Limits...
Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons.

Evans TA, Santiago C, Arbeille E, Bashaw GJ - Elife (2015)

Bottom Line: In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1).Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously.These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

ABSTRACT
During nervous system development, commissural axons cross the midline despite the presence of repellant ligands. In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1). In this study, we describe a distinct mechanism to inhibit Robo1 repulsion and promote midline crossing, in which Roundabout2 (Robo2) binds to and prevents Robo1 signaling. Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously. We show that the extracellular domains required for binding to Robo1 are also required for Robo2's ability to promote midline crossing, in both gain-of-function and rescue assays. These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.

No MeSH data available.


Related in: MedlinePlus

Robo2 can promote crossing non cell-autonomously.(A–D) Stage 17 embryos stained with anti-HRP (magenta) and anti-FasII (green). (A and B) Mis-expression of Robo1 (A) in midline cells using slit-GAL4 results in a mild ectopic crossing phenotype. In contrast, mis-expression of Robo2 (B) produces a much stronger effect, as indicated by quantification of ectopic FasII crossing in the histogram (I). (C and D) Mis-expression of either Robo2∆Ig1 (C) or Robo2∆Ig2 (D) with slit-GAL4 does not produce ectopic crossing of FasII axons. (E and F) Consistent with requirement of Robo2's first two IG domains, the chimeric protein Robo1R2IG(1+2) produces an ectopic crossing phenotype (F), whereas Robo2R1(IG1+2) has no effect (E). (G and H) Mis-expression of Robo2∆C with slit-GAL4 also results in severe ectopic crossing defects (H) that are much stronger than those observed with Robo1∆C (G), as indicated by quantification of ectopic FasII crossing (I) and fused commissures observed in anti-HRP stained embryos (J). All UAS-Robo transgenes were inserted into the same genomic location (86FB). Significance was assessed by multiple comparisons using the Student's t-test and a Bonferonni correction (*p < 0.001). Error bars represent s.e.m. n, number of embryos scored for each genotype.DOI:http://dx.doi.org/10.7554/eLife.08407.011
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fig7: Robo2 can promote crossing non cell-autonomously.(A–D) Stage 17 embryos stained with anti-HRP (magenta) and anti-FasII (green). (A and B) Mis-expression of Robo1 (A) in midline cells using slit-GAL4 results in a mild ectopic crossing phenotype. In contrast, mis-expression of Robo2 (B) produces a much stronger effect, as indicated by quantification of ectopic FasII crossing in the histogram (I). (C and D) Mis-expression of either Robo2∆Ig1 (C) or Robo2∆Ig2 (D) with slit-GAL4 does not produce ectopic crossing of FasII axons. (E and F) Consistent with requirement of Robo2's first two IG domains, the chimeric protein Robo1R2IG(1+2) produces an ectopic crossing phenotype (F), whereas Robo2R1(IG1+2) has no effect (E). (G and H) Mis-expression of Robo2∆C with slit-GAL4 also results in severe ectopic crossing defects (H) that are much stronger than those observed with Robo1∆C (G), as indicated by quantification of ectopic FasII crossing (I) and fused commissures observed in anti-HRP stained embryos (J). All UAS-Robo transgenes were inserted into the same genomic location (86FB). Significance was assessed by multiple comparisons using the Student's t-test and a Bonferonni correction (*p < 0.001). Error bars represent s.e.m. n, number of embryos scored for each genotype.DOI:http://dx.doi.org/10.7554/eLife.08407.011

Mentions: To more explicitly test whether Robo2 can promote midline crossing non-autonomously, we used slit-GAL4 to drive Robo2 expression in midline glia and neurons. We found that mis-expression of Robo2 or Robo2∆C in midline cells caused many FasII-positive axons which do not express slit-GAL4 to ectopically cross the midline, confirming that Robo2 can act non-autonomously to promote midline crossing of axons, and that this effect does not depend on the cytoplasmic domain (Figure 7). We observed a significantly milder effect with mis-expression of Robo1, suggesting that Robo2's non-cell autonomous activity is not solely a consequence of Slit titration (Figure 7). Moreover, this non-cell autonomous activity of Robo2 appears to be Ig1/Ig2-dependent, as Robo1R2Ig1+2 but not Robo2R1Ig1+2 promoted strong ectopic midline crossing when expressed using slit-GAL4 (Figure 7). In addition, expression of Robo2 variants missing either Ig1 or Ig2 with slit-Gal4 did not result in any ectopic midline crossing (Figure 7). The requirement for both Ig1 and Ig2 in this context contrasts with our findings with pan-neural mis-expression, in which Robo2∆Ig1 retained some pro-crossing activity. However, it is worth noting that the phenotype generated by elav-GAL4 mis-expression of Robo2 is stronger than that generated by slit-GAL4, perhaps because slit-GAL4 is expressed in a much smaller number of cells.10.7554/eLife.08407.011Figure 7.Robo2 can promote crossing non cell-autonomously.


Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons.

Evans TA, Santiago C, Arbeille E, Bashaw GJ - Elife (2015)

Robo2 can promote crossing non cell-autonomously.(A–D) Stage 17 embryos stained with anti-HRP (magenta) and anti-FasII (green). (A and B) Mis-expression of Robo1 (A) in midline cells using slit-GAL4 results in a mild ectopic crossing phenotype. In contrast, mis-expression of Robo2 (B) produces a much stronger effect, as indicated by quantification of ectopic FasII crossing in the histogram (I). (C and D) Mis-expression of either Robo2∆Ig1 (C) or Robo2∆Ig2 (D) with slit-GAL4 does not produce ectopic crossing of FasII axons. (E and F) Consistent with requirement of Robo2's first two IG domains, the chimeric protein Robo1R2IG(1+2) produces an ectopic crossing phenotype (F), whereas Robo2R1(IG1+2) has no effect (E). (G and H) Mis-expression of Robo2∆C with slit-GAL4 also results in severe ectopic crossing defects (H) that are much stronger than those observed with Robo1∆C (G), as indicated by quantification of ectopic FasII crossing (I) and fused commissures observed in anti-HRP stained embryos (J). All UAS-Robo transgenes were inserted into the same genomic location (86FB). Significance was assessed by multiple comparisons using the Student's t-test and a Bonferonni correction (*p < 0.001). Error bars represent s.e.m. n, number of embryos scored for each genotype.DOI:http://dx.doi.org/10.7554/eLife.08407.011
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Related In: Results  -  Collection

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fig7: Robo2 can promote crossing non cell-autonomously.(A–D) Stage 17 embryos stained with anti-HRP (magenta) and anti-FasII (green). (A and B) Mis-expression of Robo1 (A) in midline cells using slit-GAL4 results in a mild ectopic crossing phenotype. In contrast, mis-expression of Robo2 (B) produces a much stronger effect, as indicated by quantification of ectopic FasII crossing in the histogram (I). (C and D) Mis-expression of either Robo2∆Ig1 (C) or Robo2∆Ig2 (D) with slit-GAL4 does not produce ectopic crossing of FasII axons. (E and F) Consistent with requirement of Robo2's first two IG domains, the chimeric protein Robo1R2IG(1+2) produces an ectopic crossing phenotype (F), whereas Robo2R1(IG1+2) has no effect (E). (G and H) Mis-expression of Robo2∆C with slit-GAL4 also results in severe ectopic crossing defects (H) that are much stronger than those observed with Robo1∆C (G), as indicated by quantification of ectopic FasII crossing (I) and fused commissures observed in anti-HRP stained embryos (J). All UAS-Robo transgenes were inserted into the same genomic location (86FB). Significance was assessed by multiple comparisons using the Student's t-test and a Bonferonni correction (*p < 0.001). Error bars represent s.e.m. n, number of embryos scored for each genotype.DOI:http://dx.doi.org/10.7554/eLife.08407.011
Mentions: To more explicitly test whether Robo2 can promote midline crossing non-autonomously, we used slit-GAL4 to drive Robo2 expression in midline glia and neurons. We found that mis-expression of Robo2 or Robo2∆C in midline cells caused many FasII-positive axons which do not express slit-GAL4 to ectopically cross the midline, confirming that Robo2 can act non-autonomously to promote midline crossing of axons, and that this effect does not depend on the cytoplasmic domain (Figure 7). We observed a significantly milder effect with mis-expression of Robo1, suggesting that Robo2's non-cell autonomous activity is not solely a consequence of Slit titration (Figure 7). Moreover, this non-cell autonomous activity of Robo2 appears to be Ig1/Ig2-dependent, as Robo1R2Ig1+2 but not Robo2R1Ig1+2 promoted strong ectopic midline crossing when expressed using slit-GAL4 (Figure 7). In addition, expression of Robo2 variants missing either Ig1 or Ig2 with slit-Gal4 did not result in any ectopic midline crossing (Figure 7). The requirement for both Ig1 and Ig2 in this context contrasts with our findings with pan-neural mis-expression, in which Robo2∆Ig1 retained some pro-crossing activity. However, it is worth noting that the phenotype generated by elav-GAL4 mis-expression of Robo2 is stronger than that generated by slit-GAL4, perhaps because slit-GAL4 is expressed in a much smaller number of cells.10.7554/eLife.08407.011Figure 7.Robo2 can promote crossing non cell-autonomously.

Bottom Line: In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1).Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously.These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

ABSTRACT
During nervous system development, commissural axons cross the midline despite the presence of repellant ligands. In Drosophila, commissural axons avoid premature responsiveness to the midline repellant Slit by expressing the endosomal sorting receptor Commissureless, which reduces surface expression of the Slit receptor Roundabout1 (Robo1). In this study, we describe a distinct mechanism to inhibit Robo1 repulsion and promote midline crossing, in which Roundabout2 (Robo2) binds to and prevents Robo1 signaling. Unexpectedly, we find that Robo2 is expressed in midline cells during the early stages of commissural axon guidance, and that over-expression of Robo2 can rescue robo2-dependent midline crossing defects non-cell autonomously. We show that the extracellular domains required for binding to Robo1 are also required for Robo2's ability to promote midline crossing, in both gain-of-function and rescue assays. These findings indicate that at least two independent mechanisms to overcome Slit-Robo1 repulsion in pre-crossing commissural axons have evolved in Drosophila.

No MeSH data available.


Related in: MedlinePlus