Volume 4, Issue 6, Pages 903-913 (December 1999) Multiple Ephrins Control Cell Organization in C. elegans Using Kinase-Dependent and - Independent Functions of the VAB-1 Eph Receptor  Xiangmin Wang, Peter J. Roy, Sacha J. Holland, Lijia W. Zhang, Joseph G. Culotti, Tony Pawson  Molecular Cell  Volume 4, Issue 6, Pages 903-913 (December 1999) DOI: 10.1016/S1097-2765(00)80220-8

Figure 1 The C. elegans Genome Contains Four Ephrin Genes, efn-1, efn-2, efn-3, and efn-4 (A) The predicted sequences of the four Ce-ephrins are compared with mouse ephrin-B2 (GenBank P52799). Sequences were aligned using the “pileup” program of the GCG software package. Amino acid identities or residues conserved in at least four ephrins are highlighted in black or gray, respectively. The conserved domain (solid underline) and putative N-terminal secretion signal and C-terminal GPI modification signal (dashed underlines) are indicated. (B) Predicted exon (shaded boxes) and intron structure of wild-type and mutant efn-2 genes. The efn-2 gene is located on cosmid C43F9 from the left arm of chromosome IV. Mutation ev584 is a Tc1 transposon insertion into exon 4 (arrow). Mutation ev658 is a deletion of 1835 bp of genomic sequence, as indicated. (C) Predicted exon (shaded boxes) and intron structure of the wild-type and mutant efn-3 genes. The efn-3 gene is located on cosmid F15A2 from the X chromosome. Mutation ev589 is a Tc1 transposon insertion into the 5′ region of the efn-3 gene (arrow). Mutation ev696 is a deletion of 1865 bp of the genomic region. Translation initiation (ATG) and termination (TAA and TAG) codons are indicated. (B) and (C) are drawn to the same scale. Molecular Cell 1999 4, 903-913DOI: (10.1016/S1097-2765(00)80220-8)

Figure 2 Biochemical Analysis of Mutations that Affect the Tyrosine Phosphorylation of the VAB-1 Eph Receptor (A) The VAB-1 protein was immunoprecipitated from wild-type (N2) and vab-1 mutant worms (T61I, E195K, E62K, and G912E are alleles e699, e856, ju8, and e2 of vab-1, respectively) using anti-VAB-1 antibody, then blotted and probed with the same antibody to monitor loading (lower gel), or blotted with the 4G10 monoclonal anti-pTyr antibody to monitor autophosphorylation (upper gel). On Western blots, the VAB-1 protein is detected as a doublet with an approximate size of 125 kDa. Receptor tyrosine phosphorylation is reduced in mutants affecting the ligand binding domain. (B) Tyrosine residues in the juxtamembrane region are required for VAB-1 receptor autophosphorylation. Mammalian Cos-1 cells were transfected with the mammalian expression vector pCDNA3, wild-type vab-1, or a mutant vab-1 (vab-1FF) encoding phenylalanine substitutions for two tyrosines in the juxtamembrane region. The VAB-1 protein was immunoprecipitated then blotted and probed with the anti-VAB-1 antibody to monitor loading (bottom) or with the anti-pTyr antibody to monitor autophosphorylation (top). (C) Effects of ephrin mutations on the autophosphorylation of the VAB-1 receptor in vivo. The VAB-1 protein was immunoprecipitated from wild-type (N2) and mutant worms (indicated above gel) then blotted and probed, as described above, to monitor protein loading and autophosphorylation. (D) Purified EFN-2:Fc, EFN-3:Fc, EFN-4:Fc fusion proteins, Fc alone, or anti-VAB-1 antibody (as indicated above gel) were added to lysates prepared from Cos-1 cells transfected either with vector (vec) or the vab-1 gene (as indicated). The resulting protein complexes were purified using protein A-Sepharose beads, and the VAB-1 protein was detected by blotting with anti-VAB-1 antibody. The position of VAB-1 is indicated to the right. Molecular Cell 1999 4, 903-913DOI: (10.1016/S1097-2765(00)80220-8)

Figure 3 Embryonic Enclosure, Head Morphology, and Male Ray Phenotypes of vab-1 and efn-2 Mutants All animals are him-5(e1490) homozygotes. Control (him-5) (A, D, and G), efn-2(ev584) (B, E, and H), and vab-1(e2027) (C, F, and I) mutants are compared. (A–C) The efn-2 and vab-1 mutant embryos have extruded a large number of cells (arrow) from the ventral side as a result of ventral epidermal enclosure defects. Small arrows in (A) indicate head and tail of this him-5 control embryo. (D–F) efn-2 and vab-1 mutant larvae have obvious body morphology defects compared to control (him-5) animals. These defects are most noticeable in the head (arrows). (G–I) vab-1 mutants have an enlarged male tail bursa (arrow) compared to him-5 and efn-2 male tails. efn-2; efn-1 double mutants (data not shown) have similar male bursa defects. Fusions of rays 8 and 9 are also visible (8+9) in the mutants. Spicules are present (white arrows) and normal (although retracted in the mutant panels). Scale bars: 10, 25, and 10 μm in (A–C), (D–F), and (G–I), respectively. Molecular Cell 1999 4, 903-913DOI: (10.1016/S1097-2765(00)80220-8)

Figure 4 Hyp6 Fusion Defects in Ephrin and EphR Mutants All mutations are in a him-5(e1490) genetic background and carry the jam-1:gfp transgene that expresses in zonula adherens junctions between hypodermal cells. In control animals, the hyp6 cell is a contiguous collar just posterior to the tip of the head. This cell is formed by the fusion of six precursor cells into a syncytium. It often appears attached to the lateral seam (open arrowhead). In the mutant strains listed, the hyp6 precursors frequently fail to complete the fusion process, leaving a contact junction between precursor cells (arrow), which are usually separate from the lateral seam. Molecular Cell 1999 4, 903-913DOI: (10.1016/S1097-2765(00)80220-8)

Figure 5 Misarranged Tail Epidermal Cells in vab-1 and Ephrin Mutants All strains are in the him-5(e1490) background. (A, B, and C) Ventral aspect of phalloidin-stained wild-type, efn-1(e96); efn-2(ev658) double mutant, and vab-1(e2027) single mutant embryos at late stages of epiboly. Hyp8 and hyp9 cells (yellow stars) are aligned along the anteroposterior axis in the wild-type embryo (A), while the same cells have failed to arrange along this axis in the mutant embryos (B and C), even though the mutant embryos are at a later stage of development. (D and E) Oblique lateral view of a control (him-5) (D) and a vab-1(e2027) (E) hermaphrodite tail stained for epidermal cell adherens junctions as described in the Experimental Procedures. In the wild type (D), hyp7, hyp8, and hyp9 align in an anterior to posterior queue along the ventral midline, while hyp11 and hyp10 align along the dorsal midline. In vab-1 hermaphrodites (E), hyp8 and hyp9 frequently cannot be distinguished by position since the two cells align side by side (8/9), adjacent to the ventral midline instead of along it. (F and G) In a more ventral view, hyp8 and hyp9 are seen to align along the ventral midline in the control (F), whereas in vab-1(e2027) (G) the same cells align adjacent to the ventral midline. Similar defects frequently occur in efn-1(e96); efn-2(ev658) double mutant animals (see [B]). Also indicated are the lateral seam (S), anus (A), phasmid socket (Ph), and ventral epidermal P (P) cells. Molecular Cell 1999 4, 903-913DOI: (10.1016/S1097-2765(00)80220-8)

Figure 6 The EFN-2::GFP Expression Pattern Anterior is to the left in all panels. (A–F) are ventral views, and (G–L) are lateral views. At the beginning of epiboly, epidermal cells cover the dorsal part of the embryo and begin to migrate ventralward led by cells at the ventral epidermal margin (four anterior leader cells and 17 pocket cells). (A and B) Early during ventral enclosure, EFN-2::GFP is expressed in a small number of lateral putative neuroblasts underneath and anterior to the posterior epidermal pocket cells. (C–F) As enclosure proceeds and the ventral pocket (opening to the gastrulation cleft) narrows, additional anterior (red arrow) as well as posterior (white arrow) neuroblasts within the pocket express the reporter. The pattern of neuroblast expression reflects the approximate shape of the ring of leading pocket cells that line the pocket opening at this stage. (G and H) In a lateral view of an embryo that has just completed epiboly, the GFP-expressing cells are underneath the surface layer of epidermal pocket cells. Posterior and anterior neuroblasts, probably corresponding to those seen in (C) and (E), are indicated with conserved symbols (white and red arrows, respectively). The anterior part of the EFN-2::GFP-expressing neuroblast ring appears as a line of fluorescing cells (red arrow) just underneath the epidermal surface in the ventral region of the head. At this stage, a large number of more anterior cells in the head, including pharyngeal cells, expresses the reporter. (I and J) Embryos at the 2-fold stage of development continue to express in the head and also express in ventral cord motorneurons (white arrow). (K and L) In late embryonic (data not shown) and early postembryonic development, the reporter expresses in epidermal cells hyp8, hyp9, hyp10, and hyp11 (white arrows) in the tip of the tail. At this stage, hyp7 does not express high levels of EFN-2::GFP, but the hyp7 cell that flanks hyp8 reportedly expresses VAB-1::GFP during epiboly (George et al. 1998). Also indicated is the anal sphincter muscle (red arrow). Expression is prominent in the tail body muscles between the anal sphincter and the tail epidermal cells (unmarked), but not other body wall muscles. Expression persists in the tail and pharynx after hatching (data not shown). Molecular Cell 1999 4, 903-913DOI: (10.1016/S1097-2765(00)80220-8)