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

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.

Figure 1.

Molecular characterization and expression of GEF64C. (A) Genomic organization of the GEF64C locus. The location of the EP insert, the sequenced mutant alleles, and the region used for antibody generation are indicated. Coding sequences are represented by filled rectangles, UTRs by open rectangles. Colored regions of the coding sequence are as indicated in B. (B) Schematic diagram of the GEF64C and GEF64CΔC proteins. Individual domains are as indicated. The PH domain was identified by the SMART sequence analysis program, but had a very low significance score (10^e−1). Since all known Dbl domain proteins have PH domains flanking the Dbl, we have included the PH domain with a small question mark next to it. (C) Amino acid sequence of GEF64C. Sequences of identified domains are underlined in the color corresponding to each domain as indicated in B. (D–F) Stage 15–16 embryos stained with anti-GEF64C antibody. Anterior is up. (D) Wild-type. (E) UASGEF64C/ElavGal4. (F) UASGEF64CΔC/ElavGal4. These sequence data are available from EMBL/Genbank/DDBJ under accession no. AY064174.

Figure 1.

Molecular characterization and expression of GEF64C. (A) Genomic organization of the GEF64C locus. The location of the EP insert, the sequenced mutant alleles, and the region used for antibody generation are indicated. Coding sequences are represented by filled rectangles, UTRs by open rectangles. Colored regions of the coding sequence are as indicated in B. (B) Schematic diagram of the GEF64C and GEF64CΔC proteins. Individual domains are as indicated. The PH domain was identified by the SMART sequence analysis program, but had a very low significance score (10^e−1). Since all known Dbl domain proteins have PH domains flanking the Dbl, we have included the PH domain with a small question mark next to it. (C) Amino acid sequence of GEF64C. Sequences of identified domains are underlined in the color corresponding to each domain as indicated in B. (D–F) Stage 15–16 embryos stained with anti-GEF64C antibody. Anterior is up. (D) Wild-type. (E) UASGEF64C/ElavGal4. (F) UASGEF64CΔC/ElavGal4. These sequence data are available from EMBL/Genbank/DDBJ under accession no. AY064174.

Figure 2.

GEF64C loss of function. Stage 16 embryos stained with mAb BP102 to label all CNS axons. Anterior is up. Genotypes are shown below each panel. Abnormally thin or absent commissures are indicated by arrows with asterisks.

Figure 3.

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).

Figure 4.

GEF64C promotes RhoA-dependent actin stress fiber formation in fibroblasts. (A) Uninjected control cells. (B) Cells injected with a GEF64C expression construct show striking actin stress fiber formation. (B′) Injection marker for cells shown in B. (C) Cells coinjected with the GEF64C expression construct and C3 transferase protein. C3 strongly inhibits GEF64C-induced stress fiber formation. (C′) Injection marker for cells shown in C. (D) GEF exchange assays for Rac, Rho, and Cdc 42. Histogram columns are as indicated. Activity is expressed as the percent of initial ^[H3]GDP remaining bound after 25 min. The relatively weak, but significant exchange activity that we observe could be attributable to the fact that the PH domain was not included in these assays, as fusion proteins containing both the Dbl and PH domains were poorly expressed.