The overall intensity of EBAX-1::GFP peaked at the 3-fold stage, especially around the nerve

ring region ( Figure 1F, right panel) and dropped after hatching. In the fourth larval stage (L4), EBAX-1::GFP was detected in the nerve ring, the ventral nerve cord, the HSN motor neuron, and some neurons in the tail ( Figure S2A). Mos1 transposase-mediated single copy insertion (MosSCI) of Pebax-1::EBAX-1::GFP showed similar expression dynamics, albeit at a lower expression level (data not shown). EBAX-1::GFP showed a punctate pattern in the cytosol of individual neurons ( Figure S2A). A similar punctate pattern was also BI2536 observed in the soma and axons when EBAX-1::GFP was specifically expressed in mechanosensory neurons and GABAergic motor neurons (data not shown). Likewise, GFP-tagged mouse ZSWIM8 displayed a cytosolic expression pattern in cultured heterologous cells ( Figure S2B). To decipher the roles of ebax-1 in the developing selleck screening library nervous system,

we first examined the morphology of HSN motor neurons that control the egg-laying behavior, because ebax-1 mutants exhibit modest egg-laying defects that can be rescued by neuronal expression of EBAX-1 ( Figure 2B). We found that 30% of the ebax-1(ju699) mutants showed HSN axon guidance defects at 20°C ( Figures 2C and 2D). A moderate temperature rise to 25°C increased the guidance errors in wild-type and mutant animals, whereas overexpression of EBAX-1 in the wild-type background significantly improved HSN guidance accuracy ( Figure 2E). These observations suggest that the accuracy of HSN axon guidance is temperature dependent and sensitive to the level of EBAX-1. A similar temperature dependency of axon guidance defects was also observed in AVM neurons of ebax-1 mutants ( Figure S2E). Through extensive analyses of genetic interactions, we identified

a specific role Calpain for ebax-1 in the ventral axon guidance of both HSN motor neurons and AVM and PVM mechanosensory neurons (the latter two also called touch neurons). Ventral guidance of HSN and AVM/PVM axons is in response to a combination of attractive Netrin/DCC (UNC-6/UNC-40) and repellent Slit/Robo (SLT-1/SAX-3) signals ( Figure 2F) ( Desai et al., 1988, Hao et al., 2001 and Zallen et al., 1998). Mutations disrupting either pathway partially disrupt ventral guidance, whereas simultaneous loss of both pathways causes fully penetrant ventral guidance defects ( Figures 2D, 2H, and 2I; Figures S2C and S2D). In AVM neurons, ebax-1 mutants alone did not show any guidance defects at 20°C but significantly enhanced guidance defects in unc-6(ev400) or unc-40(e1430) mutants ( Figures 2H and S2C). In contrast, ebax-1 mutations did not enhance AVM axon guidance defects in the slt-1 or sax-3 mutant backgrounds ( Figures 2H and S2C). In PVM and HSN, ebax-1 showed synergistic effects with both slt-1/sax-3 and unc-6/unc-40 pathways ( Figures 2D and 2I; Figure S2D).