Coupling of apoptosis and L/R patterning controls stepwise organ looping - PubMed (original) (raw)

Coupling of apoptosis and L/R patterning controls stepwise organ looping

Magali Suzanne et al. Curr Biol. 2010.

Abstract

Handed asymmetry in organ shape and positioning is a common feature among bilateria, yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. We utilize the directional 360° clockwise rotation of genitalia in Drosophila to study LR-dependent organ looping. Using time-lapse imaging, we show that rotation of genitalia by 360° results from an additive process involving two ring-shaped domains, each undergoing 180° rotation. Our results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. We further reveal a specific pattern of apoptosis at the ring boundaries and show that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180° LR module.

Copyright © 2010 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1. Genital disc organisation and timing of genitalia circumrotation

a) The upper panel shows a schematic front view of a pupal genital disc. Segments A8, A9 and A10 are organized into concentric rings. Green, A8 anterior compartment (A8a); red, A8 posterior compartment (A8p); black stripes, MyoID expression in A8a and A8p. The lower panel shows the two rows of MyoID expression (green) with A8a and A8p outlined with white dotted lines; hhDsRed (red) marks the posterior compartments of all segments. b) Distinct stages of genitalia rotation are shown (0°, 90°, 180°, 270° and 360°). Genitalia rotation takes place during pupal development and lasts for 12–15 hrs. Upper panel, schematic representation of the progressive looping of the spermiduct (Sp) around the gut. Lower panel, 3D reconstructions of confocal images from pupal genital discs at different angles (0°, 90°, 180°, 270° and 360°). Green, armGFP; red, hhDsRed. c) Scheme of the mounting setup used for live imaging of the genital disc in pupae.

Figure 2

Figure 2. Circumrotation involves two distinct rotating domains

a,b) Still confocal images taken from AbdB>CD8-GFP; HhDsRed live pupae (Movies S1 and S2). AbdB>CD8-GFP, green; HhDsRed, red. Upper panels show whole discs. Lower panels are close-up of the region shown in dotted-lines. Right panel, summary of cell movements. The A8p/A8a boundary is indicated by a doted-line; tracking of A8p, A8a and abdominal cells is shown in red, green and blue, respectively. a) Early A8p rotation. Note the directed clockwise movement of the A8p cells (indicated by white lines and arrows). At this stage, A8a cells show a stochastic movement (see Supplemental Movie S1). b) Early A8a rotation. The A8a cells begin to move clockwise whereas cells from the abdomen remain still (see Supplemental Movie S2).

Figure 3

Figure 3. Autonomy and additive function of A8 rings during circumrotation

Absolute (a) and relative (b) movements of A8a and A8p rings in an AbdB>CD8-GFP; hhDsRed pupae. a) Still confocal images taken from Supplemental Movie S3 showing full rotation of wild type genitalia (A8a+A8p+ context). b) Relative movement of A8p. Same Movie as in (a), with the A8a domain reoriented to serve as a referential, revealing the 180° rotation of A8p. AbdB>CD8-GFP (green) marks the A8a domain and hhDsRed (red) marks the A8p, A9p and A10p domains. Dotted lines and arrows indicate the angular progression of A8a (in green) and A8p (in red). A8a and A8p domains are outlined (dotted white lines on the upper left picture). The curves on the right describe the angular variation and the angular velocity of the A8p and A8a domains. c,d) Relative movement of A8 rings in A8a−/A8p+ (c) and A8a+/A8p− (d) contexts. Upper panel, absolute movement with merged channels. Lower panels, relative movement with A8a serving as a reference. The curves on the right represent the angular variation of the A8p and A8a domains. Genotypes are as follows: UAS-CD8-GFP/+; AbdB>MyoIDRNAi/hhDsRed (c) and ptcDsRed/+; hh>MyoIDRNAi/UAS-CD8-GFP (d) Images are from Supplemental Movie S4 (c) and S5 (d). d) Colors have been inverted in the acquisition software to keep the same colour code (A8p in red, A8a in green). e) Equation showing the additive and modular nature of circumrotation.

Figure 4

Figure 4. Cell death pattern and role in circumrotation

a) Still confocal images taken from movie S6. Wild type rotation of hhDsRed genitalia. Nuclear fragmentation and cell removal are followed by manual tracking in the A8p domain. Individual dying cells as well as the region showing the highest concentration of apoptosis are outlined in white. Lower panel are higher magnification of images of the same movie showing the nuclear fragmentation of a dying cell before its removal (outlined in red). b) Still confocal images taken from movie S7, showing wild type rotation of MyoID>Apoliner /armGFP genitalia. Cells in which caspases are activated show nuclear GFP staining (region showing highest concentration of cell fragmentation and removal is outlined in white). Note the presence of dying cells at the anterior boundaries of A8p and A8a, at the beginning of their respective movements. No cell death is observed when rotation is over. Lower panels are close-up of the region shown in dotted-lines. c) Histogram presenting the percentage of the different classes of genitalia rotation phenotypes observed in wild type (black), AbdB>p35 (green), hh>p35 (red) or AbdB+hh>p35 (red and green stripes). d) Still confocal images taken from Supplemental Movie S8 showing the rotation of an AbdB>p35 genital disc. The curves on the right describe the angular variation and the angular velocity of the A8p and A8a domains (compare with wt rotation in Fig. 3b). Note that the 180° rotation is due exclusively to the movement of A8p, whereas A8a stays mostly still during the whole process. e) Model summarising the succession of events taking place in each rotating module, including early LR determination and direction through MyoID (1), and late apoptosis at the boundary of each rotating ring (2). Note that the apoptotic domain is included in the MyoID expression domain.

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