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Slit Binding via the Ig1 Domain Is Essential for Midline Repulsion by Drosophila Robo1 but Dispensable for Receptor Expression, Localization, and Regulation in Vivo.

Brown HE, Reichert MC, Evans TA - G3 (Bethesda) (2015)

Bottom Line: Biochemical structure and interaction studies support a model in which Slit binding to the first immunoglobulin-like (Ig1) domain of Robo receptors activates a repulsive signaling pathway in axonal growth cones.Here, we examine the in vivo functional importance of the Ig1 domain of the Drosophila Robo1 receptor, which controls midline crossing of axons in response to Slit during development of the embryonic CNS.These results establish a direct link between in vitro biochemical studies of Slit-Robo interactions and in vivo genetic studies of Slit-Robo signaling during midline axon guidance, and distinguish Slit-dependent from Slit-independent aspects of Robo1 expression, regulation, and activity during embryonic development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701.

No MeSH data available.


Related in: MedlinePlus

Robo1ΔIg1 cannot rescue midline crossing defects in robo1 mutants. (A–D) Stage 16 embryos stained with anti-HRP (magenta) and anti-FasII (green) antibodies. Lower images show FasII channel alone from the same embryos. FasII-positive axons cross the midline inappropriately in every segment in robo1  mutants (B, arrow with asterisk). This phenotype is completely rescued by a robo1 genomic rescue transgene expressing full-length Robo1 protein (C) but is not rescued by an equivalent rescue transgene expressing Robo1ΔIg1 (D). (E–H) Stage 13 embryos stained with anti-FasII to examine the trajectory of the pCC axon, which pioneers the medial FasII pathway. In heterozygous robo1/+ embryos (E) or robo1  mutants rescued by Robo1 (G), the pCC axon extends anteriorly and does not cross the midline (arrows in E and G). In robo1  mutants, the pCC axon inappropriately crosses the midline and fasciculates with its contralateral homolog (F, arrow with asterisk). Ectopic crossing of pCC is not rescued by expression of Robo1ΔIg1 (H, arrow with asterisk). For quantification of ectopic crossing phenotypes in the genotypes shown in (A–D), see Table 2.
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fig6: Robo1ΔIg1 cannot rescue midline crossing defects in robo1 mutants. (A–D) Stage 16 embryos stained with anti-HRP (magenta) and anti-FasII (green) antibodies. Lower images show FasII channel alone from the same embryos. FasII-positive axons cross the midline inappropriately in every segment in robo1 mutants (B, arrow with asterisk). This phenotype is completely rescued by a robo1 genomic rescue transgene expressing full-length Robo1 protein (C) but is not rescued by an equivalent rescue transgene expressing Robo1ΔIg1 (D). (E–H) Stage 13 embryos stained with anti-FasII to examine the trajectory of the pCC axon, which pioneers the medial FasII pathway. In heterozygous robo1/+ embryos (E) or robo1 mutants rescued by Robo1 (G), the pCC axon extends anteriorly and does not cross the midline (arrows in E and G). In robo1 mutants, the pCC axon inappropriately crosses the midline and fasciculates with its contralateral homolog (F, arrow with asterisk). Ectopic crossing of pCC is not rescued by expression of Robo1ΔIg1 (H, arrow with asterisk). For quantification of ectopic crossing phenotypes in the genotypes shown in (A–D), see Table 2.

Mentions: To more closely assess midline repulsion in our rescue backgrounds, we examined FasII-positive axon pathways, which provide a more sensitive readout of midline repulsion and can reveal more subtle ectopic crossing events that may be undetectable when examining the entire axon scaffold with anti-HRP. FasII-positive axons do not cross the midline in wild-type embryos, but a subset of these axons cross the midline ectopically in every segment in robo1 mutants (Figure 6, A and B). We found that the Robo1 rescue transgene was able to restore wild-type levels of midline repulsion to FasII-positive axons in robo1 mutant embryos (Figure 6C). In contrast, the Robo1ΔIg1 transgene had no effect on the ectopic midline crossing caused by the robo1 mutation, and FasII crossing defects in robo11, robo1:: robo1ΔIg1 embryos were indistinguishable from robo11 homozygous embryos (Figure 6D; Table 2).


Slit Binding via the Ig1 Domain Is Essential for Midline Repulsion by Drosophila Robo1 but Dispensable for Receptor Expression, Localization, and Regulation in Vivo.

Brown HE, Reichert MC, Evans TA - G3 (Bethesda) (2015)

Robo1ΔIg1 cannot rescue midline crossing defects in robo1 mutants. (A–D) Stage 16 embryos stained with anti-HRP (magenta) and anti-FasII (green) antibodies. Lower images show FasII channel alone from the same embryos. FasII-positive axons cross the midline inappropriately in every segment in robo1  mutants (B, arrow with asterisk). This phenotype is completely rescued by a robo1 genomic rescue transgene expressing full-length Robo1 protein (C) but is not rescued by an equivalent rescue transgene expressing Robo1ΔIg1 (D). (E–H) Stage 13 embryos stained with anti-FasII to examine the trajectory of the pCC axon, which pioneers the medial FasII pathway. In heterozygous robo1/+ embryos (E) or robo1  mutants rescued by Robo1 (G), the pCC axon extends anteriorly and does not cross the midline (arrows in E and G). In robo1  mutants, the pCC axon inappropriately crosses the midline and fasciculates with its contralateral homolog (F, arrow with asterisk). Ectopic crossing of pCC is not rescued by expression of Robo1ΔIg1 (H, arrow with asterisk). For quantification of ectopic crossing phenotypes in the genotypes shown in (A–D), see Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Robo1ΔIg1 cannot rescue midline crossing defects in robo1 mutants. (A–D) Stage 16 embryos stained with anti-HRP (magenta) and anti-FasII (green) antibodies. Lower images show FasII channel alone from the same embryos. FasII-positive axons cross the midline inappropriately in every segment in robo1 mutants (B, arrow with asterisk). This phenotype is completely rescued by a robo1 genomic rescue transgene expressing full-length Robo1 protein (C) but is not rescued by an equivalent rescue transgene expressing Robo1ΔIg1 (D). (E–H) Stage 13 embryos stained with anti-FasII to examine the trajectory of the pCC axon, which pioneers the medial FasII pathway. In heterozygous robo1/+ embryos (E) or robo1 mutants rescued by Robo1 (G), the pCC axon extends anteriorly and does not cross the midline (arrows in E and G). In robo1 mutants, the pCC axon inappropriately crosses the midline and fasciculates with its contralateral homolog (F, arrow with asterisk). Ectopic crossing of pCC is not rescued by expression of Robo1ΔIg1 (H, arrow with asterisk). For quantification of ectopic crossing phenotypes in the genotypes shown in (A–D), see Table 2.
Mentions: To more closely assess midline repulsion in our rescue backgrounds, we examined FasII-positive axon pathways, which provide a more sensitive readout of midline repulsion and can reveal more subtle ectopic crossing events that may be undetectable when examining the entire axon scaffold with anti-HRP. FasII-positive axons do not cross the midline in wild-type embryos, but a subset of these axons cross the midline ectopically in every segment in robo1 mutants (Figure 6, A and B). We found that the Robo1 rescue transgene was able to restore wild-type levels of midline repulsion to FasII-positive axons in robo1 mutant embryos (Figure 6C). In contrast, the Robo1ΔIg1 transgene had no effect on the ectopic midline crossing caused by the robo1 mutation, and FasII crossing defects in robo11, robo1:: robo1ΔIg1 embryos were indistinguishable from robo11 homozygous embryos (Figure 6D; Table 2).

Bottom Line: Biochemical structure and interaction studies support a model in which Slit binding to the first immunoglobulin-like (Ig1) domain of Robo receptors activates a repulsive signaling pathway in axonal growth cones.Here, we examine the in vivo functional importance of the Ig1 domain of the Drosophila Robo1 receptor, which controls midline crossing of axons in response to Slit during development of the embryonic CNS.These results establish a direct link between in vitro biochemical studies of Slit-Robo interactions and in vivo genetic studies of Slit-Robo signaling during midline axon guidance, and distinguish Slit-dependent from Slit-independent aspects of Robo1 expression, regulation, and activity during embryonic development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Arkansas, Fayetteville, Arkansas 72701.

No MeSH data available.


Related in: MedlinePlus