<i>pix-1</i> and <i>pak-1</i> control early elongation.

<p>A) Arrested larvae of <i>pix-1(gk41</i>6), <i>pix-1(ok982)</i>, <i>pak-1(ok448)</i> and <i>let-502(sb118ts)</i> mutants grown at 25.5°C. Bar = 25 µm. B) Box-plot representing the distribution of sizes of arrested larvae in mutant populations grown at 25.5°C. The box-plot represents the min, max, 25^th, 50^th (median) and 75^th percentile of the population. Distribution of <i>wild-type</i> animals (<i>wt</i>) has been established using N2 L1 larvae synchronized by starvation after hypochlorite treatment. C) Box-plot representing the distribution of the duration in minutes of early elongation for <i>wt</i> and mutants embryos. Embryos are collected through dissection of hermaphrodites grown at 25.5°C. Embryonic development is recorded at 23–24°C. D) Box-plot representing the distribution of the length of embryos (in μm) at the end of early elongation. The same population of embryos was used to generate data presented in panel C and D. Student's T-test <i>p-values</i> are indicated.</p

Genetic interactions of <i>pix-1</i> and <i>pak-1</i> with <i>mel-11</i> and <i>let-502</i> mutants.

<p>Early elongation arrest include embryos arresting between comma and 1.75-fold with or without rupturing.</p><p>* arrested larvae present a <i>pix-1(gk416)</i> specific morphology (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094684#pone-0094684-g001" target="_blank">Figure 1A</a>).</p><p>** arrested larvae present a <i>let-502 (sb118ts)</i> specific morphology (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094684#pone-0094684-g001" target="_blank">Figure 1A</a>).</p><p>*** late elongation arrest without hatching.</p>$<p>all arrested embryos rupture.</p>$$<p>0% rupture.</p

<i>pix-1</i> Controls Early Elongation in Parallel with <i>mel-11</i> and <i>let-502</i> in <i>Caenorhabditis elegans</i>

<div><p>Cell shape changes are crucial for metazoan development. During <i>Caenorhabditis elegans</i> embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the <i>rho-1/</i>RHOA-specific effector <i>let-502/</i>Rho-kinase and <i>mel-11</i>/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector <i>pak-1</i>. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) <i>pix-1</i>, the GTPase <i>ced-10/</i>RAC and <i>pak-1</i>.</p><p>In this study, <i>pix-1</i> is shown to control early elongation in parallel with <i>let-502/mel-11</i>, as previously shown for <i>pak-1</i>. We show that <i>pix-1</i>, <i>pak-1</i> and <i>let-502</i> control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, <i>pix-1</i> and <i>pak-1</i> are shown to control head, but not tail width, while <i>let-502</i> controls both head and tail width. This suggests that <i>let-502</i> function is required throughout the antero-posterior axis of the embryo during early elongation, while <i>pix-1/pak-1</i> function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low <i>pix-1</i> expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.</p></div

PIX-1::GFP is differentially expressed in hypodermal cells during elongation.

<p>A) Confocal lateral projections of <i>pix-1(gk416); sajEx1[pix-1p::pix-1::GFP]; mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp]</i> embryos. Dorsal-anterior (DA, upper panel), dorsal-posterior (DP, upper panel), ventral-anterior (VA, upper panel), ventral-posterior (VP, upper panel), lateral-anterior (LA, lower panel) and lateral-posterior (LP, lower panel) hypodermis are surrounded by dashed line and have been identified using <i>lin-26p::vab-10(ABD)::MCHERRY</i> hypodermal markers. B) Distributions of the dorsal-posterior/dorsal-anterior (DP/DA), lateral-posterior/lateral-anterior (LP/LA), ventral-posterior/ventral-anterior (VP/VA), dorsal-posterior/ventral (DP/V); dorsal-anterior/ventral (DA/V), dorsal-anterior/lateral (DA/L) rates of fluorescence intensity measured in <i>pix-1(gk416)</i>; <i>sajEx1[pix-1p::pix-1::GFP; rol-6]; mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp]</i> embryos between comma and 1.75-fold stages (n = 26 embryos). Similar results were also obtained in <i>pix-1(gk416)</i>; <i>sajIs1[pix-1p::pix-1::GFP; unc-119^R]</i>; <i>mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp]</i>. The box-plots represent the min, max, 25^th, 50^th (median) and 75^th percentiles of the populations. ** T-test comparing ratios to 1 <i>p</i><0.01. C) Schematic representation of <i>pix-1::GFP</i> and ABD_VAB-10 (control) constructs used to measure the DP/DA intensity ratio reported in panel D. DP/DA of animals carrying <i>mcIs40</i> (<i>lin-26p::ABD::mCh</i> expressing), <i>mcIs50</i> (<i>lin-26p::ABD::GFP</i> expressing), <i>sajIs2</i> (<i>lin-26p::pix-1::GFP</i> expressing) or <i>sajEx1</i> (<i>pix-1p::pix-1::GFP</i> expressing). ratios** T-test comparing DP/DA ratios measured on <i>pix-1::GFP</i> expressing embryos to ratio measured in ABD_VAB-10 expressing transgenics, <i>p</i><0.01. The box-plots represent the min, max, 25^th, 50^th (median) and 75^th percentiles of populations.</p

Model for signaling pathways controlling embryonic elongation.

<p>A) Schematic representation of an embryo during early elongation. Anterior is at the left and dorsal side on the top. Dorsal (white), lateral (orange) and ventral (yellow) hypodermal cells are represented. The blue plan indicates the location of the transversal sectioning of the hypodermal cells represented in panel B. B) Signaling pathways in the dorsal (white), lateral (orange) and ventral (yellow) hypodermis in the anterior part of the embryo during early elongation. In this model PIX-1 is expressed at similar level in all hypodermal cells of the anterior part of the embryo. While homogenous expression of PIX-1::GFP in these cells rescues elongation defects of <i>pix-1(gk416)</i>, we cannot exclude the possibility that <i>pix-1</i> may be required only in a subset of these cells. In PIX-1-expressing cells, PAK-1 is activated in a GTPase-dependant (through activation of CED-10 by PIX-1 and/or UNC-73) or in a GTPase-independent manner (by PIX-1 directly). LET-502 is activated only in seam cells through activation of RHO-1 by RHGF-2. PIX-1 may also activate MRCK-1 through CDC-42 upstream of MEL-11. Following this model, the contraction pressure applied on the actin cytoskeleton is similar in ventral and dorsal hypodermis and higher in seams cells. Arrows represent relative contraction forces within each cell.</p

PIX-1 is homogeneously distributed in the cytoplasm and at the cell periphery of hypodermal cells.

<p>A–I) Immunostaining of <i>pix-1(gk416); sajEx1[pix-1p::pix-1::gfp]</i> expressing embryos with MH27 antibodies (A, D, G and red in merge panel C, F, I) and anti-GFP antibodies (B, E, H and green in merge panel C, F, I). Lower panel of each view correspond to orthogonal views of embryos Z-sectioning. Position of Z-sectioning is indicated in upper panel by a yellow line in dorsal (A–C) and lateral (D–F) and ventral (G–I) hypodermis. In orthogonal views, arrows point to adherens junctions which partially colocalize with PIX-1::GFP immunostaining. Arrowheads show the decrease in PIX::GFP expression every other cell in the dorsal-posterior hypodermis (at comma stage) in picture B (upper panel); Arrow-head indicates dorsal trans-epithelial attachment structures (TEA) in picture E (upper panel). Scale bars: 10 µm.</p

Embryonic lethality and arrest of non-elongated larvae in <i>pix-1 and pak-1</i> mutants.

<p>Embryonic lethality and arrest of non-elongated larvae in <i>pix-1 and pak-1</i> mutants.</p

<i>pix-1</i> and <i>pak-1</i> control early elongation.

<p>A) Arrested larvae of <i>pix-1(gk41</i>6), <i>pix-1(ok982)</i>, <i>pak-1(ok448)</i> and <i>let-502(sb118ts)</i> mutants grown at 25.5°C. Bar = 25 µm. B) Box-plot representing the distribution of sizes of arrested larvae in mutant populations grown at 25.5°C. The box-plot represents the min, max, 25^th, 50^th (median) and 75^th percentile of the population. Distribution of <i>wild-type</i> animals (<i>wt</i>) has been established using N2 L1 larvae synchronized by starvation after hypochlorite treatment. C) Box-plot representing the distribution of the duration in minutes of early elongation for <i>wt</i> and mutants embryos. Embryos are collected through dissection of hermaphrodites grown at 25.5°C. Embryonic development is recorded at 23–24°C. D) Box-plot representing the distribution of the length of embryos (in μm) at the end of early elongation. The same population of embryos was used to generate data presented in panel C and D. Student's T-test <i>p-values</i> are indicated.</p

<i>pix-1, pak-1</i> and <i>let-502</i> control the head to tail width ratio of elongating embryos.

<p>Head (H) and tail (T) width are measured on 1.2-fold stage embryos (H1 and T1); at the end of early elongation (H2 and T2) and in arrested larvae (H3 and T3). In all panels of this figure animals were grown at 25.5°C. Embryos were collected through dissection of hermaphrodite grown at 25.5°C. Embryonic development is then recorded using 4-dimensional microscopy at 23–24°C. A) Distribution of ratio between the head and tail width of embryos at 1.2-fold stage (H1/T1; left panel), at the end of early elongation (H2/T2; middle panel) and of arrested larvae (H3/T3; right panel) in <i>wt</i>, <i>pix-1(gk416)</i>, <i>pak-1(ok448)</i> and <i>let-502(sb118ts)</i> mutants. B) Localisation of measured areas in embryos and larvae C) Distribution of the head (H1/H2) and tail (T1/T2) width reduction ratios during early elongation. The box-plots represent the min, max, 25^th, 50^th (median) and 75^th percentiles of the populations. Student's T-test <i>p-values</i> are indicated</p

<i>pix-1(gk416)</i> controls early elongation in parallel with <i>mel-11/let-502</i>.

<p>A) Morphology of <i>let-502 (sb118ts); pix-1(gk416)</i> and B) <i>mel-11(it26)</i>; <i>let-502 (sb118ts); pix-1(gk416)</i> arrested larvae grown at 25.5°C. C) Distribution of sizes of arrested larvae in mutants' populations, at 25.5°C. Distribution of wild-type animals (<i>wt</i>) has been established using N2 L1 larvae synchronized by starvation after hypochlorite treatment. D) Distribution of ratio between the head and tail width of arrested larvae in mutant populations. The box-plot represents the min, max, 25^th, 50^th (median) and 75^th percentiles of the population. Student's T-test <i>p-values</i> are indicated.</p