Effects on P7.p polarity are <i>chw-1</i>-specific.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-18(e620)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Data for strains marked with an asterisk (*) are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t001" target="_blank">Table 1</a> (<i>lin-17</i>) or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t002" target="_blank">Table 2</a> (<i>lin-18</i>). Analyses of statistical significance were performed using Fisher’s exact test.</p

Loss of CHW-1 suppresses P7.p polarity defects caused by loss of LIN-17/Fz.

<p>^a compared to N2</p><p>^b compared to <i>lin-17(n671)</i></p><p>^c compared to <i>lin-17(sy277)</i></p><p>^d compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p

Two Wnt pathways regulate VPC polarization.

<p><b>A</b>. Schematic of vulval precursor cells in the whole animal. <b>B.</b> Schematic of refined (central) and ground (posterior) Wnt signals regulating VPC polarity. <b>C.</b> 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). (<b>D-F</b>) Illustration and images of vulval lineages, model polarity signals and morphology in animals that are (<b>D</b>) wild type, (<b>E</b>) <i>egl-20(n585)</i> using only refined polarity, or (<b>F</b>) <i>lin-17(n671)</i>; <i>lin-18(e620)</i> 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.</p

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.

<p>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.</p

Loss of VANG-1 but not CAM-1/Ror in the <i>lin-17</i> (Fz) mutant background retains sensitivity to CHW-1 activity.

<p>^a compared to <i>lin-17(n671); gfp(RNAi); vang-1(ok1142)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-17(n671); chw-1(ok697); gfp(RNAi)</i></p><p>^d compared to <i>cam-1(gm122); lin-18(e620)</i></p><p>^e compared to <i>cam-1(gm122); gfp(RNAi); lin-18(e620)</i></p><p>^f compared to <i>lin-18(e620)</i></p><p>^§ Data previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.ref021" target="_blank">21</a>]</p><p>Also, loss of CAM-1 but not VANG-1 in the <i>lin-18</i> (Ryk) background retains sensitivity to CHW-1 activity. In other words, VANG-1 loss abolishes the ability of <i>lin-18</i> mutant animals to respond to <i>chw-1</i> mutation or RNAi. Animals were maintained at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p

Effects on P7.p polarity are <i>chw-1</i>-specific.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-17(n671); gfp(RNAi)</i></p><p>^c compared to <i>lin-18(e620)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Data for strains marked with an asterisk (*) are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t001" target="_blank">Table 1</a> (<i>lin-17</i>) or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133226#pone.0133226.t002" target="_blank">Table 2</a> (<i>lin-18</i>). Analyses of statistical significance were performed using Fisher’s exact test.</p

Loss of CHW-1 enhances P7.p polarity defects caused by loss of LIN-18/Ryk.

<p>^a compared to N2</p><p>^b compared to <i>lin-18(e620)</i></p><p>^c compared to <i>lin-18(n1051)</i></p><p>^d compared to <i>lin-18(e620); gfp(RNAi)</i></p><p>Animals were grown at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p

Loss of CHW-1 does not disrupt ground polarity.

<p>^a compared to <i>lin-17(n671)</i></p><p>^b compared to <i>lin-18(e620)</i></p><p>^c compared to <i>lin-17(n671); egl-20(n585)</i></p><p>^d compared to <i>egl-20(n585); lin-18(e620)</i></p><p>Animals were maintained at 23°C and scored by DIC at late L4 stage. <i>n</i> is number of animals scored. Analyses of statistical significance were performed using Fisher’s exact test.</p

CHW-1 is the <i>C</i>. <i>elegans</i> ortholog of human Wrch-1/RhoU and Chp/Wrch-2/RhoV. A.

<p>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>B.</b> An identity and similarity comparison of the GTPase-domain sequences of CHW-1 with Wrch-1, Chp and Cdc42. <b>C</b>. Gene structure of <i>chw-1</i> and an overlapping gene prediction (F22E12.3). Below, sequences deleted in <i>chw-1(ok697)</i> and sequences used for feeding RNAi are indicated. <b>D</b>. An assessment of CHW-1 activity by ectopic expression in DTCs. The <i>lag-2</i> 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.</p

Two Wnt pathways regulate VPC polarization.

<p><b>A</b>. Schematic of vulval precursor cells in the whole animal. <b>B.</b> Schematic of refined (central) and ground (posterior) Wnt signals regulating VPC polarity. <b>C.</b> 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). (<b>D-F</b>) Illustration and images of vulval lineages, model polarity signals and morphology in animals that are (<b>D</b>) wild type, (<b>E</b>) <i>egl-20(n585)</i> using only refined polarity, or (<b>F</b>) <i>lin-17(n671)</i>; <i>lin-18(e620)</i> 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.</p