An Activating Mutation in sos-1 Identifies Its Dbl Domain as a Critical Inhibitor of the Epidermal Growth Factor Receptor Pathway during Caenorhabditis elegans Vulval Development

Proper regulation of receptor tyrosine kinase (RTK)-Ras-mitogen-activated protein kinase (MAPK) signaling pathways is critical for normal development and the prevention of cancer. SOS is a dual-function guanine nucleotide exchange factor (GEF) that catalyzes exchange on Ras and Rac. Although the physiologic role of SOS and its CDC25 domain in RTK-mediated Ras activation is well established, the in vivo function of its Dbl Rac GEF domain is less clear. We have identified a novel gain-of-function missense mutation in the Dbl domain of Caenorhabditis elegans SOS-1 that promotes epidermal growth factor receptor (EGFR) signaling in vivo. Our data indicate that a major developmental function of the Dbl domain is to inhibit EGF-dependent MAPK activation. The amount of inhibition conferred by the Dbl domain is equal to that of established trans-acting inhibitors of the EGFR pathway, including c-Cbl and RasGAP, and more than that of MAPK phosphatase. In conjunction with molecular modeling, our data suggest that the C. elegans mutation, as well as an equivalent mutation in human SOS1, activates the MAPK pathway by disrupting an autoinhibitory function of the Dbl domain on Ras activation. Our work suggests that functionally similar point mutations in humans could directly contribute to disease

<i>vab-8</i> mutations that affect neuronal cell body positioning and axon outgrowth cause epidermal patterning defects.

<p>(A) <i>vab-8</i> mutations cause P8.p to adopt a vulval fate. In wild-type animals, P8.p divides once, but never forms vulval tissue. (B) <i>vab-8</i> mutations increase the frequency of P3.p becoming a vulval progenitor. 50% of the time, P3.p receives sufficient Wnt signaling to become a vulval progenitor and divides once. (C) Upper panel depicts wild-type signaling by Wnts and EGF that promotes vulval development with mirror image symmetry. MOM-2 and LIN-44 Wnts dominate over EGL-20/Wnt to polarize P7.p towards the anterior. Lower panel shows a wild-type 22-cell vulva with normal symmetry at the mid-L4 stage. (D) Upper panel depicts abnormal Wnt signaling in <i>vab-8</i> mutants that causes the formation of vulval tissue with a P-Rvl phenotype. EGL-20/Wnt dominates over MOM-2 and LIN-44 Wnts, preventing P7.p from reorienting towards the anterior. Lower panel shows a P-Rvl vulva at the mid-L4 stage. In (C) and (D), EGL-20/Wnt was overexpressed from its native promoter with the <i>muIs49</i> transgene. Scale bar is 10 µm. Colors depict Wnt signaling as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001465#pbio-1001465-g001" target="_blank">Figure 1</a>. <i>p-</i>Values were calculated using a two-tailed Fisher's exact test versus wild-type animals (A and B) or as otherwise indicated (A and D).</p

Posterior CAN axons regulate axial positioning of vulval fates and P3.p vulval progenitor frequency.

<p>Distributions of positions of CAN cell bodies (A) and furthest posterior CAN axon termini (B) in different mutants. CAN neurons were visualized in L3, Pn.px stage (A and B) or L4 stage (E) animals with the <i>kyIs4[Pceh-23::gfp]</i> transgene. <i>x</i>-Axis indicates Pn.p or Pn.px positions. <i>p-</i>Value was calculated using a two-tailed Mann-Whitney <i>U</i> test. (C) Position of CAN cell bodies and posterior axon termini relative to <i>egl-20/wnt</i>-expressing cells in L2 stage <i>vab-8</i> mutants. CANs and <i>egl-20/wnt</i>-expressing cells were marked with the <i>akEx906</i> transgenic array. The bright green signal in the head/pharynx is from the coinjected <i>Pmyo-2::cfp</i> injection marker. Scale bar is 25 µm. (D) Frequency with which P8.p adopts a vulval fate in the total <i>ceh-10</i> mutant population. (E) Correlation between the position of the furthest posterior CAN axon terminus and induction of ectopic vulval fates at P8.p in <i>ceh-10(lf)</i> mutants. For this study, an emphasis was placed on picking smaller animals to ensure that sufficient numbers of animals with short posterior CAN axons were obtained for statistical analysis. Thus, the combined frequency of ectopic vulval fates in this study is not an estimate of the actual frequency in the total population as conducted in (D). Top panels show animal 52, with normal epidermal development and normal position of furthest posterior CAN axon terminus. Bottom panels show animal 79, with an R-Pvl phenotype at P7.p and ectopic vulval fate at P8.p, and severely foreshortened furthest posterior CAN axon terminus. Scale bars are 10 µm. (F) Correlation between position of furthest posterior CAN axon terminus and frequency with which P3.p becomes a vulval progenitor in <i>ceh-10(lf)</i> mutants. In (D–F), <i>p-</i>Values were calculated using a two-tailed Fisher's exact test versus wild-type animals (D) or as otherwise indicated (E and F). H, head/pharyngeal region.</p

Wnt signaling and epidermal patterning in <i>C. elegans</i>.

<p>(A) A wild-type <i>C. elegans</i> adult hermaphrodite. Scale bar is 100 µm. (B) During the L2 larval stage, LIN-3/EGF from pre-anchor cell/ventral uterine precursor cells (not shown) cooperates with a gradient of EGL-20/Wnt (orange) from rectal cells and CWN-1/Wnt (green) from posterior muscle and neurons to cause six epidermal cells to become vulval progenitors (P3.p–P8.p). 50% of the time, P3.p does not receive sufficient Wnt signaling and adopts the “F” fate (also known as the 4° fate) and fuses with a hypodermal syncytium called hyp7. EGL-20/Wnt also polarizes P5.p and P7.p so that they face posteriorly (horizontal arrows). The epidermal cells normally touch each other, but are drawn apart to facilitate depiction of muscle and neurons. (C) At the end of the L2 larval stage, anchor cell-produced MOM-2 and LIN-44 Wnts (blue) reorient P7.p towards the anterior (horizontal arrows). During the L3 larval stage, LIN-3/EGF (purple) from the anchor cell induces the 1° vulval fate in P6.p, which is facilitated by EGL-20 and CWN-1 Wnts. P5.p and P7.p adopt 2° vulval fates because of the activation of LIN-12/Notch via a lateral signal from P6.p. (D) During the L3–L4 larval stages, vulval progenitor cells (Pn.p) divide to generate Pn.px cells, with P5.p–P7.p undergoing two additional rounds of cell division (to ultimately make Pn.pxxx cells). Because of the opposite polarities of P5.p and P7.p, their asymmetrically dividing progeny generate mirror image patterns. By the early L4 stage, a 22-cell vulva is generated. The Pn.px progeny of P3.p, P4.p, and P8.p fuse with hyp7 (3° fate).</p

VAB-8 acts in the CAN neurons to regulate epidermal patterning.

<p>(A–C) Expression of <i>vab-8s</i> and <i>CAN</i> promoters from the <i>akEx923</i> transgenic array in wild-type L2 stage animals. Scale bar is 50 µm. The CANs are a pair of neurons, CANL and CANR, located on the left and right side of each animal, respectively. (A) DsRed2 channel. (B) YFP channel. (C) Merged images from (A) and (B). (D) CAN-specific expression of VAB-8S restores inhibition to vulval fate signaling in <i>vab-8</i> mutants. <i>vab-8</i> mutations displace cell bodies and affect axon outgrowth of a subset of neurons including the CANs (blue). VAB-8S was restored to <i>vab-8</i> mutants with either the CAN-specific promoter (<i>PCAN</i>) or the control minimal <i>pes-10</i> promoter (<i>Ppes-10</i>). Vulval fates: number of vulval progenitor cells adopting vulval fates. Wild-type is 3.00. <i>p-</i>Value was calculated using a two-tailed Student's <i>t</i> test. Transgenic arrays were <i>dyEx24</i> (<i>Ppes-10::vab-8s</i>) or <i>dyEx20</i> (<i>PCAN::vab-8s</i>). CAN-specific expression of VAB-8S from the <i>dyEx20</i> transgenic array rescues patterning defects in the posterior and anterior epidermis (E and F). <i>p-</i>Value was calculated using a two-tailed Fisher's exact test.</p

CAN neurons inhibit Wnt signaling in epidermal progenitors.

<p>(A) Pn.px stage animals showing <i>syIs187</i> mCherry Wnt reporter activity. Scale bar is 20 µm. (B) Quantification of reporter data. Reporter activity was lower in P5.px–P7.px cells than in P3.px, P4.px, and P8.px cells, so data were collected using a higher brightness setting. <i>p-</i>Values were calculated using a two-tailed Fisher's exact test versus wild-type animals. (C and D) Location of <i>egl-20/wnt-</i> and <i>cwn-1/wnt</i>-expressing cells relative to epidermal cells in L3, Pn.px stage animals. Scale bar is 10 µm. (C) <i>muIs49[Pegl-20::egl-20::gfp]</i> transgenic animal. (D) <i>dyEx10[Pcwn-1::DsRed2]</i> transgenic animal. To simultaneously visualize neurons and muscle, images were taken in different focal planes, differentially colored either green or red, and merged. (E–H) Location of Wnt-producing cells relative to CAN neurons. Only one CAN cell body is visible. (G and H) Blow-up of (E) and (F), respectively. Scale bars are 50 µm (E and F) and 25 µm (G and H). Transgenic arrays were <i>akEx906</i> (E and G) and <i>akEx908</i> (F and H).</p

The CANs use the extracellular Wnt-binding domain of Ror/CAM-1 to direct epidermal patterning.

<p>(A and B) Distributions of positions of CAN cell bodies and furthest posterior CAN axon termini in <i>ror/cam-1</i> mutants and in <i>cam-1</i> mutants expressing wild-type or intracellular-domain-deleted CAM-1(ΔIntra) only in the CANs. In non-<i>ror/cam-1</i> rescue experiments, CANs were visualized with the <i>kyIs4[Pceh-23::gfp]</i> transgene. In <i>ror/cam-1</i> rescue experiments, CANs were visualized by expression of GFP-tagged Ror/CAM-1 in the CANs. In <i>ror/cam-1</i> rescue experiments, strains also harbored an <i>egf</i>/<i>lin-3(lf)</i> mutation. <i>x</i>-Axis indicates Pn.p or Pn.px positions. H, head/pharyngeal region. <i>p-</i>Values were calculated using a two-tailed Mann-Whitney <i>U</i> test. (C) Ror/CAM-1 inhibits vulval fate signaling in central vulval progenitors. (D) Transgenic CAN-specific expression of Ror/CAM-1::GFP restores inhibition of vulval development in <i>ror/cam-1</i> mutants. (E) Transgenic CAN-specific expression of a Ror/CAM-1::GFP mutant lacking the intracellular domain (ΔIntra, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001465#pbio-1001465-g006" target="_blank">Figure 6C</a>) also restores inhibition of vulval development in <i>ror/cam-1</i> mutants. In (D) and (E), scale bar is 20 µm. (F) Under physiologic conditions, the majority of Ror/CAM-1 inhibition of vulval fate signaling is mediated by the CANs. If the CAN cell bodies are anteriorly displaced and the posterior axons are severely foreshortened, loss of Ror/CAM-1 activity from all cells does not further increase P3.p progenitor frequency or the amount of vulval development in sensitized backgrounds. Drawings depict the cellular distribution of Ror/CAM-1 as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001465#pbio-1001465-g006" target="_blank">Figure 6E</a>. M, muscle cells; N, neurons; P, vulval progenitors. In (C–E), vulval fates: number of vulval progenitor cells adopting vulval fates. Wild-type is 3.00. <i>p-</i>Values were calculated using a two-tailed Student's <i>t</i> test. The <i>PCAN::cam-1::gfp</i> and <i>PCAN::</i>Δ<i>Intra::gfp</i> rescuing transgenic arrays are <i>dyEx44</i> and <i>dyEx45</i>, respectively.</p