mDISC1 in C. elegans motor neurons interacts genetically with RAC-PAK signaling pathways to regulate axon guidance. (A) Schematic diagram summarizing the known RAC-dependent and RAC-independent signaling pathways in developing C. elegans motor neurons (13). The RAC-dependent pathway involves unc-73 (a GEF gene), two functional redundant rac genes, ced-10 and mig-2, and two downstream pak genes, pak-1 and max-2. The RAC-independent pathway is simplified by showing a completely RAC-independent gene, max-1. Note that the max-2 gene also has a RAC-independent function. Solid arrows indicate direct activation, and dashed arrows indicate weaker activation. (B) The severity of axon guidance defects in mDISC1 transgenic animals is significantly suppressed by ced-10(dn) or mig-2(dn), whereas expression of wild-type ced-10 or mig-2 enhances the axon guidance defects of mDISC1 transgenic animals. (C) Single mutants of ced-10 or mig-2 suppress the axon guidance defects of mDISC1 transgenic animals. (D) Axon guidance defects in mDISC1 transgenic animals are greatly enhanced by loss of genes that can transduce signals independent of the RAC signaling pathway. (E) Double mutants of pak-1;ced-10 suppress the defects more significantly than either pak-1 or ced-10 single mutants. They also suppress the defects more significantly than pak-1;mig-2 double mutants, consistent with the preferential uses of the rac-pak genes in the motor axons. In all diagrams, bars represent the SE (n = 32–62). *P < 0.05, **P < 0.001 (Student's t test).

mDISC1 transgenic animals exhibit a similar ectopic branching phenotype seen in the transgenic animals expressing rac(gf) genes. (A) A confocal image of the representative wild-type DD motor neuron at L1 stage. The DD motor neuron first extends its short axon process along the ventral cord, from which the commissure branches out and migrates circumferentially to the dorsal side of the body. When the commissure hits the dorsal cord, it branches again and extends both anteriorly and posteriorly along the dorsal cord. The two branch points are indicated by arrows. (B) In max-1 mutant animals, although the commissural axons often fail to reach the dorsal cord, these axons exhibit two branch points as seen in the wild type (arrows). (C and D) The misguided DD axons in either mDISC1 (C) or rac(gf) (D) transgenic worms exhibit significantly more branch points (arrows). All images are taken from a single DD neuron at L1 stage. (Scale bar: 5 μm.) (E) Quantification of the branch points for each genetic background. The branch point is defined as a point with an additional neurite extending more than 1 μm. Bars represent the SE (n = 21). **P < 0.001 (Student's t test). In all pictures, anterior is to the left and dorsal is up.

Expression of mDISC1 in C. elegans motor neurons causes axon guidance defects. (A–D) Confocal images of the commissural axons from DD and VD motor neurons at L4. In wild-type animals (A and B), all of the commissures reach the dorsal nerve cord. In mDISC1 transgenic animals (C and D), the commissures exhibit guidance defects (arrowheads). Boxed areas in A and C are magnified in B and D, respectively. (Scale bars: A and C, 20 μm; B and D, 10 μm.) (E) The adult mDISC1 animals have an average of 40% axon guidance defect. The L1 transgenic animals exhibit similar percentage of defects, indicating that the phenotype is caused by a defect in the guidance of developing axons rather than to abnormal regrowth from degenerated axons. (F) The axon guidance defects caused by expressing mDISC1 are suppressed by RNAi knockdown of C. elegans homologs of known mammalian DISC1-interacting molecules (n = 25–60). col-86, negative control gene. (G) Incubation of the mDISC1 animals with a PDE4-specific inhibitor, rolipram, suppresses the guidance defects (n = 60). Bars represent the SE. *P < 0.05, **P < 0.001 (Student's t test). In all pictures, anterior is to the left and dorsal is up.

In mammalian systems, DISC1 binds to TRIO and activates RAC1 signaling. (A) Schematic diagram summarizing mDISC1 constructs used to generate His-tagged recombinants. mDISC1 contains an N-terminal globular head domain and multiple coiled-coil domains in the C terminus. The numbers indicate the beginning and ending amino acids for each construct. (B) Schematic diagram summarizing hTRIO constructs used to generate GST-tagged recombinants. hTRIO consists of a Sec14 homolog domain, a spectrin repeats (SP) domain, two GEF domains (GEF1 and GEF2), and a kinase domain. (C) Axon guidance defects in mDISC1 transgenic worms are significantly suppressed by rh40, a hypomorphic mutant allele of unc-73/trio (n = 50–69). (D) mDISC1 and hTRIO are coimmunoprecipitated as a protein complex when expressed in COS cells. (E) The SPn of hTRIO, but not the SPc or the GEF2 domain, binds to its GEF1 as shown in the GST pull-down assays. The loading inputs are 5% of the total purified His-tagged proteins. (F) hTRIO-SPn specifically inhibits the binding of human RAC1 (hRAC1) to hTRIO-GEF1 domain in the GST pull-down assays. The ratios of hRAC1 pulled down by hTRIO-GEF1 in the presence of hTRIO-SPn or hTRIO-SPc were quantified and are shown in the histogram (n = 3). The ratio is indicated as fold increase relative to that without adding hTRIO-SP proteins. (G) hTRIO pulls down more hRAC1 in the presence of mDISC1 full-length (mDISC1-FL) but not a control mDISC1 fragment (mDISC1-MD) that does not bind to hTRIO. The ratios of hRAC1 pull-down were quantified as in F (n = 3). (H) The RAC1 activities in COS cells are significantly increased when hTRIO is coexpressed with mDISC1. The activity of RAC1 was assayed by the amount of GTP-RAC1 pulled-down by GST-PAK1 70–117 (45). (I) Expression of unc-73b significantly enhances the motor axon guidance defects in mDISC1 transgenic animals (n = 54–60). In all diagrams, bars represent the SE. *P < 0.05, **P < 0.001 (Student's t test).