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doi: 10.1242/10.1242/dev.00122

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The divergent C. elegans ephrin EFN-4 functions inembryonic morphogenesis in a pathway independent of the VAB-1 Eph receptor

Ian D. Chin-Sang*,{dagger}, Sarah L. Moseley{dagger}, Mei Ding, Robert J. Harrington, Sean E. George and Andrew D. Chisholm{ddagger}

Department of Molecular, Cell, and Developmental Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA
* Present address: Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada



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  		Fig. 5. Quantitation of embryonic (A) and larval (B) lethality in vab-1 efn double mutants. vab-1; efn double mutants were constructed and lethality quantitated as described in the Materials and Methods. All vab-1; efn-4 double mutants (black bars) showed significantly enhanced lethality compared with the relevant single mutants (white bars). vab-1(0); efn-4 double mutants were fully lethal and maintained as balanced strains of genotype vab-1/mIn1 mIs14; efn-4. vab-1(e2); efn-4 and vab-1(e699); efn-4 double mutants were >96% lethal, with rare escapers; among these escapers, 30-50% were fertile with average brood sizes of 24 and 51, respectively. Data for vab-1; efn-1 double mutants, included for comparison, are taken from Chin-Sang et al. (Chin-Sang et al., 1998). efn-2 mutations significantly increase the embryonic lethality and significantly decrease the larval lethality of vab-1(0) mutants. efn-2 mutations also significantly enhanced the embryonic and larval lethal phenotypes of vab-1(kinase) mutations, a result that contrasts with the lack of enhancement of vab-1(kinase) male tail phenotypes by efn-2 (Wang et al., 1999Go). vab-1; efn-3 double mutants were not significantly different from vab-1 controls. Error bars indicate the s.e.m. Differences in penetrance between vab-1 and vab-1; efn strains were compared using ANOVA; *, P<0.05; **, P<0.01.

 


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  		Fig. 1. Structure of the efn-4 locus and sequence comparisons. (A) Genetic and physical maps of the efn-4 region. mab-26/efn-4 is located at the left end of linkage group IV. (B) Alignment of the conserved domain of EFN-4 (residues 25-170) with those of other ephrins, showing point mutations; locations of predicted secondary structure elements are based on structures of ephrin B2 (Toth et al., 2001Go; Himanen et al., 2001Go). The ephrin with highest similarity to EFN-4 within the conserved domain is EFN-1 (31% identity, 46% similarity). The EFN-4 conserved domain is approximately equally similar in sequence to the ephrin A and ephrin B subclasses of vertebrate ephrins (25-20% identity; sequences shown are human ephrin A1 and ephrin B2). Alignments were made using ClustalW; identities are in black and similarities in gray. Secondary structure elements are aligned according to Himanen et al. (Himanen et al., 2001Go). The strong allele ju134 affects a non-conserved threonine residue; the weaker allele e660 affects a conserved proline; and the weakest allele, e1746, affects a non-conserved alanine residue. When aligned according to Toth et al. (Toth et al., 2001Go) ju134 affects an exposed residue of ß-strand D, which may play a role in ephrin dimerization; e660 affects a residue at the end of ß-strand C in the tetramerization interface; and e1746 affects a buried residue between ß-strand D and {alpha}-helix E. (C) Phylogenetic tree of C. elegans, Drosophila and vertebrate ephrin sequences. Phylogenetic analysis suggests that the C. elegans ephrins diverged after the last common ancestor of vertebrates and nematodes; among the C. elegans ephrins, EFN-4 is the most rapidly evolving. Vertebrate ephrin sequences used are from humans except for ephrin A6 (chick). This tree was generated using a neighbor-joining algorithm (MEGA); bootstrap values are indicated at branches.

 


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  		Fig. 2. efn-4 embryonic and larval phenotypes. 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. Scale bar: 20 µm in A-C; 50 µm in D-G. (A) Embryogenesis of wild type (N2), ventral views. Note complete closure of the ventral gastrulation cleft (outlined) by 280 minutes. (B,C) Frames from movies of two representative efn-4(bx80) embryos, showing enlarged ventral gastrulation clefts. A total of 58 efn-4(bx80) and 36 efn-4(e36) embryos were recorded for 4D analysis; the range and penetrance of phenotypes was similar in both mutants and the data are considered together below. Some phenotypes of efn-4 embryos could be classified using the same phenotypic classes as used to classify vab-1 and efn-1 phenotypes (George et al., 1998Go; Chin-Sang et al., 1999Go). Twenty percent (19/94) of efn-4 animals displayed the Class I phenotype shown in B, in which the gastrulation cleft was enlarged and persistent (dotted outline, 230-280 minutes), the epidermis failed to enclose and internal cells ruptured during early enclosure (330 minutes); this phenotype is seen in 19% of vab-1(0) animals and 12% of efn-1 animals. Three out of 94 animals exhibited a slightly weaker phenotype resembling the vab-1 Class II phenotype (enlarged gastrulation cleft; epidermis encloses, embryo turns then ruptures). One out of 94 efn-4 embryos displayed normal gastrulation clefts and arrested during elongation (vab-1 Class III phenotype), compared with 38% of vab-1 or 16% efn-1 embryos. Sixty-one out of 94 efn-4 embryos displayed the phenotype shown in C, in which the gastrulation cleft was transiently enlarged (230-280 minutes) and later closed; epidermal enclosure and elongation occurred with normal timing, and the embryo hatched with deformations in the posterior epidermis. Ten out of 94 embryos displayed wild-type development. (D-G) L1 larvae, showing wild type (D), efn-4(e36) mutant tail morphology (arrow, E,F) and efn-1(ju1) Notch head morphology (arrow, G).

 


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  		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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  		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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  		Fig. 6. Gastrulation cleft phenotypes of mab-20/Semaphorin-2A mutants and mab-20 efn-4 double mutants. 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 except where indicated. (A,B) Embryonic morphogenesis of representative mab-20(ev574) embryos. Between 180 and 280 minutes post first cleavage, most (28/38) mab-20(ev574) embryos displayed a disorganized gastrulation cleft that was deeper and more persistent than in the wild type (A, outlined 230-280 minutes). Of these, about half (13/28) displayed gastrulation clefts that persisted until ventral enclosure; leading epidermal cells failed to enclose and the embryos ruptured at the ventral midline, similar to efn-4 mutants (compare series A 330 min with Fig. 2 series C). Sixteen percent (6/38) of animals with disorganized gastrulation clefts completed epidermal enclosure and later ruptured at the two- or threefold stage. Eight percent (3/38) closed the gastrulation cleft at the onset of epidermal enclosure, and arrested at the twofold stage of elongation (B, 500 minutes), a phenotype not observed in efn-4 mutants. Twenty-three percent (9/38) of embryos displayed an enlarged gastrulation cleft yet underwent epidermal enclosure and hatched. (C,D) Morphogenesis of mab-20(e819); efn-4(bx80) double mutant embryos. Forty-five percent (16/35) of embryos displayed aberrant gastrulation clefts and either ruptured during ventral enclosure (series C, 430 minutes), arrested during epidermal elongation, or hatched (series D). Cellular disorganization was evident at the onset of gastrulation cleft formation (D, 180 minutes). The terminal phenotypes resembled the most severe efn-4 or mab-20 single mutant phenotypes (C, 430 minutes) although the gastrulation cleft was often less open than in efn-4 (e.g. C, 230-280 minutes). Most (26/35) double mutant embryos hatched, of which one quarter (9/35) displayed gastrulation cleft defects. Typically, the gastrulation cleft remained partly open in the anterior at the onset of epidermal enclosure (outlined in D, 280 minutes) and epidermal enclosure occurred normally (D, 330 minutes). Scale bar: 20 µm.

 


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  		Fig. 7. Quantitation of genetic interactions between efn-4 and mab-20. Penetrance of embryonic lethality (A) and larval lethality (B) in mab-20 (white bars) and in mab-20 efn-4 double mutants (black bars). Error bars indicate the s.e.m. Differences in penetrance between mab-20 and mab-20; efn-4 strains were compared using ANOVA; *, P<0.05; **, P<0.01. mab-20 strains exhibited 20-30% embryonic lethality depending on the allele; in all mab-20; efn-4 strains, the penetrance of embryonic lethality was not significantly different compared with the mab-20 single mutant. Most mab-20; efn-4 double mutant strains displayed significantly increased larval lethality, consistent with additivity of mutant phenotypes.

 

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