Figure 1.
Schematic of the C. elegans EGFR/Ras/MAPK pathway, vulva development, and the rab-7 gene structure.
(A) A linear representation (left to right) of the core components of the C. elegans EGFR/Ras/MAPK pathway (black) and some negative regulators (red) of the pathway are shown above or below their presumptive targets. The mammalian homologs are shown in parenthesis below the C. elegans names. (B) The Anchor Cell (AC) in the gonad secretes a LIN-3 EGF signal (green) to the Vulva Precursor Cells (VPCs), P3.p-P8.p, most strongly activating LET-23 EGFR signaling in the closest VPC, P6.p, inducing the 1° vulva cell fate (green). Subsequently, ligands on P6.p activate LIN-12 Notch (N, blue) on the P5.p and P7.p cells inducing them to adopt the 2° vulva cell fate (yellow). P5.p, P6.p. and P7.p give rise to the 22 cells of the vulva, while P3.p, P4.p, and P8.p divide (P3.p only ∼50% of the time) and fuse with the surrounding hypodermis (Hyp). (C) A diagram representing the rab-7 W03C9.3 gene and the upstream gene W03C9.5. The genes are oriented with the 5′ end to the left and 3′ to the right with boxes representing the exons and intervening lines as introns and the 3′ UTR. The regions coding for the putative switch and nucleotide binding domains in the RAB-7 protein are shown in blue and red, respectively. The ok511 deletion that removes the first three exons of rab-7 as well as the 3′ UTR of W03C9.5 is marked with a bracket. The indicated lines represent the genomic clones used for RNAi feeding.
Figure 2.
rab-7(ok511) modulates the vulva phenotypes of mutations affecting components of the EGFR/Ras/MAPK pathway.
Representative DIC images of the vulva phenotypes of rab-7(ok511)/mIn1; let-60(n1046gf) (A), rab-7(ok511); let-60(n1046gf) (B), rab-7(ok511)/mIn1; let-60(n2021) (C), rab-7(ok511); let-60(n2021) (D), rab-7(ok511)/mIn1; dpy-20(e1282) ark-1(sy247) (E), rab-7(ok511); dpy-20(e1282) ark-1(sy247) (F), unc-101(sy108); rab-7(ok511)/mIn1 (G), unc-101(sy108); rab-7(ok511) (H), rab-7(ok511)/mIn1; lin-2(e1309) (I), and rab-7(ok511); lin-2(e1309) (J). Open circles mark the normal site of vulva cell invagination, stars mark vulva invaginations due to ectopic induction, vertical lines mark failed vulva cell inductions, and horizontal lines mark ectopic inductions that fail to invaginate. Bar, 10 µm (A).
Table 1.
rab-7 antagonizes LET-60 Ras-mediated vulval cell fate induction.
Table 2.
rab-7(ok511) is synthetic Multivulva with unc-101 and ark-1 mutants.
Figure 3.
Ectopically induced vulval cells in unc-101(-); rab-7(−) animals fail to express the vulva cell fate marker egl-17::CFP.
DIC (A, C, and E) and Epifluorescence (B, D, F and G) images of egl-17::CFP expression in the VPC nuclei of a rab-7(ok511); dpy-20(e1282) ark-1(sy247); arIs92 late L2/early L3 stage larva (A and B), and an unc-101(sy108); rab-7(ok511); arIs92 larva at the late L2/early L3 stage (C and D) and an unc-101(sy108); rab-7(ok511); arIs92 larva at the late L4 stage in which egl-17::CFP negative vulval cells were ectopically produced by P4.pp and the P8.p (E–G). An open circle marks the normal site of vulva cell invagination, and horizontal lines mark ectopic inductions that fail to invaginate. The egl-17::CFP expressing cells derived from P5.pp and P7.pa are labeled (2°) in the medial (E and F) and lateral (G) views, and four egl-17::CFP expressing cells derived from P6.p lineages are labeled (1°). Bar, 10 µm (A).
Table 3.
rab-7(ok511) suppresses the Vul phenotypes of mutations that mislocalize LET-23 but not strong alleles of lin-3 and let-23.
Table 4.
RNAi of rab-7, hgrs-1 and vps-28 suppresses the lin-2(e1309) Vul phenotype.
Figure 4.
rab-7(ok511) alters LET-23::GFP localization in the VPCs of lin-2(-) animals.
(A-L) Single section confocal images of the VPCs (lateral view) of mid-L3 stage larvae following the first round of VPC division (Pn.px stage) immunostained with anti-GFP to detect LET-23::GFP (A, D, G and J) and the MH27 monoclonal antibody to detect the AJM-1 junctional protein (B, E, H, and K) demarcating the apical/basal boundry, and (C, F, I and L) are merged images with P6.pa and P6.pp cells underlined. (A–C) wild-type larva carrying gaIs27(let-23::GFP) showing LET-23::GFP in both the basal and apical regions of P6.pa and P6.pp cells. (D–F) lin-2(e1309); gaIs27(let-23::GFP) larva with weak basal cytoplasmic and strong apical LET-23::GFP localization. (G–I) rab-7(ok511); gaIs27(let-23::GFP) larva with basal cytoplasmic and apical LET-23::GFP expression in P6.pa and P6.pp with LET-23::GFP in cytoplasmic foci. (J–L) rab-7(ok511); lin-2(e1309); gaIs27(let-23::GFP) larva with LET-23::GFP localization similar to that in rab-7(ok511); gaIs27(let-23::GFP) larvae. Bar, 10 µm (C).
Figure 5.
rab-7(−) animals accumulate LET-23::GFP-positive puncta in the hypodermis.
(A and B) Representative epifluorescence images of LET-23::GFP positive foci in the hypodermis in the mid-body of L3 stage rab-7(ok511)/+; xhIs2501 (A) and rab-7(ok511); xhIs2501 (B) larvae. (C) A scatter dot plot of the number of LET-23::GFP positive foci within a fixed area of the hypodermis of xhIs2501, rab-7(ok511)/+; xhIs2501, and rab-7(ok511); xhIs2501 L3 larvae. Error bars represent the mean +/− SEM. In an unpaired t test there is a significant difference (P value<0.0001) between the number of LET-23::GFP positive foci in rab-7(ok511) animals as compared to both rab-7(+) and rab-7(ok511)/+ animals. n = number of animals scored. Bar, 10 µm (A).