The PLEXIN PLX-2 and the ephrin EFN-4 have distinct roles in MAB-20/Semaphorin 2A signaling in Caenorhabditis elegans morphogenesis

Abstract

Semaphorins are extracellular proteins that regulate axon guidance and morphogenesis by interacting with a variety of cell surface receptors. Most semaphorins interact with plexin-containing receptor complexes, although some interact with non-plexin receptors. Class 2 semaphorins are secreted molecules that control axon guidance and epidermal morphogenesis in Drosophila and Caenorhabditis elegans. We show that the C. elegans class 2 semaphorin MAB-20 binds the plexin PLX-2. plx-2 mutations enhance the phenotypes of hypomorphic mab-20 alleles but not those of mab-20 null alleles, indicating that plx-2 and mab-20 act in a common pathway. Both mab-20 and plx-2 mutations affect epidermal morphogenesis during embryonic and in postembryonic development. In both contexts, plx-2 null mutant phenotypes are much less severe than mab-20 null phenotypes, indicating that PLX-2 is not essential for MAB-20 signaling. Mutations in the ephrin efn-4 do not synergize with mab-20, indicating that EFN-4 may act in MAB-20 signaling. EFN-4 and PLX-2 are coexpressed in the late embryonic epidermis where they play redundant roles in MAB-20-dependent cell sorting.

Figure 1.—

Structure of the plx-2 gene and PLX-2 protein. (A) Genomic structure of the plx-2 gene, location of deletions, and structure of the translational fusion construct (PLX-2∷EGFP) used in the expression analysis. (B) Structures of wild-type and mutant plexin molecules (ΔMRS-GP, Δsema, Δect) used in this study. The sema domain (sema), MRS, GP. and intracellular (IC) regions are shown below. (C) A phylogenetic tree of C. elegans (PLX-1, PLX-2), Drosophila [Plex A (DmA), Plex B (DmB)], and vertebrate plexins. The entire sequences of plexins were compared using the phylogeny analysis program PROPARS, as implemented in Joe Felsenstein's program PHYLIP 3.6 (http://evolution.genetics.washington.edu/phylip.html). The vertebrate plexin sequences used are from humans except plexin A4 (mouse).

Figure 2.—

C. elegans semaphorin 2A/MAB-20 specifically binds PLX-2. (A–C) PLX-2 bound to Ce-Sema-2a but not to Ce-Sema-1a. HEK293T cells expressing PLX-2 were reacted with Ce-Sema-2a-Fc (B) and Ce-Sema-1a-ΔC-Fc-AP (C). (D–I) PLX-2ΔMRS-GP bound to Ce-Sema-2a. HEK293T cells expressing PLX-2 (G), PLX-2ΔMRS-GP (H), and HEK293T cells transfected with the vector pCAGGS (I) were reacted with Ce-Sema-2a-Fc. (J–S) Both the sema domain and the region containing MRS–GP were required for PLX-1 to bind to Ce-Sema-1a-ΔC-Fc-AP. HEK293T cells expressing PLX-1(O), PLX-1ΔMRS-GP (P), PLX-1Δect (Q), and PLX-1Δsema (R) and HEK293T cells transfected with pCAGGS (S) were reacted with Ce-Sema-1a-ΔC-Fc-AP. The wild-type and mutant plexins were myc tagged, and their expressions on the cell surface were confirmed by reacting intact transfected cells with an anti-myc antibody. HEK293T cells expressing PLX-2 (A and D), PLX-2ΔMRS-GP (E), PLX-1 (J), PLX-1ΔMRS-GP (K), PLX-1Δect (L), and PLX-1Δsema (M) and HEK293T cells transfected with pCAGGS (F and N) were reacted with the anti-myc antibody. (T and U) Expression of full-length and mutant plexins in HEK293T cells. (T) We detected immunoreactive bands of 220 and 192 kDa for myc-PLX-1 (lane 1) and myc-PLX-2 (lane 2), respectively. (U) Expression of PLX-1ΔMRS-GP, PLX-1Δsema, PLX-1Δect, and PLX-2ΔMRS-GP (lanes 1–4). We detected bands corresponding to the predicted sizes of 157, 163, 96, and 133 kDa with anti-myc staining. (V–X) HEK293T cells expressing EFN-4 were stained with an anti-EFN-4 antibody (V), Ce-Sema-2a-Fc (W), and Ce-Sema-1a-ΔC-Fc-AP (X). Neither MAB-20 nor SMP-1 bound to EFN-4 directly.

Figure 3.—

Penetrance of embryonic and larval lethality and adult epidermal phenotypes in plx-2, mab-20, and efn-4 mutant combinations. (A) Penetrance of embryonic lethality in plx-2, mab-20, and efn-4 single mutants (open bars); mab-20; plx-2, plx-2; efn-4, and mab-20; efn-4 double mutants (shaded bars), and the mab-20; plx-2; efn-4 triple mutant (solid bars). Error bars indicate the SEM. plx-2 mutants rarely exhibit embryonic lethality. The plx-2 mutations significantly enhanced the penetrance of the embryonic lethality of the mab-20 weak alleles (wk), bx24 and bx61, and the efn-4 null allele, bx80, but not that of the mab-20 null (0) allele, ev574. bx80 also enhanced the penetrance of embryonic lethality of the mab-20 weak alleles, but suppressed that of the mab-20 null allele slightly. Differences in penetrance were compared using ANOVA and are shown only for the relevant pair being compared. *P < 0.05; **P < 0.001. (B) Penetrance of larval lethality in plx-2, mab-20, and efn-4 mutant combinations. plx-2 mutants do not exhibit larval lethality. plx-2 (tm729) enhances the larval lethality of efn-4(bx80) null mutants, but not that of the mab-20(ev574) null allele. efn-4(bx80) does not significantly enhance the larval lethality of mab-20(ev574). The numbers of animals examined are shown in parentheses below bars.

Figure 4.—

PLX-2 plays a minor role in ventral neuroblast migration. (A) DIC micrographs of C. elegans embryogenesis. Gastrulation commences ∼100 min after the first cell division with ingression of the Ea and Ep gut precursors (arrowheads). At 200 min, a transient cleft is formed on the ventral surface of the embryo (red dashed line), caused by ingression of mesodermal cells. The cleft is closed by short-range lateral movements of VNBs, which generate a substrate for the epidermis during enclosure. plx-2 mutants show a small but significant delay in cleft closure. mab-20 mutants show some disorganization during gastrulation, which leads to an apparent delay in cleft opening (the blue dotted line illustrates D granddaughters remaining on the ventral surface of the embryo) and a significant (P < 0.01) delay in cleft closure. efn-4 mutants show similar but stronger defects, with D granddaughter ingression delayed by ∼25 min. Closure of the cleft is delayed further still, with ∼10% of embryos failing to close the cleft at ventral enclosure, resulting in embryonic rupture and death. mab-20; plx-2 double mutants are similar to efn-4 single mutants, whereas mab-20; plx-2; efn-4 triple mutants show delayed cleft opening, but earlier cleft closure. mab-20; efn-4 double mutants resemble the triple mutant in phenotype (not shown). Variably penetrant morphological defects in the tail are apparent in mab-20 and efn-4 mutant combinations at the fourfold stage (arrows). (B) Gastrulation cleft duration in mab-20, plx-2 and efn-4 strains. Each single mutant shows significantly longer cleft duration compared to wild type (P < 0.05 for plx-2 and <0.01 for mab-20 and efn-4). Double and triple mutants are not significantly different from the strongest single-mutant strain.

Figure 5.—

mab-20 late embryonic epidermal rupture is recapitulated in plx-2; efn-4 double mutants. (A) Late embryonic rupture phenotype (class III arrest) characteristic of mab-20 embryos. mab-20 embryos typically enclose the epidermis at the normal time and then undergo aberrant epidermal elongation in which the posterior ventral epidermis is bulged and deformed (arrow, top) and eventually ruptures in the ventral preanal region ∼4 hr after enclosure (arrow points to extruding cells, bottom). Panels in A are from a 4D movie of mab-20(ev574). (B) Similar late embryonic rupture in the ventral posterior epidermis is seen in plx-2(tm729); efn-4(bx80) double mutants but not in the single mutants. (C) Quantitation of embryonic arrest classes. We quantified embryonic terminal phenotypes in embryos followed at 2-hr intervals using DIC microscopy (n > 50 for each genotype). Embryonic arrest stages were classified as follows: 1, arrest at epidermal enclosure due to failure to enclose epidermis (corresponding to 4D classes I and II); 2, rupture at two- to threefold stage of elongation; 3, rupture at three- to fourfold stage (shown in A and B); 4, development to hatched L1. Type 1 arrest (enclosure) is frequent in efn-4 and rare in plx-2 and mab-20. Type 3 arrest is rare in plx-2 or efn-4 single mutants but is common in efn-4; plx-2 double mutants and is the predominant mode of embryonic arrest in mab-20 single mutants.

Figure 6.—

Expression of PLX-2 and EFN-4 in embryonic neuronal and epidermal cells. PLX-2 and EFN-4 GFP transgene expression was detected by immunostaining with anti-GFP antibodies (green in all panels) ; samples are labeled with the anti-AJM-1 antibody MH27 (red) to visualize epidermal cell outlines. A and B are stained with DAPI (blue) to show cell nuclei. (A) A PLX-2∷GFP translational fusion protein is first expressed in a small number of ventral cells prior to epidermal enclosure; these are identified as ventral neuroblasts on the basis of position. (B) In late stage embryos, PLX-2∷GFP was expressed in several tail epidermal cells, including the preanal ventral epidermal cell pairs P11/12 and P9/10 (indicated as “P” with white lines). PLX-2∷GFP was also expressed in numerous neurons in the head and tail. (C and D) Expression of PLX-2 in the QV5 lateral epidermal cell at the 1.5- and 2-fold stage, detected using the Pplx-2-GFP transcriptional reporter ncIs21; images are projections of surface focal planes from a confocal z-stack. (E–H) EFN-4∷GFP (juIs109) is expressed in a subset of epidermal cells during and after epidermal enclosure. During enclosure (lateral views in E and F; ventral view in G), EFN-4∷GFP was detected in the lateral epidermal cells H0 and QV5, in the leading anterior epidermal cells (green “h”) in the head region, and in the three posterior pairs of P cells (P7/8, 9/10, 11/12) (green P cells marked in AJM-1 channel in F). (H) At the 2-fold stage, EFN-4∷GFP is seen in P9/10 and P11/12 (P, ventral view of preanal epidermis); expression also persists in the head ventral epidermis. EFN-4∷GFP expression in epidermal cells persists until late embryonic stages and was not detectable in L1 larvae. Bar, 10 μm in all panels except B (20 μm).

Figure 7.—

PLX-2 and EFN-4 promote MAB-20 function in male tail-ray development. (A–C) Ventral views of adult male tails. All genotypes contain him-5. Anterior is to the left. (A) A plx-2(nc7) tail has wild-type morphology, and nine rays can be distinguished on each side. (B) In mab-20(bx24), rays 1 and 2 on both sides (arrowheads) and rays 3 and 4 of the left side (an asterisk) are fused. (C) In a mab-20(bx24); plx-2(nc7) tail, rays 1 and 2 (an arrowhead) and rays 3 and 4 (a small asterisk) on the right side and rays 2, 3, 4, and 6 on the left side (a large asterisk) are fused. (D) The epidermal cells, R8.p and R9.p (arrows) express GFP in a third-larval-stage tail of a male carrying the partial PLX-2∷GFP translational fusion. (F) Tail seam cells (SET) express GFP (arrow) in the early fourth-larval-stage male tail. The corresponding DIC images are shown in E and G, respectively. Bar, 10 μm. (H–M) Lateral view of the larval male tail to visualize epidermal cells with AJM-1∷GFP. Anterior is to the left. (H) Wild-type epidermal cells and (I) a schematic to show Rn.p's and ray precursor clusters 1, 2, 3, 4, 6, and 8. (J) In a bx61 larva, ray precursor clusters 2 and 3 (an arrowhead) and 4 and 6 (an arrow) aggregate. In animals with mab-20 and/or efn-4 mutations, in addition to the arrangement defects of ray precursor clusters, the morphology of Rn.p's is often abnormal. (K) R4.p (asterisk) adopted a triangular shape (class II) rather than a wild-type rectangular shape. (L) R1/2.p (asterisk) is larger or irregular compared with wild type (class III). (M) The morphology of all Rn.p's is severely affected (class V). (N) Aggregation of ray precursor clusters 1 and 2, 2 and 3, 3 and 4, and 4 and 6 was scored. plx-2(tm729) enhances the aggregation in weak mab-20 alleles, whereas no aggregation is detected in any ray precursor cluster of plx-2(tm729) mutants (n = 50). (O) Frequencies of Rn.p morphological classes in plx-2, mab-20, and efn-4 mutant combinations. The number of animals examined is shown in parentheses.

Figure 8.—

Model for function of PLX-2 and EFN-4 in MAB-20 signaling. (A) In early embryonic movements of ventral neuroblasts, MAB-20 and EFN-4 may act in a common pathway that could promote adhesion between neighboring neuroblasts or between neuroblasts and unknown substrate cells. (B) In later embryonic development, MAB-20 prevents ectopic cell contacts between epidermal cells; EFN-4 and PLX-2 have redundant functions in this process. EFN-4 may also play an antagonistic role as the frequency of late embryonic arrest is reduced in mab-20 efn-4 double mutants relative to mab-20 single mutants. (C) In postembryonic development of male tail rays, MAB-20 and EFN-4 play nonredundant roles in preventing inappropriate adhesion of ray cells; PLX-2 plays a cryptic role that is revealed only in mab-20 hypomorphic backgrounds.