Fig. 3. Expression pattern of EFN-4. (A-C) EFN-4 expression during and after epidermal enclosure, detected using the EFN-4-GFP transgene juIs109 or in animals overexpressing EFN-4 from the transgene juEx350. Genotype in A is lin-15(n765); juIs109[EFN-4::GFP, lin-15(+)], stained with anti-GFP (green) and the monoclonal antibody MH27 to illustrate epidermal adherens junctions (red); ventral view. (B,C) EFN-4 overexpressing embryos, stained with anti-EFN-4 (green) and MH27 (red). B is a confocal midline section at about the same stage as A; C shows a lateral view confocal projection at comma stage, after enclosure. Note the broad distribution of EFN-4-positive cells in the head. Genotype for D-I is lin-15(n765);EFN-4-GFP(juIs109);EFN-1(juIs53), stained with anti-GFP, anti-EFN-1 and MH27. (D-F) In early embryo (100-cell stage; ventral views). Both EFN-4 and EFN-1 are broadly expressed. The expression of EFN-4 and EFN-1 appears slightly complementary: some cells expressing high levels of EFN-4 (circled) express lower levels of EFN-4, and vice versa. (G-I) Expression of EFN-1 and EFN-4 during late epidermal enclosure, subventral views. EFN-4 expression (green) partly overlaps (yellow) that of EFN-1 (blue). (J-L) Double staining (midline confocal section) of EFN-4-GFP (green) and VAB-1 (purple) during epidermal enclosure. Circle indicates pharyngeal cell expressing EFN-4-GFP and VAB-1. Genotype is n765; juIs109; juEx445[vab-1(+)], also stained with MH27 antibodies. (M) EFN-4-GFP in L1 stage larva. Double staining with anti-GFP and MH27 antibodies (red). (N,O) EFN-4-GFP expression in late larvae. Note localization of EFN-4-GFP to nerve ring (arrowhead, N) and ventral cord (arrowhead, O). Genotype for M-O is juIs109. Scale bar: 10 µm (A-L); 20 µm (M); 50 µm (N,O).

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