Limits...
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.

Show MeSH

Related in: MedlinePlus

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 (10e−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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199320&req=5

fig1: 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 (10e−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.

Mentions: Expression of EP3035 dramatically enhances the axon guidance defects of the Robo-DCC chimera, leading to a significant increase in ectopic midline crossing (unpublished data). Molecular characterization of the genomic region adjacent to EP3035 revealed a large transcription unit that encodes a novel member of the Dbl family of guanine nucleotide exchange factors (GEFs) (Cerione and Zheng, 1996) specific for the Rho family of small GTPases (Fig. 1), GEF64C. In addition to the canonical Dbl and pleckstrin homology (PH) domains, GEF64C also contains several proline-rich motifs, including a sequence similar to the Enabled EVH1 domain binding site (LPLPP) (Niebuhr et al., 1997) (Fig. 1). RNA in situ analysis on EP3035/ElavGal4 embryos confirms that EP3035 drives overexpression of the GEF64C transcript. In addition, the genetic enhancement of Robo-DCC by EP3035 can be phenocopied by expressing a UAS GEF64C transgene, confirming that the enhancement is due to GEF64C expression (unpublished data). Protein expression analysis in wild-type embryos, using an mAb to GEF64C, reveals broad, low level expression of this GEF, with some enrichment in the CNS (Fig. 1 D). The specificity of the GEF64C mAb is demonstrated by comparing embryos expressing full-length UASGEF64C under control of elavGAL4, with those expressing a COOH-terminal truncation, UASGEF64CΔC, which removes the mAb epitope (Fig. 1 B). Robust CNS expression can be seen in animals with the wild-type transgene, while only the low-levels characteristic of wild-type expression can be seen in animals with the truncated transgene (Fig. 1, E and F).


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)

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 (10e−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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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 (10e−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.
Mentions: Expression of EP3035 dramatically enhances the axon guidance defects of the Robo-DCC chimera, leading to a significant increase in ectopic midline crossing (unpublished data). Molecular characterization of the genomic region adjacent to EP3035 revealed a large transcription unit that encodes a novel member of the Dbl family of guanine nucleotide exchange factors (GEFs) (Cerione and Zheng, 1996) specific for the Rho family of small GTPases (Fig. 1), GEF64C. In addition to the canonical Dbl and pleckstrin homology (PH) domains, GEF64C also contains several proline-rich motifs, including a sequence similar to the Enabled EVH1 domain binding site (LPLPP) (Niebuhr et al., 1997) (Fig. 1). RNA in situ analysis on EP3035/ElavGal4 embryos confirms that EP3035 drives overexpression of the GEF64C transcript. In addition, the genetic enhancement of Robo-DCC by EP3035 can be phenocopied by expressing a UAS GEF64C transgene, confirming that the enhancement is due to GEF64C expression (unpublished data). Protein expression analysis in wild-type embryos, using an mAb to GEF64C, reveals broad, low level expression of this GEF, with some enrichment in the CNS (Fig. 1 D). The specificity of the GEF64C mAb is demonstrated by comparing embryos expressing full-length UASGEF64C under control of elavGAL4, with those expressing a COOH-terminal truncation, UASGEF64CΔC, which removes the mAb epitope (Fig. 1 B). Robust CNS expression can be seen in animals with the wild-type transgene, while only the low-levels characteristic of wild-type expression can be seen in animals with the truncated transgene (Fig. 1, E and F).

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