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. 2012;7(3):e33089.
doi: 10.1371/journal.pone.0033089. Epub 2012 Mar 21.

Mechanical force alters morphogenetic movements and segmental gene expression patterns during Drosophila embryogenesis

Abhishek Kumar  1 , G V Shivashankar
Affiliations

Mechanical force alters morphogenetic movements and segmental gene expression patterns during Drosophila embryogenesis

Abhishek Kumar et al. PLoS One. 2012.

Abstract

The development of an organism is accompanied by various cellular morphogenetic movements, changes in cellular as well as nuclear morphology and transcription programs. Recent evidence suggests that intra and inter-cellular connections mediated by various adhesion proteins contribute to defining nuclear morphology. In addition, three dimensional organization of the cell nucleus regulate the transcription programs. However the link between cellular morphogenetic movements and its coupling to nuclear function in a developmental context is poorly understood. In this paper we use a point perturbation by tissue level laser ablation and sheet perturbation by application of force using magnetic tweezers to alter cellular morphogenetic movements and probe its impact on nuclear morphology and segmental gene expression patterns. Mechanical perturbations during blastoderm stage in a developing Drosophila embryo resulted in localized alterations in nuclear morphology and cellular movement. In addition, global defects in germ-band (GB) extension and retraction are observed when external force is applied during morphogenetic movements, suggesting a long-range physical coupling within the GB layer of cells. Further local application of force resulted in redistribution of non muscle myosin-II in the GB layer. Finally these perturbations lead to altered segmental gene (engrailed) expression patterns later during the development. Our observations suggest that there exists a tight regulation between nuclear morphology and cellular adhesive connections during morphogenetic movement of cells in the embryo. The observed spatial changes in patterning genes, with perturbation, highlight the importance of nuclear integrity to cellular movement in establishing gene expression program in a developmental system.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nuclear morphology during morphogenetic movement- large scale collective movement of germ band positions cells in different regions of the embryo.
(A) Maximum intensity z-projected confocal time lapse images of single live embryo showing movement of nuclei, marked by H2B-EGFP, at the dorsal side. White arrows indicate the position of germ band front at different times (Cellular blastoderm stage, during GBE, two-third extension of GBE, GBR, dorsal closure and segmented embryo respectively, corresponding time points are indicated at the top of each image). Scalebar = 100 µm. A-P denotes anterior and posterior axis of embryo. (B) Panel of images showing change in nuclear morphology starting from cellular blastoderm stage Scalebar = 10 µm. Region of interest (ROI) was chosen in the posterior half at the dorsal side of the embryo. (C) Typical plot of GB front displacement with time, measured from the posterior side. GB front moves towards the anterior side during elongation and comes towards the posterior during retraction leaving a sheet cells called amnioserosa (1, 2 and 3 are the positions when external perturbations are applied). Inset shows quantification of length of embryo and the extent of GB elongation measured (N = 32). EL = egg length (D) Two dimensional tracks of individual nuclei tracked during GBE measured by their centroid positions from Fig. 1A. Nuclei from posterior end were tracked. Direction of movement of cells is bottom to top. (E) Change in mean circularity and its standard deviation (SD) for nuclei at the dorsal side in a single embryo with time. 0′ in all these cases correspond to blastoderm stage.
Figure 2
Figure 2. Perturbation techniques used and its effect on global movement of nuclei inside the embryo.
(A) Schematic of perturbation technique used: Tissue level ablation at the dorsal side using 835 nm NIR (near infra red) multiphoton laser and application of external force (1.5 amp for 1.7 min) using a magnetic tweezers on embryo injected with 100 nm paramagnetic beads. (B) Time lapse images of GB front position post application of external force during GBE. Scalebar = 50 µm. 0 min' corresponds to start of GBE at the dorsal side. (C) Time lapse images of GB front position post application of external force post 2/3rd extension of GB respectively. Scalebar = 100 µm. 0 min corresponds to time just before when GBR starts. White arrows in (B) and (C) indicate the position of GB front at times indicated on each image (D) Quantification of GBE, post ablation of a group of cells (15–20) at 1/3rd 2/3rd egg length and control embryo. (E) and (F) Comparison of GBE and GBR with the control embryos when force of applied during GBE (a typical plot) and post 2/3rd extension of GB respectively (black arrows indicate times at which force is applied). N≥3. Error bars are standard deviations.
Figure 3
Figure 3. Reversible and irreversible modulation of nuclear size using force exerted by magnetic tweezers.
(A) Panel shows the effect of application of external force (electromagnet was placed at the left side of the image) on nuclear size and (B) position (corresponding XY nuclear trajectory). Scalebar = 10 µm. (C) Normalized nuclear area is plotted with time (shaded region shows the time for which external force is applied). (D) Nuclear displacement with time on application of force is plotted indicating that external force displaces the nuclear position inside the embryo. For reversible case, N = 3; for irreversible, N = 4. ROI was chosen in the posterior half at the dorsal side of the embryo.
Figure 4
Figure 4. Localization and dynamics of non muscle myosin II during morphogenesis and on application of external force.
(A) Panel shows the images pre bleaching 0″ and post bleaching- 10″, 20″, 45″ and 120″ at different stages of Drosophila embryo development as well as different regions in the embryo. First row: Pre-blastoderm stage (Bownes stage -4), second row: Blastoderm stage (Bownes stage -5), third row: anterior region during slow phase of GBE (early Bownes stage -9), fourth row: posterior region during slow phase of GBE (early Bownes stage -9). Circle and arrow highlights the region where photo-bleaching is performed. Scale bar = 5 µm (B) Plot shows the FRAP curves of EGFP tagged non muscle myosin II at different stages and regions inside a live embryo. (Filled Triangle: stage 4, Filled square: anterior stage 9, Filled circle: posterior stage 9 and open circle: posterior stage 9 after application of force) (C) Bar graphs show fractional recovery after 110 sec of bleaching in all the above cases. Curves and bar graph shown here are average of 7,13, 18, 27 and 8 curves for preblastoderm, blastoderm, GBE-anterior, GBE- posterior and GBE-posterior after force respectively. Single plane at the dorsal side was imaged for FRAP.
Figure 5
Figure 5. Effect of perturbation on engrailed expression and symmetry in patterning.
(A) Panel shows the effect of application of external force during different stages of development from the posterior end on engrailed pattern 20 hour after egg laying. First row shows nuclear images marked by H2B tagged with EGFP. Second row is the engrailed pattern in the same embryo visualized by mRFP (;en-Gal4-UAS-myr-mRFP;). Third row is the merge of the above two. First column shows engrailed pattern in control embryo injected with 100 nm paramagnetic beads but without application of force while other columns show engrailed pattern post application of force during blastoderm stage (second and third column), during GBE (fourth column) and post 2/3rd GBE (fifth column). For reversible case, N+/Ntotal = 17/21; irreversible case, N+/Ntotal = 76/89; during GBE, N+/Ntotal = 7/9; post 2/3rd, N+/Ntotal = 8/10. Scalebar = 100 µm. (B) Panel shows differential effect of application of force on engrailed patterning. Force is applied post 2/3rd GBE and force protocol is as shown (1.5 amp for 1.7 min) either from right (N+/Ntotal = 4/7) or left (N+/Ntotal = 3/5) side of the embryo (corresponding schematic shown on left). All the embryos were imaged at the dorsal side and force was applied from the posterior side.
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References

    1. Houchmandzadeh B, Wieschaus E, Leibler S. Establishment of developmental precision and proportions in the early Drosophila embryo. Nature. 2002;415:798–802. - PubMed
    1. Keller R. Cell migration during gastrulation. Curr Opin Cell Biol. 2005;17:533–541. - PubMed
    1. Leptin M. Drosophila gastrulation: from pattern formation to morphogenesis. Annu Rev Cell Dev Biol. 1995;11:189–212. - PubMed
    1. Struhl G, Johnston P, Lawrence PA. Control of Drosophila body pattern by the hunchback morphogen gradient. Cell. 1992;69:237–249. - PubMed
    1. McMahon A, Supatto W, Fraser SE, Stathopoulos A. Dynamic analyses of Drosophila gastrulation provide insights into collective cell migration. Science. 2008;322:1546–1550. - PMC - PubMed

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