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A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion.

Bashaw GJ, Hu H, Nobes CD, Goodman CS - J. Cell Biol. (2001)

Bottom Line: Curr.Opin.Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA. gbashaw@mail.med.upenn.edu

ABSTRACT
The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509-514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173-180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103-110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

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Related in: MedlinePlus

GEF64C gain of function overcomes Robo repulsion and is suppressed by the RhoA dominant negative. Stage 16 embryos stained with mAb BP102 to label all CNS axons. Anterior is up. Genotypes are shown below each panel. Note the more wild-type appearance of commissural and longitudinal axon bundles in the embryo coexpressing GEF64C and the RhoA dominant negative (B), relative to GEF64C alone (A).
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fig3: GEF64C gain of function overcomes Robo repulsion and is suppressed by the RhoA dominant negative. Stage 16 embryos stained with mAb BP102 to label all CNS axons. Anterior is up. Genotypes are shown below each panel. Note the more wild-type appearance of commissural and longitudinal axon bundles in the embryo coexpressing GEF64C and the RhoA dominant negative (B), relative to GEF64C alone (A).

Mentions: In contrast to the modest effects of loss of GEF64C function, pan-neural overexpression of GEF64C (using EP3035 or UASGEF64C) results in a dramatic, dose-dependent, gain of function phenotype, in which many axons abnormally project across the midline. The commissures are thicker and there is a commensurate reduction in the longitudinal axon tracts (Fig. 3 A). This phenotype suggests that GEF64C expression promotes axon attraction to the midline. The point mutations in GEF64C were introduced on the EP3035 chromosome, allowing for GAL4 overexpression of the mutant alleles. None of the mutant alleles, nor the UASGEF64CΔC transgene (a deletion of the Dbl and PH domains), were capable of generating the gain of function phenotype, indicating that the abnormal midline crossing is due to GEF64C expression, and that this effect requires the intact Dbl and PH domains. Examination of gain of function embryos with antibodies to Wrapper (Noordermeer et al., 1998), a marker for midline glia, indicates that the guidance defects caused by GEF64C overexpression are not a secondary consequence of nonautonomous perturbations of midline glial cell survival or migration (unpublished data).


A novel Dbl family RhoGEF promotes Rho-dependent axon attraction to the central nervous system midline in Drosophila and overcomes Robo repulsion.

Bashaw GJ, Hu H, Nobes CD, Goodman CS - J. Cell Biol. (2001)

GEF64C gain of function overcomes Robo repulsion and is suppressed by the RhoA dominant negative. Stage 16 embryos stained with mAb BP102 to label all CNS axons. Anterior is up. Genotypes are shown below each panel. Note the more wild-type appearance of commissural and longitudinal axon bundles in the embryo coexpressing GEF64C and the RhoA dominant negative (B), relative to GEF64C alone (A).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199320&req=5

fig3: GEF64C gain of function overcomes Robo repulsion and is suppressed by the RhoA dominant negative. Stage 16 embryos stained with mAb BP102 to label all CNS axons. Anterior is up. Genotypes are shown below each panel. Note the more wild-type appearance of commissural and longitudinal axon bundles in the embryo coexpressing GEF64C and the RhoA dominant negative (B), relative to GEF64C alone (A).
Mentions: In contrast to the modest effects of loss of GEF64C function, pan-neural overexpression of GEF64C (using EP3035 or UASGEF64C) results in a dramatic, dose-dependent, gain of function phenotype, in which many axons abnormally project across the midline. The commissures are thicker and there is a commensurate reduction in the longitudinal axon tracts (Fig. 3 A). This phenotype suggests that GEF64C expression promotes axon attraction to the midline. The point mutations in GEF64C were introduced on the EP3035 chromosome, allowing for GAL4 overexpression of the mutant alleles. None of the mutant alleles, nor the UASGEF64CΔC transgene (a deletion of the Dbl and PH domains), were capable of generating the gain of function phenotype, indicating that the abnormal midline crossing is due to GEF64C expression, and that this effect requires the intact Dbl and PH domains. Examination of gain of function embryos with antibodies to Wrapper (Noordermeer et al., 1998), a marker for midline glia, indicates that the guidance defects caused by GEF64C overexpression are not a secondary consequence of nonautonomous perturbations of midline glial cell survival or migration (unpublished data).

Bottom Line: Curr.Opin.Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, University of Pennsylvania, Philadelphia, PA 19104, USA. gbashaw@mail.med.upenn.edu

ABSTRACT
The key role of the Rho family GTPases Rac, Rho, and CDC42 in regulating the actin cytoskeleton is well established (Hall, A. 1998. Science. 279:509-514). Increasing evidence suggests that the Rho GTPases and their upstream positive regulators, guanine nucleotide exchange factors (GEFs), also play important roles in the control of growth cone guidance in the developing nervous system (Luo, L. 2000. Nat. Rev. Neurosci. 1:173-180; Dickson, B.J. 2001. Curr. Opin. Neurobiol. 11:103-110). Here, we present the identification and molecular characterization of a novel Dbl family Rho GEF, GEF64C, that promotes axon attraction to the central nervous system midline in the embryonic Drosophila nervous system. In sensitized genetic backgrounds, loss of GEF64C function causes a phenotype where too few axons cross the midline. In contrast, ectopic expression of GEF64C throughout the nervous system results in a phenotype in which far too many axons cross the midline, a phenotype reminiscent of loss of function mutations in the Roundabout (Robo) repulsive guidance receptor. Genetic analysis indicates that GEF64C expression can in fact overcome Robo repulsion. Surprisingly, evidence from genetic, biochemical, and cell culture experiments suggests that the promotion of axon attraction by GEF64C is dependent on the activation of Rho, but not Rac or Cdc42.

Show MeSH
Related in: MedlinePlus