Progressive depletion of actin over time in C. elegans gonads and embryos. (A) Fixed gonads stained with anti-actin antibody in wild-type and actin(RNAi) treated animals at successive timepoints after injection of dsRNA, visualized with FITC-conjugated secondary antibody. The distal end of the gonad is shown. For each timepoint, the maximum signal intensity was quantified in each of three sampled areas per gonad, and the average of these was then compared to WT to obtain a measure of relative intensity. Scale bar is 10 μm. (B) Graph of actin depletion over time in actin(RNAi) gonads based on data shown in (A). Brackets indicate approximate time intervals during which the four classes of actin(RNAi) phenotypes were observed in the early embryo for this batch of dsRNA. Average maximum intensities and standard deviations are based on cumulative data from 3–5 gonads and three sampled areas per gonad (i.e. 9–15 data points) for each timepoint. Numbers on the vertical axis represent units of intensity as measured by Openlab 3.1.7 software. (C) Time-course depletion of actin and requirements for early embryogenesis. Each row, labeled by class, shows DIC images from a time series analysis spanning from just after the completion of meiosis to the second mitotic division. For each timepoint, hallmark events in wild-type (top row) are listed above each column. actin(RNAi) Class I phenotypes (n = 11 embryos): Loss of cortical contractions, no pseudocleavage, and synchronous second cell division. actin(RNAi) Class II phenotypes (n = 12): Pronuclei meet more centrally, loss of spindle displacement, and parallel and synchronous second cell division. Note the increase in distance between the paternal nucleus and the posterior cortex. actin(RNAi) Class III phenotypes (n = 12): First cell division is incomplete with nuclear reformation close to contractile ring, and multiple nuclei in subsequent divisions. actin(RNAi) Class IV phenotypes (n = 14): Failure to extrude polar bodies, loss of cytokinesis and multiple nuclei. Arrows point to polar bodies (where observed). Scale bar is 10 μm.