Epithelial Morphogenesis: Apoptotic Forces Drive Cell Shape Changes

Studying how cells produce and transmit forces that drive morphogenesis is critical to understanding organismal development. A new paper by Monier et al. (2015) identifies an apicobasal actomyosin cable that characterizes apoptotic cells and contributes force(s) for cell sheet bending.

The cellular and molecular mechanisms of morphogenesis remain key extant questions in developmental biology. Insight into how cells produce, transmit, or otherwise respond to forces that drive the cellular shape changes that establish the organization of cells, tissues, and organs is essential for understanding development (Davidson, 2011).

A new contribution by Monier and colleagues (2015) uses leg morphogenesis during pupal development in Drosophila to investigate how apoptosis contributes forces for epithelial sheet folding. Each leg is born as an imaginal disk. During larval stages, disc cells proliferate and each disc morphs into a capped cylindrical structure that, during pupal stages, takes on its final adult form. The legs are segmented, and the joints between segments are characterized by specialized regions of cuticle that are secreted by cells that reside in the folds that prefigure the position of the joints. Monier et al. have focused on the fold responsible for the t4-t5 joint (between tarsal segments 4 and 5), which forms during pupal stages.

Apoptosis plays an important role in morphogenesis (Meier et al., 2000). Previous work from Toyama et al. (2008) demonstrated that apoptosis provides key functions during Drosophila dorsal closure: in addition to removing unwanted cells, it provides an “apoptotic force” in the amnioserosa that helps drive closure. The absence of apoptosis slows closure, whereas increases in apoptosis speed closure. Laser cutting experiments indicate that approximately one-third of forces produced in the amnioserosa are attributable to apoptosis. Subsequent studies (Muliyil et al., 2011) indicate that rates of closure can be uncoupled from apoptosis but do not rule out a role for an apoptotic force that contributes to closure. More recently, the Suzanne group showed that apoptosis is necessary for proper leg segmentation (Manjón et al., 2007).

Here, the Suzanne group combines experimental observation with in silico modeling to provide new evidence for apoptotic force(s). They correlate the position, progression, and depth of the epithelial folds that generate the t4-t5 joint with apoptosis: apoptosis begins on the ventral side of the leg (where the rate of apoptosis, measured as the number of cells that complete apoptosis per unit time, is highest) and proceeds dorsally (where the fold invaginates last). Using a FRET-based apoptosis biosensor, they also confirm that the dying cells exhibit key hallmarks of apoptosis: cells shrink, bleb, fragment, and express caspases. Of key functional importance, they also demonstrate that inhibiting apoptosis, either genetically or pharmacologically, inhibits fold formation.

Interestingly, they document two key structures in the dying cells: an apical “adhesion peak” comprised of aggregated junctional proteins, and a transiently formed, apicobasal cable of actomyosin that extends from the adhesion peak basally (Figure 1). Morphological folds initiate at apoptotic cells that have both the adhesion peak and the apicobasal, actomyosin-rich cable. Ectopic induction of apoptosis in wing tissue also results in an apicobasal myosin cable and ectopic folds. Such folds depend on myosin: when a dominant-negative (DN) form of myosin (Franke et al., 2010) is induced along with apoptosis, the number of cells that apically deform is dramatically reduced. Once apoptotic cells fragment, the cable, the cell’s attachment to the apical surface of the epithelium, and the local deformation of the surface all disappear. The authors conclude that apicobasal actomyosin cables are a fundamental property of epithelial cells undergoing apoptosis, that such cables drive local deformation of the epithelial surface, and that they in turn contribute to fold formation.

Figure 1.

Cell Dynamics during the Early Stages of Apoptotic Force Production

The schematics show an apoptotic cell (red), the formation of an adhesion peak (light blue), apicobasal actomyosin cables (green), the direction of the force it produces (green arrow), and the relaxation of the cell surface following apoptotic cell fragmentation. Reprinted with permission from Monier et al. (2015), Nature, Macmillan Publishers, copyright 2015.

Also of importance is that cells neighboring the apoptotic cells have additional myosin recruited to their apical ends and undergo characteristic shape changes that further contribute to fold formation. Together, the apoptotic cells and their neighbors constitute a fold domain. Cells in the fold domain become increasingly asymmetric, decrease in area, and orient along the dorsal ventral axis. Concomitant with the accumulation of myosin and these shape changes, tension in the fold increases, as revealed by recoil rates following laser cutting. These shape changes in neighboring cells are induced by the dying cells—inhibition of apoptosis blocks such characteristic changes in the non-dying cells.

Finally, the authors construct a 3D model for fold formation based on the 2D vertex model devised by Farhadifar et al. (2007) and the experimental data presented here. The model retains many of the features of the2Dmodel but introduces a volume “buffer” to account for the thickness of the cells and the apicobasal myosin cable. In the model, each folding domain is initially represented by three rings of cells centered on a cylinder over 30 cells long and 50 cells around (thus, the model has 150 fold-domain cells centered on a cylinder of _1,500 cells). The starting condition for the model provides a simplification of the structure of the leg near where the t4-t5 fold is to form, and the forces that Monier et al. apply during in silico morphogenesis produce qualitatively satisfying folds under some of the conditions tested. A detailed description of the model is available online.

The virtue of the model is that it makes simplifying assumptions about the forces that contribute tension to the epithelium, establishes how such forces act on cell boundaries to cause cell shape changes, and illuminates how such local cell shape changes can drive fold formation. Using the model, Monier et al. investigated fold formation under several conditions, including the absence of apoptosis, the presence of 30 apoptotic cells with or without a cell-autonomous apoptotic force (due to the apicobasal actomyosin cable), and the presence of cell nonautonomous forces (due to shape changes in cells neighboring the apoptotic cells). In each case, key parameters (cell asymmetry, area, and orientation) were assessed in silico. Unfortunately, the relationship between the in-silico-generated folds and the folds generated in vivo is difficult to evaluate quantitatively, in part because the in silico cylinder only roughly approximates the overall morphology of the developing leg and in part because the morphological changes observed experimentally may not be sufficiently reproducible. As a consequence, it is not obvious which combinations of forces applied in silico best mimic morphogenesis in vivo. Nevertheless, the model confirms that apoptosis alone cannot drive fold formation and instead establishes that a combination of cell-autonomous and cell-non-autonomous apoptotic forces must contribute to joint morphogenesis.

This research poses a number of very interesting questions. First, what about the apoptotic process induces the formation of the apicobasal actomyosin cable, and what are the other protein components of the cable necessary for its function? At a molecular level, it will be interesting to identify the proteins that anchor the cable at its apical and basal ends and the signal or signals that cause the cable to contract. Furthermore, how do dying cells induce characteristic cell shape changes in their neighbors? And finally, do junctional belts of actomyosin and apical medial arrays of actomyosin, which would also be inhibited by the DN myosin construct used in this study, also contribute to the apoptotic cell-autonomous apoptotic force?

Overall, this provocative research identifies new structures that link apoptosis to morphogenesis and raises a treasure trove of questions for both experimental and theoretical developmental biologists to investigate.