Developmental characterization of the microRNA-specific C. elegans Argonautes alg-1 and alg-2.

The genes alg-1 and alg-2 (referred to as "alg-1/2") encode the Argonaute proteins affiliated to the microRNA (miRNA) pathway in C. elegans. Bound to miRNAs they form the effector complex that effects post-transcriptional gene silencing. In order to define biological features important to understand the mode of action of these Argonautes, we characterize aspects of these genes during development. We establish that alg-1/2 display an overlapping spatio-temporal expression profile and shared association to a miRNAs set, but with gene-specific predominant expression in various cells and increased relative association to defined miRNAs. Congruent with their spatio-temporal coincidence and regardless of alg-1/2 drastic post-embryonic differences, only loss of both genes leads to embryonic lethality. Embryos without zygotic alg-1/2 predominantly arrest during the morphogenetic process of elongation with defects in the epidermal-muscle attachment structures. Altogether our results highlight similarities and specificities of the alg-1/2 likely to be explained at different cellular and molecular levels

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

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

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