Timeline of excretory system development. (A) Schematics of excretory canal cell (red, ABplpappaap), duct (yellow, ABplpaaaapa), G1 (blue, ABprpaaaapa), G2 (green, ABplapaapa) and W (green, ABprapaapa) at different developmental stages, based on Sulston et al. (Sulston et al., 1983), prior electron microscopy (Stone et al., 2009) and this work. Dark black lines, apical junctions; dotted line, duct auto-fusion; arrow, pore autocellular junction; arrowhead, duct-canal cell intercellular junction; bracket, duct cell body. Not shown are the non-essential excretory gland cells, which also connect to the duct-canal junction (Nelson et al., 1983; Nelson and Riddle, 1984). (B-E) Progressively older ventral enclosure stage embryos. (B-C,E) Ventral views. GFP::MLS-2 marks the presumptive duct and G1 pore nuclei. DLG-1::GFP marks epidermal cell junctions in B and E, which are confocal projections. (B) The presumptive duct and G1 initially lack junctions. (C) The presumptive duct is closer to the canal cell than is the presumptive G1. (D) TEM of a wild-type embryo at a similar stage to C, with cells pseudo-colored as in A. Transverse anterior view. The presumptive duct and G1 have met at the ventral midline. The duct makes extensive contact with the canal cell, while G1 is excluded. No epithelial junctions or lumen are detectable. (E) G1 moves ventrally. The asterisk indicates the site of future G1 pore opening between G2 and W epidermal cells (see also Fig. S1 in the supplementary material). (F-L) Left lateral views. (F) 1.5-fold stage embryo immunostained for DLG-1, showing newly formed autocellular junctions (inset) just before duct auto-fusion. (G-K) L1 larvae. The box in G′ indicates the region magnified in H. AJM-1::GFP marks junctions. The duct no longer has an autocellular junction. (I) lin-48p::mcherry marks the duct. (J) dct-5p::mcherry marks the duct and G1 pore in early L1 and (K) the duct and G1 in late L1 after G1 withdrawal and G2 entry. (L) Adult canal cell marked with vha-1p::GFP. Note that the canal cell elongates extensively.