Fig 1.
Two Wnt pathways regulate VPC polarization.
A. Schematic of vulval precursor cells in the whole animal. B. Schematic of refined (central) and ground (posterior) Wnt signals regulating VPC polarity. C. A schematic of polarized 2°-1°-2° VPC lineages (P5.p (left), P6.p (center), and P7.p (right)). P5.p and p7.p lineages are asymmetric and mirror one another around a central axis (dotted line). (D-F) Illustration and images of vulval lineages, model polarity signals and morphology in animals that are (D) wild type, (E) egl-20(n585) using only refined polarity, or (F) lin-17(n671); lin-18(e620) using only ground polarity. Arrows represent putative Wnt polarity signals received by VPCs, grayed arrows are inactivated polarity signals, and dotted lines represent central vulval axis.
Fig 2.
CHW-1 is the C. elegans ortholog of human Wrch-1/RhoU and Chp/Wrch-2/RhoV. A.
Sequence alignment of CHW-1, human Wrch-1, and human Chp. Identical residues have a black background, conservative changes have a gray background, and non-conservative changes have a white background. CHW-1 lacks the N-terminal extension found in Wrch-1 and Chp (gray bracket). CHW-1 also contains an atypical residue (alanine; arrowhead) at position 18 (analogous to position 12 in Cdc42 and most Rho and Ras family members). The core effector domain (black bracket) is most similar to that of Wrch-1, with one conservative variant (V→I) between CHW-1 and Wrch-1. Indicated with a gray caret are the atypical A residue at position 18 and the typical Q residue at position 69, both of which we mutated for our DTC locomotion studies in panel D. B. An identity and similarity comparison of the GTPase-domain sequences of CHW-1 with Wrch-1, Chp and Cdc42. C. Gene structure of chw-1 and an overlapping gene prediction (F22E12.3). Below, sequences deleted in chw-1(ok697) and sequences used for feeding RNAi are indicated. D. An assessment of CHW-1 activity by ectopic expression in DTCs. The lag-2 promoter was used to drive ectopic CHW-1 expression in DTCs and migration defects were analyzed by DIC microscopy. A total of 200 DTC migrations were analyzed for each construct. Expression of wild-type CHW-1 caused significantly more frequent migration defects than did expression of GFP or CHW-1(A18G). Expression of CHW-1(A18V) or CHW-1(Q61L) caused significantly more DTC migration defects than did expression of WT CHW-1. Tests of statistical significance were performed using Fisher’s exact test.
Table 1.
Loss of CHW-1 suppresses P7.p polarity defects caused by loss of LIN-17/Fz.
Animals were grown at 23°C and scored by DIC at late L4 stage. n is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.
Table 2.
Loss of CHW-1 enhances P7.p polarity defects caused by loss of LIN-18/Ryk.
Animals were grown at 23°C and scored by DIC at late L4 stage. n is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.
Table 3.
Effects on P7.p polarity are chw-1-specific.
Animals were grown at 23°C and scored by DIC at late L4 stage. n is number of animals scored. Data for strains marked with an asterisk (*) are from Table 1 (lin-17) or Table 2 (lin-18). Analyses of statistical significance were performed using Fisher’s exact test.
Table 4.
Loss of CHW-1 does not disrupt ground polarity.
Animals were maintained at 23°C and scored by DIC at late L4 stage. n is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.
Fig 3.
chw-1 promoter-driven GFP is absent from induced VPCs during vulval development, but is expressed in uninduced, 3° cells after induction.
A, B. Fluorescent images (600x) of a reIs3[Pchw-1::gfp]-bearing animals. C, D. DIC images corresponding to A and B, respectively. A and C show an animal at the Pn.p (undivided) stage of vulval development prior to. VPCs (Pn.p cells) are indicated by white arrows in A and black arrows in C, and the anchor cell is indicated by a black arrowhead. B and D show an animal at the Pn.pxx (2 divisions) stage during ingression onset of induced VPC progeny, just prior to the last round of division. Differentiated vulval progeny starting to coalesce and ingress are indicated by a white bar in B and a black bar in D. Uninduced 3° cells have divided once and are indicated by solid white arrows in B and solid black arrows in D. Note that 3° cells express GFP, with stronger nuclear GFP signal due to a single nuclear localization signal in the construct. Induced vulval lineages (1°s and 2°s) do not express GFP. In both animals anterior is right, ventral is down.
Fig 4.
CHW-1 regulates relative LIN-17/Fz and LIN-18/Ryk signaling in refined (central) polarity, and LIN-17/Fz and LIN-18/Ryk execute a specific repressive program to exclude activity of the ground (posterior) polarity system.
Shown is a schematic of P7.p with anterior, the direction of refined (central) polarization, to the left and posterior, the direction of ground (posterior) polarization to the right.
Table 5.
Loss of VANG-1 but not CAM-1/Ror in the lin-17 (Fz) mutant background retains sensitivity to CHW-1 activity.
Also, loss of CAM-1 but not VANG-1 in the lin-18 (Ryk) background retains sensitivity to CHW-1 activity. In other words, VANG-1 loss abolishes the ability of lin-18 mutant animals to respond to chw-1 mutation or RNAi. Animals were maintained at 23°C and scored by DIC at late L4 stage. n is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.