Figure 6. SAX-7 interacts with RIG-6.

(A) Distribution of a mCherry-tagged presynaptic marker RAB-3 in HOA axon of wild type or sax-7(nj48) mutants. Arrowheads indicate gaps between the presynaptic puncta. (B) Number of mCherry::RAB-3 puncta in sax-7 mutants was counted and compared to wild type (n = 30). Error bars are SEM. (C) Images of axon placement of HOA, AVG and PHC in sax-7 and rig-6;sax-7 double mutants. Axon fasciculation between each neuronal pair in the dashed box region and schematic of axon fasciculation are shown on the right. Arrowheads indicate the region where two axons are detached from each other. (D) Percentage of axon-axon contact between each neuronal pair in wild type or mutant animals (n = 40). Each dot represents individual animal. Red bar represents the median. (E) Percentage of axon-axon contact between each neuronal pair in animals expressing the short isoform sax-7s or long isoform sax-7l cDNA in HOA or PHC in sax-7 mutant background (n = 30). In (C–E), the data for wild type and rig-6 mutants are the same as shown in Figure 2. (F) Co-immunoprecipitation of RIG-6 and SAX-7S. (G) Images of axon placement of HOA and AVG in PHC-ablated animals expressing sax-7s cDNA in HOA (Ex[HOAp::sax-7s]). Arrowheads indicate the region where the HOA axon is detached from the AVG axon. (H) Percentage of axon-axon contact between HOA and AVG in PHC-ablated animals either expressing or not expressing sax-7s cDNA in HOA. The number of animals analyzed is indicated. The data for PHC-ablated wild type animals are the same as shown in Figure 2I. Scale bars, 20 μm. *p<0.05; **p<0.01; ***p<0.001; ns, not significant (by Mann-Whitney test). For the data and statistics, see Figure 6—source data 1.

Figure 6—figure supplement 1. sax-7 is expressed in PHC, but not in HOA or AVG.

A fosmid-based reporter line carrying sax-7::gfp was crossed with animals carrying eat-4^fosmid::mCherry or unc-47^fosmid::mCherry and examined for expression in PHC, HOA or AVG in L4 or adult males. Note that SAX-7 is localized to the cytoplasm and plasma membrane in neuronal cell bodies, while mCherry-tagged proteins are targeted to the nucleus due to the nuclear localization signal of H2B. Location of PHC, HOA or AVG cell body is indicated as an arrow. Identities of eat-4 or unc-47 expressing cells in males are previously described (Gendrel et al., 2016; Serrano-Saiz et al., 2017b). SAX-7 was expressed in PHC both in L4 and adult males, but in the L4 stage it was easier to detect the signal than in the adult stage where SAX-7 expression in male-specific ray neurons near PHC was more intense. The image for unc-47^fosmid::mCherry was overexposed to show AVG expression. Scale bars, 10 μm.