gex mutant embryos fail to undergo morphogenesis but do differentiate specific tissues. (Top panels) Nomarski images. All other panels are fluorescent images using monoclonal antibodies to pharynx (3NB12 and 9.2.1) and intestine (J126). Anterior is left, and dorsal is up. Wild-type N2 embryos at this stage have the internal organs of the pharynx and intestine fully enclosed by the hypodermis. Black-tipped arrows point to the anterior of the pharynx, and white-tipped arrows point to the anterior of the intestine. Antibodies 9.2.1 and J126 illustrate that the pharynx and intestine form elongated tubular structures. In gex-2 and gex-3 embryos the hypodermis never encloses the embryo and instead constricts on the dorsal side. Embryos end embryogenesis with internal organs that lie on the outside and do not form the correct elongated tubular structures.

gex-3(zu196) zygotic phenotypes. Anterior is left, and dorsal is up. (Top panels) The DTC defect: In gex-3(zu196) embryos the DTCs (distal tip cells) often show migration defects including incorrect dorsal/ventral polarity. The black arrows below the photographs trace the migration of the anterior DTC. One or both DTCs can show defects in gex-3(zu196) animals. (Bottom panels) The vulval phenotype: gex-3(zu196) larvae form what appear to be wild-type mid-L4 vulvae, yet 100% of adult worms become Egl (Egg laying defective). (Middle panels) gex-3(zu196) mid-L4 vulvae have the Christmas tree structure and flattened-out thin laminar process (black arrowhead) separating the vulva and uterus, just as in wild-type, suggesting normal cell differentiation and cell fusions. Whereas the wild-type N2 adult worm contains one row of embryos, all at early stages, the adult gex-3 worm is beginning to fill up with unlaid embryos, many at late stages of development. The white arrowheads show the position of the vulva.

Direct interaction between GEX-2 and GEX-3. (A) Two-hybrid scheme including deletion constructs. The GEX-2 deletion series fused to the GAL4 activator domain and full-length GEX-3 fused to the GAL4 DNA-binding domain were tested in the two-hybrid assay. Only the combination of full-length GEX-2 and GEX-3 shows a direct interaction. Reciprocally, the GEX-3 deletion series fused to the GAL4 DNA-binding domain and full-length GEX-2 fused to the GAL4 activator domain were tested for interaction in the two-hybrid assay. Full-length and GEX-3 slightly deleted at the C terminus could bind to full-length GEX-2 in the two-hybrid assay. (B) Immunoprecipitation from yeast. Yeast lysates expressing HA–GEX-2 and myc–GEX-3 were prepared as described in Materials and Methods. Left lysate panels shows the expression of HA–GEX-2 and myc–GEX-3 recombinant proteins in yeast. These lysates were immunoprecipitated with anti-myc antibody (upper panels) and anti-HA antibody (lower panels). Coprecipitation was observed in the precipitate of the lysate prepared from yeast expressing both HA–GEX-2 and myc–GEX-3 as detected by anti-GEX-2 and anti-GEX-3 antibodies. (IB) Immunoblot; (IP) immunoprecipitate. (C) Immunoprecipitation from C. elegans. C. elegans lysates were immunoprecipitated with antibodies to GEX-3 and probed with anti-GEX-2 and anti-GEX-3 rabbit polyclonal antibodies. The GEX-3 immunoprecipitation includes a protein that can be detected by the GEX-2 antibody.

(A) GEX-2 is the C. elegans Sra-1 homolog. The C. elegans Sra-1 homolog GEX-2 maps to cosmids F56A11 and C18H7 on the left arm of Chromosome IV, and the F56A11.1 gene product predicted by Gene Finder is the C. elegans homolog of mammalian Sra-1. The GEX-2 cDNA is composed of 10 exons. Amino acids of GEX-2 (Ce, Caenorhabditis elegans) and Sra-1 (Hs, Homo sapiens) are compared. Identical amino acids are shown boxed. (*) The approximate genetic position of gex-2 is indicated. (B) Cloning gex-3 indicates it is the HEM2/NCKAP1/KETTE homolog. gex-3(zu196) maps to the right arm of Chromosome IV at ∼+5.7 map units. gex-3 is rescued by YAC Y39H4 and by a PCR product consisting of the GEX-3 genomic region plus 1 kb of upstream and downstream DNA. C. elegans (Ce) GEX-3/HEM2 is aligned with Drosophila (Dm) HEM2/KETTE and human (Hs) NCKAP1 proteins. Identical amino acids are shown boxed. The zu196 allele contains a Tc1 insertion in exon 10 at amino acid 982, between the A and T, which causes a frameshift at the C terminus of the protein. An arrowhead marks the insertion spot.

Time course to illustrate the hypodermal Gex defect using antibody MH27 (a,b,k,l) and AJM-1::GFP (c–j). gex-2(RNAi) embryos are shown. Similar results were obtained with gex-3(zu196) and gex-3(RNAi) embryos. (a–f,h,j) Dorsal views; (g,i,k,l) lateral views, because wild-type embryos undergo a rotation as they finish morphogenesis. (a,b) The hypodermal cell junction marker AJM-1 (shown with antibody MH27) is present. For the AJM-1::GFP series, the wild-type embryos are shown at 20-min intervals, and the gex embryos are shown at 60-min intervals. (c,d) By 280 min after first cleavage, the gex embryo shows hypodermal abnormalities, having failed to initiate dorsal intercalation and ventral migrations. Comparing i with f, both at 340 min, shows that in gex embryos the hypodermis fails to become organized. In gex embryos the hypodermis fails to form rows (as is apparent in c,e,g,i), the cells do not change shape or intercalate (as the white arrowheads indicate in e), and the lateral cells do not migrate ventrally (as the white-tailed arrow shows in e,g). Instead, the disorganized cells begin to constrict on the dorsal side (f,h,j; e.g., follow the two gray arrows in h and j and the two white arrows in d–j). (Panel l) An embryo at a similar stage as the terminal embryos shown in Figure 2. l (MH27 antibody) shows the hypodermis constricted on the dorsal side (up; white arrow) and the internal organs exposed on the ventral side (down).

Antibodies to GEX-2 and GEX-3 show localization to the boundaries of all cells starting at the two-cell stage. GEX-2 has a distinct localization pattern compared with AJM-1. Wild-type embryos are shown with dorsal up and anterior to the left. (a,b) Antibodies to GEX-3 on embryos at the 8-cell (a) and at the early comma stage (b) show GEX-3 expression in the hypodermis as well as at the boundaries of other cells. (c–f) Antibodies to GEX-2 on embryos at the 32-cell (c), 1.5-fold stage (d), and at the late comma stage (e,f) show GEX-2 is found at cell boundaries in the hypodermis, which has enclosed the embryo and is beginning to constrict to squeeze the embryo into a worm (d,e). (g,h) Antibodies to AJM-1 reveal that GEX-2 does not colocalize with the AJM-1 junctional domain. (i,j) Merged image. The focal plane in e–f is though the center of the embryo. f, h, and j are close-ups of a dorsal section of the images in e, g, and i.

ced-10(n3417) mutant embryos show morphogenesis defects similar to Gex defects. Black-tipped arrows point to the anterior of the pharynx, and white-tipped arrows point to the anterior of the intestine in a, b, e, and f. The embryos in a–d are at an earlier stage and are shown from a different angle from that of the embryos in e and f. (a,c) gex-2(RNAi) embryo (a) shown from the ventral side shows unenclosed pharynx and intestine and (c) shown from the dorsal side shows disorganized epidermis. (b,d) A ced-10(n3417) embryo similarly shows (b) unenclosed pharynx and intestine on the ventral side, but the dorsal view (d) reveals some organization of the dorsal epidermis, including dorsal intercalation (white-tipped arrow). (e) A gex-3(zu196) embryo shown laterally reveals unenclosed pharynx and intestine on the ventral (bottom of the image) side. (f) A ced-10(n3417) embryo, also shown laterally, at a later stage than the embryo in b and d has ended embryogenesis with a terminal phenotype resembling gex-2 and gex-3 (also compare with the embryos in Fig. 2b,c). The embryo in a shows several spherical refractile cells (to the left of the black arrows) that resemble persistent corpses found in ced-10 mutants, whereas similar cells in the other panels are out of the plane of focus.