Additionally, the mean L1 staining intensity did not differ between the KO and the WT spinal cord sections. Quantification of the normalised floor plate commissure thickness (Figure S2) revealed no significant difference between srGAP3 KO and WT cords (cervical level: WT (n = 22)  = 0.0013, KO (n = 68)  = 0.00142, P = 0.215), indicating that the enlarged ventral funiculus is not the result of more axons crossing the midline.


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)

The ventral funiculus of srGAP3 KO spinal cords is significantly thicker compared to WT.A–B: L1 staining on transverse sections of srGAP3 WT (A) and srGAP3 KO (B) spinal cords revealed an enlargement of the ventral funiculus in srGAP3 KO cords compared to WT (white arrows). C: The area of the L1-positive ventral funiculus was quantified in WT and srGAP3 KO cord sections at cervical, thoracic and caudal levels and the ventral funiculus of srGAP3 KO spinal cords was found to be significantly thicker at all levels using the Independent Samples T-test (P<0.0001). D: The L1 positive lateral funiculus area was also quantified in the same sections and was found to be significantly thinner in srGAP3 KO cervical sections (P = 0.0204) compared to WT and srGAP3 KO thoracic sections (P = 0.0335) compared to WT. The lateral funiculus was also found to be slightly thinner in caudal sections of srGAP3 KO spinal cords compared to WT, but this difference was not significant (P = 0.339). E: The number of embryos and sections analysed is provided in the table.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104994&req=5

pone-0019887-g004: The ventral funiculus of srGAP3 KO spinal cords is significantly thicker compared to WT.A–B: L1 staining on transverse sections of srGAP3 WT (A) and srGAP3 KO (B) spinal cords revealed an enlargement of the ventral funiculus in srGAP3 KO cords compared to WT (white arrows). C: The area of the L1-positive ventral funiculus was quantified in WT and srGAP3 KO cord sections at cervical, thoracic and caudal levels and the ventral funiculus of srGAP3 KO spinal cords was found to be significantly thicker at all levels using the Independent Samples T-test (P<0.0001). D: The L1 positive lateral funiculus area was also quantified in the same sections and was found to be significantly thinner in srGAP3 KO cervical sections (P = 0.0204) compared to WT and srGAP3 KO thoracic sections (P = 0.0335) compared to WT. The lateral funiculus was also found to be slightly thinner in caudal sections of srGAP3 KO spinal cords compared to WT, but this difference was not significant (P = 0.339). E: The number of embryos and sections analysed is provided in the table.
Mentions: We used L1 staining to visualise the longitudinal axon tracts in E12.5 transverse cryosections (Figure 4A–B). We observed and were able to quantify a thickening of the ventral funiculus next to the floor plate in srGAP3 KO spinal cords, which was absent in WT sections at the same rostro-caudal level (Figure 4A–B, white arrows). We quantified the area of the ventral funiculus (Figure 4C) and the increase in the ventral funiculus was found to be 16% in cervical sections (P  =  <0.0001), 11% in thoracic sections (P  =  <0.0001) and 10% in caudal sections (P  =  <0.0001). Additionally, we quantified the area of the lateral funiculus (Figure 4D) and found a significant thinning of the lateral funiculus in cervical (8% decrease, P = 0.0204) and thoracic (8% decrease, P = 0.0335) sections of srGAP3 KO spinal cords. The lateral funiculus was also thinner in caudal KO sections compared to caudal WT sections but this was not statistically significant, probably because the lateral funiculus is still in early stages of development at the caudal level (4% thinner, P = 0.339). The total normalised ventrolateral funiculus area was not significantly different between srGAP3 KO and WT spinal cords (Cervical level: WT (n = 38)  = 0.0942, KO (n = 72)  = 0.0937, P = 0.8; Thoracic level: WT (n = 41)  = 0.0859, KO (n = 58)  = 0.0843, P = 0.52; Caudal level: WT (n = 30)  = 0.0784, KO (n = 61)  = 0.0799, P = 0.6). Additionally, the mean L1 staining intensity did not differ between the KO and the WT spinal cord sections. Quantification of the normalised floor plate commissure thickness (Figure S2) revealed no significant difference between srGAP3 KO and WT cords (cervical level: WT (n = 22)  = 0.0013, KO (n = 68)  = 0.00142, P = 0.215), indicating that the enlarged ventral funiculus is not the result of more axons crossing the midline.

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