Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jul 1;143(13):2417-30.
doi: 10.1242/dev.131334. Epub 2016 May 25.

Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity

Bing He  1 , Adam Martin  2 , Eric Wieschaus  2
Affiliations

Flow-dependent myosin recruitment during Drosophila cellularization requires zygotic dunk activity

Bing He et al. Development. .

Abstract

Actomyosin contractility underlies force generation in morphogenesis ranging from cytokinesis to epithelial extension or invagination. In Drosophila, the cleavage of the syncytial blastoderm is initiated by an actomyosin network at the base of membrane furrows that invaginate from the surface of the embryo. It remains unclear how this network forms and how it affects tissue mechanics. Here, we show that during Drosophila cleavage, myosin recruitment to the cleavage furrows proceeds in temporally distinct phases of tension-driven cortical flow and direct recruitment, regulated by different zygotic genes. We identify the gene dunk, which we show is transiently transcribed when cellularization starts and functions to maintain cortical myosin during the flow phase. The subsequent direct myosin recruitment, however, is Dunk-independent but requires Slam. The Slam-dependent direct recruitment of myosin is sufficient to drive cleavage in the dunk mutant, and the subsequent development of the mutant is normal. In the dunk mutant, cortical myosin loss triggers misdirected flow and disrupts the hexagonal packing of the ingressing furrows. Computer simulation coupled with laser ablation suggests that Dunk-dependent maintenance of cortical myosin enables mechanical tension build-up, thereby providing a mechanism to guide myosin flow and define the hexagonal symmetry of the furrows.

Keywords: Actomyosin network; Cellularization; Cortical myosin recruitment; Cytokinesis; Dunk.

PubMed Disclaimer

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Biphasic recruitment of myosin to the invagination front during cellularization. (A-C) Schematics showing the midsagittal view (top) and the en face view (bottom) of a cellularizing embryo. Red, actomyosin; blue, nucleus; green, basal adherens junction. Actin and myosin first accumulate at the invagination front and form a hexagonal network (A). As cellularization proceeds, the actomyosin network reorganizes into individual actomyosin rings (B) which then constrict to close the basal side of the newly formed cells (C). (D) 3D rendering of Sqh-GFP movies demonstrating cortical myosin flow during early cellularization. Shown is a single pair of daughter nuclei. Color coding corresponds to the depth of myosin structures from the apical surface. (E) Projections of Sqh-GFP at the invagination front that correspond to the 3D images in D. Red dotted lines highlight the outline of the two daughter nuclei. Yellow dotted lines correspond to the axes that are parallel or perpendicular to the newly formed edge between the two daughter nuclei. Arrows in D and E indicate the position of the new furrow. Scale bar: 5 µm. (F) Schematics demonstrating the separate phases of myosin recruitment during cellularization. Red, myosin; blue, nucleus. Arrows on the apical surface indicate the direction of myosin flow. (G) Kymographs corresponding to the yellow lines in E, showing myosin flow perpendicular (left) or parallel (right) to the edge. Scale bars: 5 min (x); 5 µm (y). (H) An example of particle image velocimetry (PIV) analysis of cortical Sqh-GFP during the flow phase. The velocity map corresponds to the boxed region in the image above. (I) Measurement of the width of the myosin band at newly formed edges (n=4 edges). Gray area indicates ±s.d. (J) Measurement of total myosin intensity at the invagination front (n=3 embryos). Gray area indicates ±s.d.
Fig. 2.
Fig. 2.
The actomyosin network is under tension. (A) Laser ablation of a newly formed cleavage furrow in an embryo expressing Sqh-GFP. Red dotted lines mark the outline of the previous mitotic figure. The red cross marks the site of laser ablation. Arrows highlight the retraction of tissues from the incision site. Scale bar: 5 µm. (B) Laser ablation at the apical cortex in an embryo at cycle 13 anaphase. Red dotted lines mark the outline of the mitotic figure. Scale bar: 5 µm. (C) An example of PIV analysis of Sqh-GFP immediately after laser ablation. The velocity map corresponds to the images shown in A. (D) Average velocity map immediately after laser ablation. N=24 ablations in eight embryos. (E) Average velocity distribution along the boxed regions in D. Error bars indicate s.d.
Fig. 3.
Fig. 3.
dunk is transiently transcribed at the onset of cellularization. In situ hybridization of wild-type embryos with an antisense dunk probe. Note that dunk is transiently induced immediately before cellularization starts and rapidly diminishes during late cellularization. Arrows indicate the depth of invagination font. Boxed regions are enlarged to the right. Scale bar: 100 µm.
Fig. 4.
Fig. 4.
dunk1 mutant embryos fail to maintain myosin at the cortex during the flow phase. (A-C) Immunostaining (A,C) or phalloidin staining (B) showing localization of myosin (Zipper) (A), F-actin (B) and Rho1 (C) in cross-sections (top) and projections of confocal sections at the invagination front (bottom). In dunk1 mutant embryos at early cellularization, myosin shows inhomogeneous distribution at the invagination front, being preferentially enriched at the vertices (red arrows) but depleted from the edges (green arrows). By contrast, the distributions of F-actin (B) and Rho1 (C) at the invagination front remain homogeneous. During mid-cellularization, although actin and myosin can still form individual rings, the rings are less rounded and frequently highly angular. Scale bars: 25 µm (top); 10 µm (bottom). (D) Projections of confocal sections showing Sqh-GFP at the invagination front in wild-type and dunk1 mutant embryos over time. Note that myosin distribution is abnormally inhomogeneous in dunk1 mutant embryos at t=8 min. Myosin preferentially accumulates at vertices (red arrows) and is depleted from edges (green arrows). Scale bars: 10 µm. (E) Quantification of total myosin intensity at the invagination front. Error bars indicate s.d. (F) Left panel: ratio between vertex- and edge-myosin intensities in wild-type (blue) and dunk1 mutant (red) embryos. Error bars indicate s.d. Right panel: schematic showing quantification of myosin intensity at vertices (magenta) and edges (yellow).
Fig. 5.
Fig. 5.
Myosin flow is destabilized in dunk1 mutant embryos. (A,B) Two examples demonstrating distinct myosin behaviors at newly formed edges in dunk1 mutant embryos. In each example, the left panel shows projections of confocal sections at the invagination front, and the right panel shows the 3D rendering of the corresponding Sqh-GFP movies. Color coding corresponds to the depth of the myosin structures from the apical surface. Red dotted lines outline the two daughter nuclei. Yellow dotted lines correspond to the axes that are parallel and perpendicular to the newly formed edge between the two daughter nuclei. Arrows in A and B demonstrate the stretching and shortening of the new furrow, respectively. Scale bars: 5 µm. (C,D) Kymographs corresponding to the yellow lines in A and B, respectively. Arrows highlight the divergence (C) or convergence (D) of myosin trajectories in the direction parallel to the new furrow. Scale bars: 5 min (x); 5 µm (y). (E,F) PIV analysis of Sqh-GFP movement shown in A and B, respectively. The velocity map corresponds to the boxed region in the image above. (G) Correlation between the length of edge and the myosin intensity along the edge at different times after the onset of cellularization. Wild type: n=3 embryos; dunk1 mutant: 5 embryos. (H) Immunostaining of Armadillo (Drosophila β-catenin homolog) showing the distribution of basal adherens junctions in wild-type and dunk1 mutant embryos at early cellularization. Scale bars: 10 µm.
Fig. 6.
Fig. 6.
Computer simulation demonstrates the link between cortical myosin dynamics and the anisotropy of the myosin flow. (A) Implementation of an interconnected contractile network. The contractile force is applied along each interface between neighboring myosin nodes. Within the network, myosin nodes undergo dynamic turnover with a recruitment rate constant of kon and a dissociation rate constant of koff. (B) The model configuration at t=8000 simulation steps when kon≫koff ([kon, koff]=[0.05, 0.001]). Myosin nodes undergo anisotropic flow similar to that observed in the wild-type embryos. (C) The model configuration at t=8000 simulation steps when kon/koff∼1 ([kon, koff]=[0.003, 0.001]). The simulation recapitulates the destabilized myosin flow and vertical accumulation of myosin observed in dunk1 mutant embryos. (D,E) Projections of confocal sections showing Sqh-GFP at the invagination front in a wild-type (D) and dunk1 mutant (E) embryo at t=2 min (top) and t=12 min (bottom). Scale bars: 5 µm.
Fig. 7.
Fig. 7.
Dunk colocalizes with myosin at the invagination front during early and mid-cellularization. (A) Immunostaining of sqhAX3; sqh-GFP embryos with anti-Dunk and anti-GFP antibodies. Dunk co-localizes with Sqh-GFP at the invagination front during early and mid-cellularization but becomes undetectable at late cellularization. Left panels: mid-sagittal view; right panels: en face view. Arrowheads indicate the colocalization of Dunk and myosin at the old furrows surrounding the previous mitotic figure. Arrows indicate the enrichment of Dunk at the furrows between neighboring nuclei where myosin puncta are also enriched. Scale bars: 10 µm. (B) Immunostaining of wild-type (top) and dunk1 mutant (bottom) embryos with anti-Dunk and anti-Zipper (MHC) antibodies. Arrows highlight the colocalization of Dunk and myosin as myosin becomes enriched at the furrows at the onset of cellularization. Scale bars: 10 µm. (C) Immunostaining of wild-type embryos with anti-Dunk, anti-Zipper and anti-DE-cadherin antibodies. Dunk and myosin colocalize at the invagination front (arrows); the basal adherens junctions localize immediately apical to Dunk and myosin (arrowhead). Scale bars: 10 µm. (D-F) Ectopically expressed Dunk-3HA localizes to contractile actomyosin structures (arrows) such as the apical actomyosin network in posterior midgut (D) or ventral furrow (E), and cytokinetic rings in dividing cells (F). Scale bars: 30 µm (D,E); 5 µm (F).
Fig. 8.
Fig. 8.
Slam and Dunk function in different phases of myosin recruitment during cellularization. (A) Projections of confocal sections showing Sqh-GFP at the invagination front (Sqh-GFP) in slam and slam dunk1 mutant embryos during the recruitment phase. Arrows highlight edges that remain devoid of myosin throughout cellularization. Scale bars: 10 µm. (B) Quantification of total myosin intensity at the invagination front as percentage of intensity at t=2 min. Error bars indicate s.d. (C) Immunostaining showing localization of myosin (Zipper) and the plasma membrane marker Neurotactin in cross-sections (top) and projections of confocal sections at the invagination front (bottom) in slam and slam dunk1 mutant embryos. Arrowheads highlight the discrete myosin foci in slam dunk1 mutant embryos. Arrows highlight the partially connected actomyosin network in slam single mutant embryos. Scale bars: 50 µm (top); 25 µm (bottom).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Acharya S., Laupsien P., Wenzl C., Yan S. and Großhans J. (2014). Function and dynamics of slam in furrow formation in early Drosophila embryo. Dev. Biol. 386, 371-384. 10.1016/j.ydbio.2013.12.022 - DOI - PubMed
    1. Adam J. C., Pringle J. R. and Peifer M. (2000). Evidence for functional differentiation among Drosophila septins in cytokinesis and cellularization. Mol. Biol. Cell 11, 3123-3135. 10.1091/mbc.11.9.3123 - DOI - PMC - PubMed
    1. Afshar K., Stuart B. and Wasserman S. A. (2000). Functional analysis of the Drosophila diaphanous FH protein in early embryonic development. Development 127, 1887-1897. - PubMed
    1. Beach J. R. and Egelhoff T. T. (2009). Myosin II recruitment during cytokinesis independent of centralspindlin-mediated phosphorylation. J. Biol. Chem. 284, 27377-27383. 10.1074/jbc.M109.028316 - DOI - PMC - PubMed
    1. Behrndt M., Salbreux G., Campinho P., Hauschild R., Oswald F., Roensch J., Grill S. W. and Heisenberg C.-P. (2012). Forces driving epithelial spreading in zebrafish gastrulation. Science 338, 257-260. 10.1126/science.1224143 - DOI - PubMed

LinkOut - more resources