Two distinct phases of junction remodelling.

<p>(A-A’) Seam cell adherens junctions of an early (A) and late elongation (A’) <i>C</i>. <i>elegans</i> embryo marked by the CRISPR HMR-1/E-cadherin GFP-fusion reporter. Seam cells are shaded in yellow. Yellow lines show approximately the embryo contour. Note that in A’, the embryo is like a tube folded within the eggshell, only 5 μm on top of the embryo was projected and the five first seam cells are visible. Scale bar, 5 μm. (A”) Schematic representation of seam (magenta) and dorsal-ventral epidermis (green) based on the embryo in (A). The position of the seam cells from H0 to V3 is shown; anterior to the left, dorsal up. A, P, D and V represent the anterior, posterior, dorsal and ventral junctions of the cell examined. (B) Measurements of seam cell perimeter showing slight changes up to the 2-fold stage (around 80–90 μm in embryo length) then a clear increase after this stage. Curves are quadratic fit to show the variation trend. (C) Length measurements of V1 and V2 dorsal (D) and ventral (V) junctions showing a linear increase in dorso-ventral junction length. Solid lines show linear fit. (D) Length measurements of seam-seam junctions showing a variable decrease rate until they all reach a similar junction length at the 2-fold stage. The seam-seam junction length decreased only slightly beyond that stage. Curves are single exponential decay fit to show the variation trend. (E) Scheme showing two phases of seam cell shape changes: with a constant perimeter from lima-bean to 2-fold, then an increasing perimeter due to elongation of only dorso-ventral junctions in the antero-posterior axis.</p

HMP-1 maintains the junction mechanical integrity whereas VAB-9 anchors actin bundles at seam-dorsal/ventral junctions.

<p>(A-P) Time-lapse sequence of embryos carrying the CRISPR construct HMR-1::GFP (green) and an integrated actin reporter (ABD::mCherry, magenta) in control (A-D), <i>vab-9(e1744)</i> (E-H), <i>hmp-1(zu278)</i> (I-L) and <i>hmp-1(zu278); vab-9(e1744)</i> (M-P) embryos. (A’-P’) Close-up images of the dashed rectangle in (A-P). (A”-P”) Single channel HMR-1::GFP of images (A’-P’). Arrows show the extension of HMR-1::GFP at seam-dorsal junctions in the dorsal direction; arrowheads show spots of HMR-1::GFP in the dorsal epidermal cell cytoplasm, some of which are still connected to where the bulk of the junctions through thin HMR-1-labelled extensions. Note the change of the V4 cell shape in late <i>hmp-1(zu278)</i> embryos compared to earlier stage (blue shading, K-L, O-P). Scale bar, 5 μm.</p

ALG-1/2 function is required to maintain muscles and the epidermis.

<p>(<b>A–B</b>) Embryos collected between 6–8 hours post egg-laying at 25°C and stained with the antibodies MH27 (adherens junction). The <i>alg-1; alg-2</i> mutant embryo did not progress beyond the two-fold stage, yet has grossly normal junctions. (<b>C–E</b>) Embryos collected between 6–8 hours post egg-laying at 25°C and stained with the antibodies 4F2 (VAB-10A; C–E) and NE8/4C6 (muscle; C′–E′); merge picture (C″–E″). Arrow, are where the fibrous organelle (D–E) and muscle (D′–E′) pattern is partially interrupted; in addition, muscles do not closely follow the body wall in this area (they should be closer to the blue dotted line; see blue arrowheads). Embryos did not elongate beyond the two-fold stage.</p

Summary of ALG-1 and ALG-2 expression in different organs and tissues.

<p>Fluorescence intensity is indicated as + to indicate a discernible signal, and – if signal was not clearly discernible from background fluorescence.</p

Profile of ALG-1 and ALG-2 expression in larval stage.

<p>Expression profile of GFP::ALG-2 and RFP::ALG-1 as seen in the L1 larval stage. Subsequent larval stages (L2 to L4) and adults display the same expression profile (not shown). Scale bar 20 µm.</p

Relative microRNA association with ALG-1 and ALG-2.

<p>Heatmaps representing the ratios of ALG-1 to ALG-2 associated miRNAs at the larval stages indicated as estimated from miRNA microarrays (towards red: stronger association to ALG-2, towards green: stronger association to ALG-1). miRNA expression data were filtered for robustly expressed miRNAs. Ratios were log2 transformed, centered and normalized for each column. The distance of the solid blue line from the center of each color-cell is proportional to the ratio. Mean ratio indicated as dotted blue line.</p

Developmental timing of double <i>alg-1/2</i> mutant and siblings.

<p>Time-lapse microscopy of <i>C. elegans</i> embryos. Both embryos proceed through development at similar rates until the morphogenetic phase (375 min) were the <i>alg-2(ok304); alg-1(gk214)</i> double mutant arrest (asterisk). Viable siblings were able to proceed development normally. The double mutant embryos are not paralyzed and are able to twitch (see Movie S1).</p

ALG-1 and ALG-2 expression profile in adult worms.

<p><b>Top panel:</b> GFP::ALG-2 and RFP::ALG-1 are co-expressed in most tissues including vulva (V) and spermatheca (S). <b>Bottom panel:</b> Predominant GFP::ALG-2 expression is seen in a set of head neurons (HN) and tail cells (T) while RFP::ALG-1 is strongly expressed in the pharynx (P) and some tail cells (T). Scale bar 20 µm.</p