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. Author manuscript; available in PMC: 2014 Mar 4.
Published in final edited form as: Neuron. 2013 Sep 4;79(5):903–916. doi: 10.1016/j.neuron.2013.06.035

Figure 2. EBAX-1 Regulates the Ventral Axon Guidance of AVM and PVM Neurons.

Figure 2

(A) Illustration of the genomic structure of ebax-1. In the ebax-1(ju699) allele, a region of 1548 bp covering the SWIM domain and the domain A is deleted. In the ebax-1(tm2321) allele, a region of 551 bp in exon 5 is deleted.

(B) ebax-1 mutants show mild egg-laying (Egl) defects. The Egl phenotype can be fully rescued by ebax-1 genomic DNA drive by a pan-neuronal promoter (Prgef-1::EBAX-1), suggesting EBAX-1 functions in neurons that control the egg-laying behavior. Data are shown as means (SD); statistics, t-test. **p<0.01 and ##p<0.01 relative to WT.

(C) Morphological defects of HSN axons labeled by Punc-86::myr::GFP(kyIs262) in WT and ebax-1(ju999) animals. Starting from the L2 stage, the HSN axon migrates ventrally, and then extends along the ventral nerve cord. At the L4 stage, the axon forms synapses with the VC4 motor neuron and vulva muscles (c1). A significant proportion of ebax-1(ju699) animals showed various HSN guidance defects, including parallel or directionless axon growth (c3-c4), or axons reaching the ventral cord but missing the vulva midline (marked by asterisk in c2). Scale, 5 μm.

(D) Genetic interactions between ebax-1 and the unc-6/unc-40 and slt-1/sax-3 pathways in HSN guidance. The ebax-1 mutation enhanced the guidance defects in both unc-6(ev400) and sax-3(ky123) mutant backgrounds. Note that in the unc-6 mutants, HSN neurons already showed highly penetrant guidance defects; loss of ebax-1 increased the penetrance of defects to 100%. Data represent means ± SEM. *p<0.05 and ***p<0.001 relative to indicated controls; t-test.

(E) The effects of EBAX-1 on HSN guidance defects at 25°C. The guidance errors increased in wild-type animals at 25°C. The guidance defects in WT was further increased by the ebax-1(ju699) mutation but suppressed by overexpression of EBAX-1. *p<0.05 and **p<0.01 relative to WT.

(F) Schematic illustration of guidance signals for the ventral growth of AVM, PVM and HSN axons. The repellent guidance cue SLT-1/Slit is secreted by dorsal muscles, and the attractive guidance cue UNC-6/Netrin is secreted by the ventral nerve cord. Growth cones expressing SAX-3/Robo and UNC-40/DCC receptors are guided to grow ventrally.

(G) Representative images of AVM neurons in WT, ebax-1(ju699), unc-6(ev400), and ebax-1(ju699); unc-6(ev400) mutants. ALMR is another touch neuron adjacent to AVM. Scale, 20 μm.

(H-I) Quantitative analysis of AVM (H) and PVM (I) guidance defects in various genetic backgrounds. EVA-1 is a co-receptor of SAX-3 (Fujisawa et al., 2007). Data represent means ± SEM. *p<0.05, **p<0.01 and ***p<0.001 relative to indicated controls; t-test.

(J) Rescue and mosaic analysis of Pebax-1::EBAX-1 in unc-6; ebax-1 and unc-40; ebax-1 mutants. The numbers of scored animals are shown in the bar graph. Data represent means ± SEM. ##p<0.01 and ***p<0.001 relative to indicated controls; one-way ANOVA.