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.


robo2 mRNA is transiently expressed in midline cells.Fluorescent in situ for robo2 mRNA (green). robo2 is transiently expressed in midline glia (magenta) during Stages 12 and 13 but is no longer detected there by Stage 14. Midline glia are labeled with an antibody to Wrapper.DOI:http://dx.doi.org/10.7554/eLife.08407.013
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fig8s1: robo2 mRNA is transiently expressed in midline cells.Fluorescent in situ for robo2 mRNA (green). robo2 is transiently expressed in midline glia (magenta) during Stages 12 and 13 but is no longer detected there by Stage 14. Midline glia are labeled with an antibody to Wrapper.DOI:http://dx.doi.org/10.7554/eLife.08407.013

Mentions: Robo2 can promote midline crossing when expressed in a subset of embryonic neurons and glia, and endogenous robo2 contributes to midline crossing of commissural axons. During embryogenesis, robo2 expression is dynamically regulated: it is broadly expressed in neurons during early stages of CNS development, including transient expression in a number of ipsilateral pioneer neurons, and later becomes restricted to neurons whose axons form longitudinal pathways in the lateral regions of the neuropile (Simpson et al., 2000a). To gain additional insight into Robo2's role in promoting midline crossing of commissural neurons, we examined robo2 mRNA and protein expression in embryos during the early stages of axon path finding, when the first commissural axons are crossing the midline (stages 12–13). Using fluorescent mRNA in situ hybridization, we were able to detect robo2 mRNA expression in cells labeled by slit-GAL4 in late stage 12 embryos, around the time that pioneer commissural axons are crossing the midline (Figure 8B). robo2 mRNA expression persists through the end of stage 13, but is no longer detectable by stage 14; thus, midline expression of robo2 coincides with the time when most commissural axons are crossing the midline (Figure 8, Figure 8—figure supplement 1). Moreover, a robo2-GAL4 enhancer-trap insertion is expressed in midline glia at this time, as detected by anti-GFP staining in robo2-GAL4, UAS-TauMycGFP embryos (Figure 8A). Expression of UAS-HARobo2 with robo2-Gal4 and detection of transgenic Robo2 with anti-HA reveals an expression pattern that closely resembles the endogenous pattern of Robo2 protein, suggesting that the robo2-GAL4 faithfully reports Robo2 expression (data not shown). In addition, we could detect weak expression of Robo2 protein produced by an HA-tagged knock-in allele of robo2 (Spitzweck et al., 2010) in a subset of slit-GAL4 expressing cells at stage 12, confirming that Robo2 protein is produced in midline cells during the stages of commissural axon path finding, and raising the possibility that Robo2 endogenously acts in these cells to promote midline crossing of commissural axons (Figure 8B).10.7554/eLife.08407.012Figure 8.robo2 is expressed in midline cells during commissural axon path finding, and over-expressing robo2 with slit-GAL4 restores midline crossing in robo2, fra double mutants.


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 mRNA is transiently expressed in midline cells.Fluorescent in situ for robo2 mRNA (green). robo2 is transiently expressed in midline glia (magenta) during Stages 12 and 13 but is no longer detected there by Stage 14. Midline glia are labeled with an antibody to Wrapper.DOI:http://dx.doi.org/10.7554/eLife.08407.013
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4505356&req=5

fig8s1: robo2 mRNA is transiently expressed in midline cells.Fluorescent in situ for robo2 mRNA (green). robo2 is transiently expressed in midline glia (magenta) during Stages 12 and 13 but is no longer detected there by Stage 14. Midline glia are labeled with an antibody to Wrapper.DOI:http://dx.doi.org/10.7554/eLife.08407.013
Mentions: Robo2 can promote midline crossing when expressed in a subset of embryonic neurons and glia, and endogenous robo2 contributes to midline crossing of commissural axons. During embryogenesis, robo2 expression is dynamically regulated: it is broadly expressed in neurons during early stages of CNS development, including transient expression in a number of ipsilateral pioneer neurons, and later becomes restricted to neurons whose axons form longitudinal pathways in the lateral regions of the neuropile (Simpson et al., 2000a). To gain additional insight into Robo2's role in promoting midline crossing of commissural neurons, we examined robo2 mRNA and protein expression in embryos during the early stages of axon path finding, when the first commissural axons are crossing the midline (stages 12–13). Using fluorescent mRNA in situ hybridization, we were able to detect robo2 mRNA expression in cells labeled by slit-GAL4 in late stage 12 embryos, around the time that pioneer commissural axons are crossing the midline (Figure 8B). robo2 mRNA expression persists through the end of stage 13, but is no longer detectable by stage 14; thus, midline expression of robo2 coincides with the time when most commissural axons are crossing the midline (Figure 8, Figure 8—figure supplement 1). Moreover, a robo2-GAL4 enhancer-trap insertion is expressed in midline glia at this time, as detected by anti-GFP staining in robo2-GAL4, UAS-TauMycGFP embryos (Figure 8A). Expression of UAS-HARobo2 with robo2-Gal4 and detection of transgenic Robo2 with anti-HA reveals an expression pattern that closely resembles the endogenous pattern of Robo2 protein, suggesting that the robo2-GAL4 faithfully reports Robo2 expression (data not shown). In addition, we could detect weak expression of Robo2 protein produced by an HA-tagged knock-in allele of robo2 (Spitzweck et al., 2010) in a subset of slit-GAL4 expressing cells at stage 12, confirming that Robo2 protein is produced in midline cells during the stages of commissural axon path finding, and raising the possibility that Robo2 endogenously acts in these cells to promote midline crossing of commissural axons (Figure 8B).10.7554/eLife.08407.012Figure 8.robo2 is expressed in midline cells during commissural axon path finding, and over-expressing robo2 with slit-GAL4 restores midline crossing in robo2, fra double mutants.

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.