Fig 1.
Cross-section of wing imaginal disc along the anterior-posterior axis.
(A) Imaginal wings discs in 3rd instar ap>GFP larvae. (B) Top view of a wing disc at 96 hours after wing disc in B stained for actin (phalloidin), P-Myosin II, and β-Integrin. (C, D) Patterns of Actin, P-Myosin II, and β-Integrin in the cross-section that follows the major axis of the wing disc pouch. (E) Multiple cell types within the wing disc. The wing disc is composed of squamous peripodial cells (top, blue), columnar pouch cells (bottom center, red), and cuboidal marginal cells (sides, grey). An aqueous lumen is enclosed by the apical surface of the two cell layers. The basal surface is constrained by the extracellular matrix (ECM). (F) Structural components of the Drosophila wing disc columnar cells (left). Schematic showing for actomyosin contractility (right, top). An actin mesh provides structural support to the cell membrane. Actin filaments are pulled together by phosphorylated non-muscle Myosin II (P-Myosin II) resulting in increased membrane tension. Actomyosin driven tension opposes the internal pressure in cells and acts to constrict the membrane (right, bottom).
Fig 2.
Quantitative analysis of wing disc shapes and nuclear positions along the major axis of imaginal wing discs at 96 hours AEL.
(A) Representative pre-processed image of a wing disc cross-section stained with DAPI (nuclei) and phalloidin (staining F-actin). These images were used segment nuclei and surfaces defining the wing disc shape. B) The fractional position between the apical and basal surface serves as a metric for quantifying relative nuclear positions. Note: heights of columnar pouch cells were calculated as the sum of LA and LB. C) Representative segmentation of nuclei. Blue and red lines correspond to LA and LB in B, respectively. D) Basal surface segmentation for multiple samples in different colors (N = 13). E) Composite representation of wing disc morphology generated from samples in D. F) Quantification of global and local curvatures. Fig A and Fig B in S4 Text provide additional details on the image processing pipeline. G) Representative image highlighting average curvature quantification used for H, the curvature was found using a circumcircle approach. H) Curvature quantification of regions of interest along the pouch as well as intensity quantification of integrin along the basal side of the pouch.
Fig 3.
Potential mechanisms of wing disc bending.
(A) The patterned ECM tension hypothesis. In this hypothesis, the level of passive tension of the ECM is higher next to the columnar cells compared with ECM next to squamous cells. (A’) Graphical illustration of potential hypothesis explaining how high ECM tension at the basal side of the columnar cells compresses the tissue and contributes to curvature profile of wing disc. (B) The patterned actomyosin contractility hypothesis. The columnar cells were stained for actin by Phalloidin and an antibody to P-Myosin II. (B’) Schematic of hypothesized mechanism for generating epithelial bending and asymmetrical nuclear positioning in the wing imaginal disc. Actomyosin contractility beneath the nucleus of columnar cells drives wing disc bending.
Fig 4.
Computationally testing the hypothesis of patterned ECM tension.
In these simulations, the passive tensile stress of the ECM associated with squamous cells (FECMs) is lower than the passive tensile stress of the ECM associated with columnar cells (FECMc). Extremely different tensile forces, FECMs v.s. FECMc, are needed to bend the tissue, (A) Initial and final frames of a representative computational simulation showing how higher tension in ECM associated with columnar cells in comparison with the tension in the ECM associated with squamous cells leads to slightly curved shape profile of the wing disc. Comparison of in-silico prediction of impacts of different levels of ECM tension on (B) curvature profile of experimental (n = 16) and simulated (n = 1) wing discs. Experimental data are shown with mean ± standard deviation. The final global curvature of the tissue is uniquely determined in the simulation with a specific set of input parameters; (C) the relative position of nuclei, (D) the height of columnar cells. In C and D, boxplots show minimum, first quartile, mean, third quartile, maximum, and outlier of simulated (n = 65) and experimental (n = 1064) columnar cells. “Baseline,” here, corresponds to the condition that the whole ECM is initiated without any tension in the simulation.
Fig 5.
Quantitative and qualitative comparisons of experimental data with simulation results for different levels of simulated basal actomyosin contractility.
(A) Initial and final frames of simulation result where actomyosin contractility leads to curved profile shape. (B) Global curvature of apical pouch surface and basal peripodial surface for experimental (n = 16) and simulated (n = 1) wing discs. Experimental data are mean ± standard deviation. (C) Relative nuclei positions of pouch cells. Note, the relative nuclei position has a value of zero at the basal surface and one at the apical surface. (D) Height of pouch cells. In C and D, boxplots show minimum, first quantile, mean, third quartile, maximum and outlier of simulated (n = 65), and experimental (n = 1064) columnar cells. Measurements for columnar cell heights experimentally and computationally were taken as the summation of the length of straight lines from apical to centers of segmented nuclei centers and from segmented nuclei centers to the basal side of the cells. (E) Comparison of local curvature profiles and shapes obtained from experimental data and computational results.
Fig 6.
Rho1 promotes tissue bending and regulation of cell height.
(A-B”) MS1096>RyRRANi was used as a control (n = 5) for the comparisons. (A-A”) Representation of immunohistochemistry data using standard deviation z plane projections along with cross-sectional views along a line parallel to the AP axis. (C-D”) Loss of function of Rho1 in the wing imaginal disc was introduced using the Rho1RNAi line (BL# 27727). (C-C”) Data corresponding to A-A”. (E) Rho1RNAi (n = 8) in the wing imaginal discs leads to an increased columnar cell height as compared to control discs (n = 5). (F) Inhibition of Rho1 in the wing imaginal discs leads to flattening of the discs quantified through Menger curvature. A sample size of 5 and 8 was used for the control and Rho1RNAi discs. (p-values of a student’s t-test included in plots) (G-I”) MS1096-Gal4 is expressed preferentially in the dorsal compartment of the wing imaginal disc. (G-H”‘) Representation of immunohistochemistry data using standard deviation z plane projections along with cross-sectional views along a line parallel to the D-V boundary. (H–I”‘) Cross-sectional views of the wing disc along lines parallel to the D-V boundary and located above and below the DV boundary as indicated by the yellow +δ and—δ lines in G.
Fig 7.
A comparison of experimental and simulated profiles demonstrates that ECM is sufficient to maintain the bent profile of the tissue.
(A) Experimental profile before adding Latrunculin A to inhibit actomyosin. The wing disc was stained with CellMask. Note that the imaging conditions required for live-imaging do not provide as fine resolution as for fixed images. (A’) Computational model after the bent profile of the wing disc is formed with higher levels of basal actomyosin contractility. (B) Experimental profile three hours after the addition of 4 μM Latrunculin to inhibit actomyosin. (B’) Computational profile of wing disc showing that bend profile of wing disc is preserved even after removal of basal actomyosin contraction.
Fig 8.
Enzymatic degradation of Collagen IV in 96 h wing discs.
(A-D) Enzymatic degradation of the wing disc substantially changes the curvature profile of wing disc. (A’-D’) Computational simulation of acute removal of ECM and removal of actomyosin contractility components below the nuclear qualitatively match the experimental results. Wing disc was stained with CellMask for experimental imaging.
Fig 9.
Enzymatic degradation of Collagen IV and actomyosin contractility in 96 h wing discs.
(A-D) Enzymatic degradation of the wing disc in the presence of ROCK inhibitor to inhibit actomyosin contractility results in loss of adhesion between the squamous cells (top layer) and the central columnar cells. (A’-D’) Computational simulation of acute removal of ECM, removal of adhesion between cell layers, and removal of actomyosin contractility components below the nuclear qualitatively match the experimental results. The experimental wing disc is stained with CellMask.
Fig 10.
Diagram of the subcellular element (SCE) model of the cross-sectional profile of the wing along the major anterior-posterior axis.
(A) Columnar cell submodel with its potential energy functions describing interactions between adjacent cells, intracellular interactions, and cell-ECM interactions. (B) Marginal boundary cell submodel. (C) Squamous cell submodel. (D) Diagram of the cross-sectional profile of the wing along the anterior-posterior axis which includes columnar cells, boundary cells, squamous cells, and ECM. (E) Diagram of the submodel of the ECM divided into separate sections: ECMc, ECM associated with columnar cells in the wing pouch; ECMbc, ECM associated with the marginal boundary cells at the lateral region of the disc, and ECMs, ECM associated with squamous cells.
Table 1.
Potential energy functions used in the subcellular element model.
Table 2.
Calibration of the model using experimental data over 96 hours of development of the cross-sectional profile of the wing along the anterior-posterior axis.
Fig 11.
Temporal evolution of global curvature (blue) and its asymptotic fitting regression curve (red).
Simulation leading to a nearly steady state shape in the wild-type simulation. Simulation runs on a dedicated GPU for 192 hours.