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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01828

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Distinct roles for two C. elegans anillins in the gonad and early embryo

¹ Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
² Scionics Computer Innovation, Pfotenhauerstrasse 110, Dresden 01307, Germany

View larger version (46K): [in a new window]   Fig. 1. C. elegans anillin-related proteins. (A) Schematics comparing features of the human and Drosophila anillins with the three C. elegans anillin homologs: C-terminal PH domains (grey); anillin homology (AH) regions (red); actin-binding and -bundling domains (blue); myosin-binding domains (green). For sequence comparisons, see Fig. S1B,C in the supplementary material. Worms were injected or soaked with dsRNA that targeted each of the anillin homologs. Embryos laid by the treated hermaphrodites between 48 and 72 hours after injection, or between 72 and 96 hours after the start of soaking, were counted (brood size, B) and the viability of the embryos (C) was measured. Data were normalized to the number and viability of embryos laid by worms treated with a control RNA against a yeast sequence not present in the C. elegans genome. Asterisks: significantly different from control (P<0.05, t-test; n=4-7 worms; 100 embryos/worm). (D) Extracts from worms injected with ani-1 dsRNA or soaked in ani-2 dsRNA were analyzed by western blotting. Numbers above control lanes indicate percentage of amount loaded in 100% lane. The same blots were probed for -tubulin as a loading control. (E) ANI-1 and ANI-2 are enriched in embryos and adult worms, respectively. Arrowheads indicate presumptive ANI-1 and ANI-2 bands. High-speed supernatants prepared from extracts of isolated embryos or whole worms were western blotted and probed with antibodies to ANI-1 and ANI-2. Blots were probed for -tubulin as a loading control. Identical results were obtained with crude extract (data not shown). (F) A C. elegans embryo undergoing cytokinesis was fixed and stained with Hoechst to label DNA and specific antibodies to myosin II (NMY-2), ANI-1 and ANI-2. Scale bar: 10 µm.

View larger version (129K): [in a new window]   Fig. 2. Polar body extrusion, ruffling and pseudocleavage fail in ani-1(RNAi) embryos, but cytokinesis appears normal. Schematic drawings of C. elegans early embryonic development (left column) and selected images from timelapse DIC sequences of control (A-E) and ani-1(RNAi) (A'-E') embryos. Embryo anterior is on the left; posterior (defined by the site of sperm entry) is on the right. Arrow indicates a polar body nucleus that has entered the embryo. Scale bar: 10 µm.

View larger version (96K): [in a new window]   Fig. 3. (A) ANI-1 is required to target the septins, but not myosin II to the contractile ring. Control, ani-1(RNAi), unc-61(e228) and nmy-2(RNAi) telophase embryos were fixed and stained for DNA, ANI-1, myosin II (NMY-2) and the septin UNC-59. Images are equivalently scaled. Only very low amounts of residual ANI-1 are detected in ani-1(RNAi) embryos. The contractile ring at the tip of the ingressing furrow (red arrowheads) and the new cell-cell boundary, which forms behind the ingressing furrow and consists of two juxtaposed cortical surfaces (white arrows), are indicated. The furrow does not ingress in nmy-2(RNAi) embryos. Scale bar: 10 µm. (B) Insets from the images in A are magnified twofold. The new cell-cell boundary (black arrows) and contractile ring (C.R., red arrowheads) are indicated. (C) Schematic showing location and orientation of magnified view in D. (D) Embryos undergoing polar body extrusion were fixed and stained for ANI-1, NMY-2 and UNC-59. In control embryos, all three proteins localize to a ring around the site of polar body extrusion. Images have been rotated so that this ring is viewed end on. Scale bar: 10 µm.

View larger version (117K): [in a new window]   Fig. 4. ANI-1 is required for the formation of cortical patches containing myosin II and the septins. (A) Wild-type embryos and embryos depleted of ANI-1, both UNC-59 and UNC-61, or NMY-2 were fixed prior to pronuclear migration (when the anterior cortex normally ruffles) and stained for ANI-1, NMY-2 and UNC-59. Ruffles are not preserved by the fixation. In wild-type embryos, all three proteins concentrate in an inhomogeneous network of cortical patches. In ani-1(RNAi) embryos, UNC-59 and NMY-2 are diffuse on the cortex. Scale bar: 10 µm. (B) Schematic showing region of embryo magnified in C. (C) Magnified view of the cortex from control and ani-1(RNAi) embryos in A. Scale bar: 5 µm.

View larger version (125K): [in a new window]   Fig. 5. ANI-1 depletion does not inhibit cortical or cytoplasmic flows or the establishment of polarity. (A) Kymographs of yolk granule flow generated from timelapse DIC sequences of control and ani-1(RNAi) embryos collected between meiosis II and pronuclear meeting. Red line: cytoplasmic yolk granules in the center of the embryo flow towards the posterior. Green line: cortical yolk granules in the posterior flow towards the anterior. Vertical scale bar represents 5 minutes; horizontal scale bar represents 10 µm. (B) Left: selected images from timelapse sequences of control and ani-1(RNAi) embryos expressing NMY-2:GFP (see also Movies 4 and 5 in the supplementary material). Right: kymographs showing the behavior of cortical NMY-2:GFP over time (8 minutes, 20 seconds preceding pronuclear meeting). The maximum fluorescence intensity for each point across a 2 µm wide box (broken lines) is displayed as a strip (x-axis) for each timepoint and placed sequentially to generate kymographs (time is on the y-axis). NMY-2:GFP patches (control) and smaller fluorescence discontinuities [ani-1(RNAi)] in the embryo posterior moved towards the anterior. Times are given in minutes:seconds. Scale bar: 10 µm.

View larger version (118K): [in a new window]   Fig. 6. ANI-2 localizes to the lining of the rachis of the syncytial gonad. Wild-type C. elegans syncytial gonads were fixed and stained for DNA, NMY-2 and ANI-2. Images were collected every 0.4 µm through the volume of the gonad. (A) Images of the bottom half of the gonad were projected to visualize the inside surface of the rachis and the openings to the developing oocytes (arrows in A,D). Right: schematic view of the central section of a C. elegans gonad. (B) Schematic of a cross-sectional view of the rachis comparing the localization of NMY-2 and ANI-2. Inset box shows region and orientation of view in C. (C) Higher magnification view of the pachytene region of the gonad. Nuclei are regularly spaced. The ANI-2 staining on the surface of the rachis is visible along the edges in cross-section (arrows). (D) Higher magnification view of the bend in the gonad where oocytes increase dramatically in size. The images in C and D are taken from a different gonad from the one shown in A. Scale bars: 10 µm.

View larger version (100K): [in a new window]   Fig. 7. Depletion of ANI-2 disrupts gonad architecture (compare with wild type in Fig. 6). (A,B) Gonads from worms depleted of ANI-2 by soaking RNAi were extruded, fixed, stained and imaged as for Fig. 6. Scale bars: 10 µm. (C) Higher magnification view representing the indicated region of the gonad in B. Images of a different gonad from that shown above. Scale bar: 10 µm. Arrows indicate abnormally small oocytes in the oviduct; arrowheads indicate tightly packed developing oocytes in the distal gonad.

View larger version (95K): [in a new window]   Fig. 8. Oocytes become disconnected from the rachis prematurely in ani-2(RNAi) worms. (A) Fluorescent dextran was injected into the gonad rachis of control or ani-2(RNAi) worms. Injected worms were imaged by DIC and fluorescence microscopy to visualize the dextran. Schematics summarize results; numbers label oocytes, from the proximal end of the gonad adjacent to the spermatheca (green asterisks). Scale bar: 50 µm. (B) Embryos from control and ani-2(RNAi) worms were imaged by DIC microscopy. Scale bar: 10 µm. (C) The lengths of the embryos in B were measured and the distribution plotted for control and ani-2(RNAi) worms (n>85 for each condition).

View larger version (42K): [in a new window]   Fig. 9. Model for the role of ANI-1 in the formation of cortical patches. (1) ANI-1 at the plasma membrane binds F-actin. (2) F-actin bound to ANI-1 recruits additional ANI-1 to form a cortical meshwork. (3) Patches of ANI-1 and F-actin recruit myosin II. (4) Myosin II contractility produces ingressions. ani-1(RNAi): patches of cortical myosin II and ingressions fail to form, but flows are normal. nmy-2(RNAi): ANI-1-containing patches still form, but do not ingress and cortical flows fail.