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First published online 6 May 2008
doi: 10.1242/jcs.025171

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Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization

¹ Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA
² Howard Hughes Medical Institute, Princeton University, Washington Road, Princeton, NJ 08544, USA

View larger version (69K): [in this window] [in a new window]   Fig. 1. Furrow canals are established as discrete cortical compartments at the onset of cellularization. (A) Illustration showing somatic buds over each nucleus (N) extending their margins to form cellularization furrows. At early cellularization, F-actin and Myosin 2 (Myo-2) furrow canals assemble at the furrow tips and E-cadherin (E-cad) and β-catenin (Arm) coalesce into adjacent basal junctions. Basal junctions mark the boundary between furrow canal compartments and the growing lateral plasma membrane (PM). Furrow canals and basal junctions travel in register at the tip of the ingressing furrow until late cellularization, when furrow canals contract to close off the basal ends of the cells. (B,D,E) Confocal images of cellularizing embryos. (B) Cross-sections showing overlap between the PM component Dlg (red) and F-actin (green) as furrows first form. These markers are resolved once furrows reach a length of 5 µm (arrow in B) with Dlg restricted to the lateral PM and F-actin concentrating in the furrow canals. This compartmentalization is maintained throughout late cellularization. (C) TEM image of an early cellularization furrow with arrows indicating the apical (top) and basal (bottom) boundary of the lateral PM. The furrow canal compartment appears as a broadening at the furrow tip. (D) Cross-sections showing Nullo first concentrating with F-actin in early furrow canals and then concentrating at the basal junction regions slightly later. (E) Cross-sections showing Arm accumulating in basal junctions at the basal-most region of the lateral PM (Nrt, red) in wild-type embryos as compared with spreading along the PM in nulloX embryos. Scale bars: 5 µm in B,D,E and 500 nm in C.

View larger version (77K): [in this window] [in a new window]   Fig. 2. Furrow canal compartments are compromised in nulloX embryos. (A,B) Confocal images of cellularizing embryos. (A) Cross-sections showing tear-shaped F-actin furrow canals (green) that lack Dlg staining (red) in wild-type embryos as compared with flattened furrow canals with significant Dlg staining in nulloX embryos. (B) Cross-sections showing tear-shaped Myosin 2 furrow canals (Myo-2, green) that lack Nrt staining (red) in wild-type embryos as compared with flattened furrow canals with significant Nrt staining in nulloX embryos. (C,D) TEM images showing furrows ingressing in cellularizing nulloX embryos. Arrows indicate the position of furrow canal, shown at higher magnification in the right-hand images. (C) Furrow canal broadenings are often collapsed at the furrow tip (arrowheads in the higher-magnification image). (D) Alternatively, furrow canals may contain PM blebs and show accentuated basal flattening (arrowheads). Scale bars: 5 µm in A,B and 2 µm in C,D.

View larger version (88K): [in this window] [in a new window]   Fig. 3. The absence of Myosin 2 from furrow canals precipitates furrow regression in nulloX embryos. (A,B,D) Confocal images of PM furrows (Nrt, red) and Myosin 2 furrow canals (Myo-2, green) ingressing between adjacent nuclei in cellularizing embryos. (A) En face images from a single plane at the level of the furrow canals showing that in nulloX embryos, some furrows are missing (arrows) or have no Myosin 2 in the furrow canals (arrowheads). (B) Projected z-section from a nulloX embryo showing adjacent nuclei separated by: (1) a furrow with Myosin 2; (2) a furrow with no Myosin 2; or (3) a missing furrow. (C) The average percentage of disrupted furrows at progressive stages of cellularization in nulloX embryos grown at 23°C or 29°C (total disrupted furrows, blue; furrows with no Myosin 2, red; furrows missing, green). Each point represents 4-6 cellularizing embryos of the given furrow length, with 200 interfaces scored per embryo. Error bars represent s.d. (D) Cross-sections at progressive phases of cellularization showing furrows with Myosin 2 between all nuclei in wild-type embryos. In nulloX embryos, some furrows are missing (arrows) or have no Myosin 2 in the furrow canals (arrowhead). (E) Kymographs showing furrow dynamics (GFP-Spider) in live cellularizing embryos (taken from supplementary material Movies 1 and 2). Three types of furrow dynamics are seen in nulloX embryos: (1) furrows that ingressed 40 µm at rates comparable to those of the wild type; (2) furrows that ingressed to lengths >5 µm then regressed; and (3) furrows that ingressed to a length of only 5 µm then regressed. Arrows follow the tip of the regressing furrows. (F) En face confocal image from a single plane at the level of the furrow canals showing PM furrows (Nrt, red) and Anillin furrow canals (green) in cellularizing nulloX embryos. Some furrows are missing (arrows) or have no Anillin in the furrow canals (arrowheads). Scale bars: 5 µm in A,B,D,F.

View larger version (109K): [in this window] [in a new window]   Fig. 4. Basal junctions do not assemble in arm043A01 embryos, but functional furrow canal compartments are established and maintained. (A-C) Confocal images of PM (Nrt, red) and basal junctions (Arm, green) in cellularizing embryos. (A) En face images from a single plane at the level where basal junctions should be, showing that truncated Arm fails to accumulate in basal junctions in arm043A01 mutants. (B) Cross-sections of a wild-type embryo showing furrows ingressing between all nuclei and Arm accumulating at basal junctions (arrowheads). (C) Cross-sections of an arm043A01 mutant showing furrows ingressing between all nuclei, but no Arm accumulating at basal junctions. Truncated Arm instead accumulates apically (arrows). (D,E) TEM images showing furrows ingressing in cellularizing embryos. Arrows indicate the furrow shown at higher magnification in the right-hand images, and arrowheads indicate the position where the basal junctions should be. (D) In wild-type embryos, adjacent PMs are tightly apposed. (E) In arm043A01 embryos, adjacent PMs gape apart. Furrow tips broaden into furrow canals despite the absence of basal junctions. (F,G) Confocal images of Myosin 2 furrow canals (Myo-2, green) in cellularizing embryos. (F) En face images from a single plane at the level of the furrow canals at progressive phases of cellularization showing that furrow canals form around all nuclei(Early) and later constrict (Late). (G) Cross-section of arm043A01 mutant showing tear-shaped furrow canals that lack staining for a lateral PM probe (Nrt, red). (H) Cross-sections showing F-actin (phalloidin) in cellularizing embryos. Images collected at the same settings show no difference between F-actin levels for wild-type versus arm043A01 embryos. (I) F-actin levels quantified in furrow canals of wild-type (black) versus arm043A01 (red) embryos at progressive phases of cellularization, as measured by fluorescent intensity of phalloidin staining. Each point represents one embryo in which 75-100 furrow canals were analyzed. Error bars represent s.d. Scale bars: 5 µm in A-C,F-H and 2 µm in D,E.

View larger version (66K): [in this window] [in a new window]   Fig. 5. Furrow canal compartments are established and maintained in anillin-deficient embryos. (A-E) Confocal images of cellularizing embryos derived from anillinHP/RS or wild-type mothers. (A) En face images from a single plane, showing that Septin (green) fails to accumulate in furrow canals [Myosin 2 (Myo-2), red] of anillin-deficient mutants. (B) Cross-section of an anillin-deficient mutant showing that furrow canals (Myo-2, green) lack staining for a lateral PM probe (Dlg, red). (C) Cross-section of an anillin-deficient mutant showing that collapsed furrow canals (Myo-2, green) lack staining for a lateral PM probe (Nrt, red). (D) En face images from a single plane, showing that Nullo (green) accumulates normally in furrow canals of anillin-deficient mutants. (E) Cross-sections showing F-actin (phalloidin) in cellularizing embryos. Images collected at the same settings show no difference between F-actin levels for wild-type versus anillin-deficient embryos. (F) F-actin levels quantified in furrow canals of wild-type (black) versus anillin-deficient (red) embryos at progressive phases of cellularization, as measured by fluorescent intensity of phalloidin staining. Each point represents one embryo in which 75-100 furrow canals were analyzed. Error bars represent s.d. Scale bars: 5 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 6. F-actin disruption mimics nulloX phenotypes. (A-E) Confocal images of PM furrows ingressing between adjacent nuclei in cellularizing wild-type embryos treated with Cyto-D. (A) En face images collected in a single plane at the level of the furrow canals showing that in embryos treated with a low dose of Cyto-D, some furrows (Dlg, red) are missing (arrows) or have no Myosin 2 (Myo-2, green) in the furrow canals (arrowheads). The furrow canal network is severely disrupted in embryos treated with higher doses of Cyto-D. (B) En face image collected in a single plane at the level of the furrow canals showing that in embryos treated with 1 µg/ml Cyto-D, some furrows (Nrt, red) are missing (arrows) or have no Anillin (green) in the furrow canals (arrowheads). (C) Cross-section from an embryo treated with 1 µg/ml Cyto-D showing Myosin 2 furrow canals (green) are absent from some furrows (Dlg, red; arrowheads). (D) Cross-section from an embryo treated with 1 µg/ml Cyto-D showing Dlg (red) spreading into some Myosin 2 furrow canals (green; arrowheads). (E) Cross-section from an embryo treated with 1 µg/ml Cyto-D showing some furrows (Nrt, red) are missing between adjacent nuclei (arrow) or have no Anillin (green) in the furrow canals (arrowhead). (F,G) Confocal images of PM furrows (Nrt, red) and basal junctions (Arm, green) in cellularizing wild-type embryos treated with 1 µg/ml Cyto-D. (F) Cross-sections showing basal junction defects, ranging from furrows that have some basal Arm accumulation (arrowhead) to those that have none (arrows). (G) En face images from a single plane at the level of the basal junctions showing basal junction defects, ranging from Arm plaques at some furrows (arrowheads) to diffuse Arm puncta at others (arrows). (H) En face images from a single plane, showing that Nullo (green) accumulates normally in furrow canals of Cyto-D-treated embryos. Scale bars: 5 µm.

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