Fig. 4. Embryogenesis of efn-4; vab-1 and efn-4 ptp-3 double mutants and vab-1 ptp-3 efn-4 triple mutant. Frames from 4D Nomarski DIC movies of individual embryos are shown; all embryos are shown as ventral views; times are relative to first cleavage and are the same for all genotypes. (A) Typical vab-1(0) efn-4(0) double mutant; open gastrulation cleft at 230-280 minutes is outlined. Nineteen vab-1(0) efn-4(0) double mutant embryos were recorded from strain CZ2251, genotype vab-1(e2027)/mIn1 mIs14; efn-4(bx80). Twelve out of 19 were more severely defective than the typical vab-1 Class I phenotype: the gastrulation cleft was deeper and did not close; cells also appeared loose and disorganized throughout the embryo, and the epidermis failed to enclose. Six out of 16 displayed enlarged gastrulation clefts, underwent epidermal enclosure and ruptured at 1.5-fold stage of elongation; one out of 19 displayed a vab-1 Class III phenotype (normal gastrulation cleft, rupture at twofold stage). (B) vab-1(kinase) efn-4 double mutant, genotype vab-1(e118); efn-4(bx80), showing transiently enlarged gastrulation cleft and elongation stage arrest; this phenotype resembles a combination of the most common efn-4 phenotype and the vab-1 Class III phenotype. Seventeen vab-1(kinase) efn-4 double mutant embryos were recorded. The embryo shown is from strain CZ2252, genotype vab-1(e118)/mIn1 mIs14; efn-4(bx80). Four non-GFP-expressing embryos were recorded, of which one displayed a severe Class I phenotype; three displayed milder gastrulation cleft defects and arrested at the twofold stage. Embryos were also recorded from strain CZ1944, genotype vab-1(e116); efn-4(bx80); juEx350[vab-1(+) efn-4(+)]. Thirteen embryos were recorded that had lost the array (judged from absence of GFP expression). Of the thirteen, five displayed a typical Class I phenotype, six arrested at the 1.5-fold stage (Class II-like), and two arrested later in elongation (Class III). We also recorded four vab-1(ecd) efn-4 double mutants from strain CZ2249, genotype vab-1(e200)/mIn1 mIs14; efn-4(e36). Three out of four displayed a severe Class I phenotype and one displayed a Class IV-like phenotype. (C) ptp-3(op147); efn-4(bx80) double mutant, showing Class I phenotype. Twelve out of 12 ptp-3(op147); efn-4(bx80) embryos recorded showed similar phenotypes. Measured as a percentage of the embryo at the maximum extent of the cleft, the gastrulation clefts in the double mutants extended to 30-50% of embryonic width; 50-100% of embryonic length, and were on average 8 µm deep at their maximum extent. (D) vab-1(dx31) ptp-3(op147); efn-4(bx80) triple mutant, illustrating extreme Class I phenotype and disorganization of embryonic blast cells during gastrulation. All of the 16 such embryos showed the phenotypes illustrated. Gastrulation clefts in these triple mutants extended to 50-75% of embryonic width; 75-90% of embryonic length, and were on average 10 µm deep. Scale bar: 20 µm.

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