Semaphorin-1 and netrin signal in parallel and permissively to position the male ray 1 sensillum in Caenorhabditis elegans

Abstract

Netrin and semaphorin axon guidance cues have been found to function in the genesis of several mammalian organs; however, little is known about the underlying molecular mechanisms involved. A genetic approach could help to reveal the underpinnings of these mechanisms. The most anterior ray sensillum (ray 1) in the Caenorhabditis elegans male tail is frequently displaced anterior to its normal position in smp-1/semaphorin-1a and plexin-1/plx-1 mutants. Here we report that UNC-6/netrin and its UNC-40/DCC receptor signal in parallel to SMP-1/semaphorin-1a and its PLX-1/plexin-1 receptor to prevent the anterior displacement of ray 1 and that UNC-6 plus SMP-1 signaling can account entirely for this function. We also report that mab-20/semaphorin-2a mutations, which prevent the separation of neighboring rays and cause ray fusions, suppress the anterior displacements of ray 1 caused by deficiencies in SMP-1 and UNC-6 signaling and this is independent of the ray fusion phenotype, whereas overexpression of UNC-40 and PLX-1 cause ray fusions. This suggests that for ray 1 positioning, a balance is struck between a tendency of SMP-1 and UNC-6 signaling to prevent ray 1 from moving away from ray 2 and a tendency of MAB-20/semaphorin-2a signaling to separate all rays from each other. Additional evidence suggests this balance involves the relative adhesion of the ray 1 structural cell to neighboring SET and hyp 7 hypodermal cells. This finding raises the possibility that changes in ray 1 positioning depend on passive movements caused by attachment to the elongating SET cell in opposition to the morphologically more stable hyp 7 cell. Several lines of evidence indicate that SMP-1 and UNC-6 function permissively in the context of ray 1 positioning.

Figure 1

Mutants of semaphorin-1a and UNC-6 mediated signaling components share the same ray 1 anterior displacement phenotype. In all panels, anterior is to the left, dorsal is up. (A and B) Ray 1 and 2 process clusters are indicated by black and white arrowheads, respectively, for adult wild-type (A) and unc-40(e1430) (B) mutant males. Note the increased distance between ray 1 and the next adjacent ray (ray 2) in unc-40 mutants. (C) Schematics of stages 1–3 of ray development (stage 4 is shown in Figure 6) (adapted from Emmons 2005). The eff-1 promoter drives expression in Rn.p cells (n = 1–9) (yellow cells with a red outline) at the onset of Rn.p cell fusion (stages 2–4) (Mohler et al. 2002). The lin-32 promoter drives expression in all ray neurons (white cells with blue outline) and in the ray structural cells (green cells) during stages 1 and 2, and diminishes in later stages (Portman and Emmons 2000). The ram-5 promoter drives expression in all ray structural cells starting in stage 1 and onward (Yu et al. 2000). The schematized cells are sandwiched between dorsal and ventral portions of hyp 7 (white background). The body seam is shown in part (blue cell). (D–F) Epifluorescence microscopy images of male ray development in animals carrying the ajm-1::GFP gene reporter. Arrows indicate the seam–SET junction. For each strain, stage 1 (S1), stage 2 (S2), and stage 3 (S3) of male development are shown. No significant differences can be found between wild-type and mutant strains at stage 1 (D–G). However, plx-1(ev724) (E, S2) and unc-6(ev400) (F, S2) single mutants have their ray 1 cell groups displaced anterior to normal at late stage 2 as compared to wild type (D, S2). unc-40(e1430); plx-1(ev724) double mutants show a more severe anterior ray 1 cell group displacement at stages 2 and 3 (G, S2 and S3) and as compared to wild type (D, S2 and S3), plx-1(ev724) (E, S2 and S3), and unc-6(ev400) (F, S2 and S3) single mutants. In this case, the ray 1 cell group either contacts the seam–SET boundary (G, S2) or localizes close to it (G, S3).

Figure 2

UNC-6, UNC-5, and UNC-40 function in one pathway and plexin-1/PLX-1 functions in a parallel pathway to prevent anterior displacement of ray 1. The frequencies and standard errors for severe (solid bar) and mild (cross-hatched bar) anterior ray 1 displacements are shown for a variety of unc-5, unc-6, unc-40, and plx-1 single, double, and triple null mutants, all in a him-5(1490) (control WT) genetic background. Standard errors for percentages of animals manifesting the anterior ray 1 phenotype were calculated assuming a binomial distribution of the same sample size and the observed proportion as mean.

Figure 3

Semaphorin-1a and UNC-6 signaling components are localized to ray cells. All panels show him-5 males (“wild-type” controls) with anterior to the left and dorsal up. Epifluorescence microscopy (A) and laser confocal microscopy (B–D) images show the localization of smp-1p::smp-1::gfp (in a plx-1(ev724) mutant) (A), unc-6::venus (B), and unc-40p::unc-40::gfp (C and D). (A) plx-1(ev724) male carrying extrachromosomal array evEx170 (pVGS1AGFP), which encodes a smp-1p::smp-1::gfp rescuing construct (Dalpe et al. 2004), is shown. The GFP signal is observed at the cell membrane of all ray cell groups (small white arrowheads), all Rn.p cells (large white arrowheads), and in the body seam hypodermis (black arrowhead). (B) L4 stage males carrying an extrachromosomal array of a genomic unc-6::venus (Asakura et al. 2007) rescuing construct show localization on all ray structural cell processes and their tips (white arrowheads). (C and D) In wild-type males carrying the genomically integrated evIs103 [unc-40p::unc-40::gfp] transgene array, the GFP signal (white arrowheads) is observed at the cell membrane of all dividing ray precursors during third larval (L3) stage (C) and in all ray structural cell processes and their tips at the L4 stage (D). Bars for A and B–D, 25 μm and 16 μm, respectively.

Figure 4

Cell-type–specific rescue of smp-1, unc-6, and plx-1 mutant ray 1 defects. The percentages of severe (solid bar) and mild (cross-hatched bar) anterior ray 1 displacements are shown for a variety of smp-1, unc-6, and plx-1 mutants strains—all in a him-5 (e1490) (control WT) genetic background. (A) plx-1 mutant ray 1defects are rescued by expression of plx-1 in all the rays (driven by lin-32 promoter) or the ray structural cell (driven by the ram-5 promoter), but not by expression in Rn.p cells (driven by the eff-1 promoter). (B) Percentage of wild type (WT = him-5) and unc-40 mutant ray 1 anterior displacement defects are shown (B, a and b) to compare with the same mutant expressing an unc-40cDNA driven by (B, c) the ram-5 promoter (evEx411), or (B, d) the lin-32 promoter (evEx412) or (B, e) the eff-1 promoter (evEx413) or (B, f) both the ram-5 and eff-1 promoter (evEx414) or (B, g) both the lin-32 and eff-1 promoter (evEx415). (B, h) ray 1 anterior displacement defects induced by evIs103 (an unc-40::gfp fusion driven by the unc-40 promoter) in a wild-type background, and (B, i) the effects of halving the dose of the evIs103 transgene on ray 1 defects. (B, j) ray 1 anterior displacement defects in an unc-40 mutant homozygous for the evIs103 transgene array, or (B, k) carrying the evEx416 extrachromosomal array of plasmid pSC11 comprising the entire unc-40 coding sequence plus regulatory sequence previously shown to rescue an unc-40 mutant (Chan et al. 1996). (B, l) plx-1 mutant displacement defects—shown to compare with (B, m) the same mutant carrying evIs103. (B, n) unc-6 mutant ray 1 anterior displacement defects—shown to compare with (B, o) the same mutant homozygous for evIs103 or (B, p) evEx412, or (B, q) evEx411. Standard deviations for percentages of the anterior ray 1 phenotype were calculated assuming a binomial distribution of the same sample size and the observed proportion as mean. Statistical tests were carried out using a standard (two tailed) comparison of two proportions (Moore and McCabe 1998). Joined horizontal lines designate data used for statistical comparisons to determine whether specific transgene arrays rescue unc-40 or unc-6 mutant anterior ray 1 displacement defects. In all cases rescue was significant (P < 0.005).

Figure 5

Model for the role of semaphorin-1a and UNC-6 signaling pathways during ray 1 positioning. SMP-1, SMP-2, and PLX-1 are part of a single pathway that functions in parallel to a pathway comprising UNC-6, UNC-40, and UNC-5. SMP-1 and UNC-6 ligands send a permissive signal to PLX-1 and UNC-40 receptors, required cell autonomously in ray structural cells. In wild-type males, individual ray cell groups form and separate on the anterior–posterior axis. This process is dependent on MAB-20–mediated signaling (green) (as well as the PLX-2 receptor and other unknown components—see Ikegami et al. 2004) since ray cell groups tend to cluster with adjacent cell process groups in mab-20 mutants. This MAB-20/PLX-2–dependent ray separation (green) is normally inhibited by the semaphorin-1a and UNC-6 pathways (red). Loss of function in semaphorin-1a and UNC-6 signaling pathways results in an effective gain of function of MAB-20–mediated signaling in the ray 1 structural cells, which increases separation between ray 1 and ray 2 cell groups.

Figure 6

Possible adhesive functions for PLX-1 in ray 1 positioning. The SET remains abnormally attached to ray 1 in young adult plx-1 mutant males (B), but not in comparably staged wild-type males (A) or mutant animals with normally positioned rays (not shown). Cell boundaries are revealed by the ajm-1::gfp reporter for adherens junctions (Mohler et al. 2002). Bar, 16 μm for both panels.

Figure 7

Heat-shock promoter-driven rescue of smp-1 and unc-6 mutant ray 1 defects. Percentages of severe (solid bar) and mild (cross-hatched bar) anterior ray 1 displacements are shown for smp-1 and unc-6 mutants strains—all in a him-5 (control WT) genetic background. (A) Ray 1 defects of heat-shocked (HS) and nonheat-shocked (no HS) smp-1(ev715) males carrying evEx406 [an extrachromosomal of a wild-type smp-1 gene driven by a heat-shock promoter (hsp16.41p)] are shown. Ray 1 defects of heat-shocked and nonheat-shocked unc-6(ev400) males carrying evEx407 [an extrachromosomal of a wild-type unc-6 gene driven by a heat-shock promoter (hsp16.41p)] are also shown. Statistical analyses were performed as described in the legend to Figure 4. Joined horizontal lines designate data used for statistical comparisons to determine whether specific transgene arrays rescue unc-40 or unc-6 mutant anterior ray 1 displacement defects. In each case, the heat-shock promoter-driven gene (smp-1 or unc-6) causes significant rescue (P < 0.0005) when heat shocked, as compared to heat-shocked mutant controls with no transgene array.