Cell Sheet Morphogenesis: Dorsal Closure in Drosophila melanogaster as a Model System

Abstract

Dorsal closure is a key process during Drosophila morphogenesis that models cell sheet movements in chordates, including neural tube closure, palate formation, and wound healing. Closure occurs midway through embryogenesis and entails circumferential elongation of lateral epidermal cell sheets that close a dorsal hole filled with amnioserosa cells. Signaling pathways regulate the function of cellular structures and processes, including Actomyosin and microtubule cytoskeletons, cell-cell/cell-matrix adhesion complexes, and endocytosis/vesicle trafficking. These orchestrate complex shape changes and movements that entail interactions between five distinct cell types. Genetic and laser perturbation studies establish that closure is robust, resilient, and the consequence of redundancy that contributes to four distinct biophysical processes: contraction of the amnioserosa, contraction of supracellular Actomyosin cables, elongation (stretching?) of the lateral epidermis, and zipping together of two converging cell sheets. What triggers closure and what the emergent properties are that give rise to its extraordinary resilience and fidelity remain key, extant questions.

Keywords: Actomyosin; amnioserosa; biomechanics; ingression; morphogenetics; oscillation.

Figure 1

Overview of Drosophila embryogenesis from extended germ band to completion of dorsal closure. (a–d) Low-resolution confocal images of a Drosophila embryo expressing a protein that fuses GFP to the Actin binding domain of Drosophila Moesin (GFP-Moe-ABD), which labels F-Actin (the embryo is otherwise essentially wild type; U.S. Tulu & D.P. Kiehart, unpublished). (a′--d′) Schematics traced from panels a to d. Red tissue is amnioserosa. (a,a′) Extended germ band stage. (b,b′) End of germ band retraction. Canthi are not yet formed. Arrows depict the direction of movement of the leading edge of the lateral epidermis. Forces from either flank are equal; arrows from the far side of the embryo depicted are shorter because their origins lie behind the bulging amnioserosa. (c,c′) Canthi have formed (ACa and PCa denote anterior and posterior canthus, respectively), and the position of the purse strings (PS) near the leading edge of the advancing dorsal-most epidermal cells is shown. (d,d′) Late closure. The lateral epidermal sheets have met at the dorsal midline; the amnioserosa has been internalized; and a seam remains, as the purse strings have not yet completely disassembled.

Figure 2

Integrins mediate close attachment of the amnioserosa (AS) to the surface of the yolk and the rearrangement of the interface between the dorsal-most epidermal (DME) and peripheral-most amnioserosa (PAS) cells. (a) The AS as it adheres to the surface of the yolk during four stages of germ band retraction (redrawn with permission from Reed et al. 2004). The yolk is in red, the germ band is in blue, and the AS is in black. (b) Micrographs show successive stages of the AS (arrow) adhering to the surface of the yolk (reproduced with permission from Reed et al. 2004). (c) Schematic showing the rearrangement of the relationship between the lateral epidermis (blue) and the AS (yellow) at the DME/PAS interface (arrowhead) in wild-type embryos. (d) Schematic drawn from panel e showing the interface between the AS (yellow) and the lateral epidermis (blue) at the DME/PAS interface (arrowhead) in wild-type (upper panel) and Integrin mutant (bottom panel) embryos. The AS, the DME cell, and the rest of the lateral epidermis do not adhere properly to the yolk in Integrin mutant embryos (double arrow). (e) Micrograph (reproduced with permission from Narasimha & Brown 2004) from which panel d was drawn. The position of the DME cell is shown.

Figure 3

Shape of the dorsal opening and canthus formation. Confocal micrographs of living embryos that are essentially wild type other than carrying a ubiquitin-driven transgene that encodes GFP-Cadherin. Anterior is left and posterior is right in all panels. (a) At the end of germ band retraction, the dorsal hole has a blunt anterior end and a rounded posterior end. (b–d) Canthus formation at the anterior end. (e–g) Canthus formation at the posterior end. Panel a is from one embryo, and panels b through g are from another embryo. Seams that are obvious in embryos labeled for F-Actin or Myosin are not so obvious in GFP-Cadherin embryos (see the beginning of anterior seam formation in panel c and compare it to the formed, posterior seam shown in Figure 4d). Images were provided by Stephanie Fogerson.

Figure 4

The leading edge accumulates F-Actin to form the supracellular purse string in the dorsal-most epidermal (DME) cells and is transformed from scalloped to smoothly curved. Shown are confocal images of a Drosophila embryo expressing a protein that fuses GFP to the Actin binding domain of Drosophila Moesin (GFP-Moe-ABD), which labels F-Actin (the embryo is otherwise essentially wild type). Posterior is to the left and anterior is to the right in all panels. (a) Near the end of germ band retraction, but before the onset of dorsal closure. The leading edge of the lateral epidermis is scalloped, and a few DME cells have begun to accumulate F-Actin (arrowheads show accumulation). (b) Just prior to the onset of closure, F-Actin has begun to accumulate (arrowheads). (c) By the onset of closure, F-Actin has accumulated in virtually all cells. (d) A robust purse string (arrowhead) has formed. The leading edge also accumulates Myosin in a pattern akin to bars on a string (not shown; see Young et al. 1993, Franke et al. 2005). Previously unpublished images provided by R. Montague, V. Williams & D.P. Kiehart.

Figure 5

Dorsal-most epidermal (DME) cells remain associated with peripheral-most amnioserosa (PAS) cells throughout the duration of closure. (a–e) Micrographs depict the morphogenesis of the amnioserosa (AS) from mid- to late closure. The AS is brightly labeled with a protein that fuses GFP to the Actin binding domain of Drosophila Moesin (GFP-Moe-ABD), which labels F-Actin and whose expression is driven by a mostly AS-specific promoter/enhancer complex (Gal4-C381). The arrows in panel a depict the location of the leading edges of the DME cells. Some cells in the lateral epidermis also weakly express the F-Actin label GFP-Moe-ABD. Panels a–e reproduced with permission from figure 1 (panels g, h, i, j, and l) in Rodriguez-Diaz et al. (2008). (f) Schematic of a cross section of the DME, PAS, and bulk of the AS during closure. The progression of closure (early is at the top) and corresponding tissue arrangements are shown in successive time points during closure. The schematic illustrates the relationship between the DME cells and the PAS cells (cells labeled 1 and 9, respectively), which remain tightly associated with one another as dorsal closure proceeds. The cells in the bulk of the AS (cells labeled 2 through 8) progressively ingress. Red arrows indicate the positions of the purse strings. Panel f reproduced with permission from figure 3b in Lu et al. (2015).

Figure 6

Newtonian force vectors. (a) An embryo, midway through closure, labeled with GFP-Cadherin. The white line highlights the curvature of the bulk of the leading edge of the DME cells and the purse string that they contain. (b) Newtonian force vectors for σ_LEds (green), σ_ASds (blue), and T (red) and Tκ (red). The vertical arrows are drawn approximately to scale such that σ_LE ≈ σ_AS + Tκ. The curvature of the leading edges of the DME cells is reproduced from panel a and is shown in black. Micrograph provided by Stephanie Fogerson.