pix-1 controls early elongation in parallel with mel-11 and let-502 in Caenorhabditis elegans

Abstract

Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans 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 rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. 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) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.

Figure 1. pix-1 and pak-1 control early elongation.

A) Arrested larvae of pix-1(gk416), pix-1(ok982), pak-1(ok448) and let-502(sb118ts) 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 wild-type animals (wt) 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 wt 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 p-values are indicated.

Figure 2. pix-1, pak-1 and let-502 control the head to tail width ratio of elongating embryos.

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 wt, pix-1(gk416), pak-1(ok448) and let-502(sb118ts) 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 p-values are indicated

Figure 3. pix-1(gk416) controls early elongation in parallel with mel-11/let-502.

A) Morphology of let-502 (sb118ts); pix-1(gk416) and B) mel-11(it26); let-502 (sb118ts); pix-1(gk416) 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 (wt) 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 p-values are indicated.

Figure 4. PIX-1 is homogeneously distributed in the cytoplasm and at the cell periphery of hypodermal cells.

A–I) Immunostaining of pix-1(gk416); sajEx1[pix-1p::pix-1::gfp] 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.

Figure 5. PIX-1::GFP is differentially expressed in hypodermal cells during elongation.

A) Confocal lateral projections of pix-1(gk416); sajEx1[pix-1p::pix-1::GFP]; mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp] 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 lin-26p::vab-10(ABD)::MCHERRY 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 pix-1(gk416); sajEx1[pix-1p::pix-1::GFP; rol-6]; mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp] embryos between comma and 1.75-fold stages (n = 26 embryos). Similar results were also obtained in pix-1(gk416); sajIs1[pix-1p::pix-1::GFP; unc-119^R]; mcIs40 [lin-26p::ABDvab-10::mcherry + myo-2p::gfp]. 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 p<0.01. C) Schematic representation of pix-1::GFP and ABD_VAB-10 (control) constructs used to measure the DP/DA intensity ratio reported in panel D. DP/DA of animals carrying mcIs40 (lin-26p::ABD::mCh expressing), mcIs50 (lin-26p::ABD::GFP expressing), sajIs2 (lin-26p::pix-1::GFP expressing) or sajEx1 (pix-1p::pix-1::GFP expressing). ratios** T-test comparing DP/DA ratios measured on pix-1::GFP expressing embryos to ratio measured in ABD_VAB-10 expressing transgenics, p<0.01. The box-plots represent the min, max, 25^th, 50^th (median) and 75^th percentiles of populations.

Figure 6. High expression of PIX-1::GFP in dorsal posterior hypodermis is detrimental for elongation rate of embryos.

A) Confocal projections of pix-1(gk416); sajEx1[pix-1p::pix-1::GFP, rol-6] at t = 0 min and t = 14 min. The DP/DA fluorescence intensity ratio and elongation rate (in μm/min) are indicated. Arrows indicate the dorsal-posterior hypodermis. Scale bar: 10 µm. B) Distribution of elongation rate in μm/min of wild-type (wt), pix-1(gk416), and pix-1(gk416) animals carrying sajEx1[pix-1p::pix-1::GFP, rol-6] or sajIs2[lin-26p::pix-1::GFP, unc-119^R] during early elongation. Elongation rate was measured from 4-dimensional recording of embryonic development between comma and the end of early elongation upon DIC illumination. Box-plots represent the min, max, 25^th, 50^th (median) and 75^th percentile of the populations. ** T-test p<0.01 vs wt. C) Scatter plot representing the relationship between the dorsal-posterior/dorsal-anterior (DP/DA) intensity ratio of PIX-1::GFP and the elongation rate in μm/min during early elongation of pix-1(gk416) embryos carrying sajEx1[pix-1p::pix-1::GFP, rol-6] or sajIs2[lin-26p::pix-1::GFP, unc-119^R] (n = 20 for each line). The spearman correlation (R2) between the elongation rate and the DP/DA ratio are indicated, as well as the p-values rejecting the null hypothesis being that the two values are not significantly correlated. Similar results were obtained for pix-1(gk416) animals carrying sajIs1 and sajIs3 (see methods). D) DP/DA, lateral-posterior/lateral-anterior (LP/LA), ventral-posterior/ventral-anterior (VP/VA), dorsal-posterior/ventral (DP/V), dorsal-anterior/ventral (DA/V) and dorsal-anterior/lateral (DA/L) fluorescence intensity ratio measured for pix-1(gk416); sajEx1[pix-1p::pix-1::GFP, rol-6] embryos elongating at a wt-rate (elongation≥288nm/min) or elongating slower (elongation<288nm/min) during early elongation. Bar correspond to the mean and error bars to the standard deviation. ** T-test p-value<0.01. E) PIX-1::GFP fluorescence intensity (AU) was measured in DA and DP hypodermal cells of embryos elongating at a wt-rate (elongation≥288nm/min) or elongating slower (elongation<288nm/min) during early elongation (see methods). ** T-test p-value<0.01.

Figure 7. Model for signaling pathways controlling embryonic elongation.

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 pix-1(gk416), we cannot exclude the possibility that pix-1 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.