Fig 1.
Sdk is a novel component of apical vertices in epithelia.
(A) Cartoon summarising the geometry of tricellular vertices and the proteins known to localise specifically there in epithelia of different types. (B,C) Immunostaining of fixed ventral ectoderm of stage 7 Drosophila embryos for Sdk-YFP and the lateral marker Dlg. (B) Maximum projection. Scale bar = 5 μm. (C) Z-reconstruction based on confocal slices taken from confocal stack in B. Scale bar = 20 μm. (D–G) Super-resolution SIM imaging of fixed embryos immunostained with Sdk-YFP and either DE-Cad to show the position of AJs (D,E) or Gap43-mCherry (F) to label the whole membrane. This reveals the localisation of Sdk in ‘strings’ at apical vertices in embryonic epithelia. Images are maximum projection (labelled XY) and z-reconstruction (labelled XZ) from z-slices of the apical side of the cells. Scale bars, apical views (top panels) = 5 μm; lateral views (bottom panels) = 2 μm. (G) 3D reconstruction to show the z-component of the strings. Scale bars = 5 μm. (H) Example of an sdkΔ15 clone (absence of nls-RFP signal) in the follicular epithelium (stage 6 egg chamber) stained for Sdk-YFP and actin phalloidin. Right panels show a close-up of the four-cell clone (asterisks) with individual channels on the left to show Sdk-YFP and actin, respectively, and the merge. Scale bars = 20 μm. (I) Cartoon summarising the results of the clone experiment shown in H. Vertex localisation of Sdk requires Sdk proteins in at least two of the three cells forming a vertex. AJ, adherens junction; DE-Cad, DE-cadherin; Dlg, Discs large; Gap43, growth-associated protein 43; ILDR, Immunoglubulin-like domain-containing receptor; LSR, Lipolysis-stimulated lipoprotein receptor; nls, nuclear localisation signal; RFP, red fluorescent protein; Sdk, Sidekick; SIM, Structured Illumination Microscopy; YFP, yellow fluorescent protein.
Fig 2.
Sdk localises differently at shortening versus elongating junctions during polarised cell intercalation.
(A,B) Sdk localisation during a T1 transition imaged over 20 minutes in live embryos labelled with Sdk-YFP and DE-Cad-mCherryKI. Time is indicated in seconds since the start of the intercalation event. Each image is a maximum intensity projection over 3 z-slices spanning 1.5 μm. This movie is representative of behaviour found in all of n = 9 complete intercalation events, n = 8 junction shrinkages, and n = 6 junction growths. (B) Cartoon illustrating the behaviour of Sdk-YFP shown in A. (C–H) Analysis of Sdk-YFP string localisation at shortening and elongating junctions by super-resolution SIM. Embryos are fixed and stained for GFP and E-Cad. Scale bars = 1 μm. (C–E) Representative SIM super-resolution images of DV-oriented junctions at late stage 6 (C) and at stage 7 (D) and of an AP-oriented junction at stage 8 (E). Orientation is within 20o of AP or DV axis. For each example, the string classification used in F is shown. (F) Quantification of string morphologies based on 3D reconstructions in stage 7 and stage 8 embryos. Morphologies where divided into three classes: vertical, planar, and step-like (stage 7: total n = 40, step = 19, planar = 13, vertical = 8; stage 8: total n = 47, step = 15, planar = 11, vertical = 21). Statistical significance calculated by chi-squared test. (G) Quantification of string lengths at shrinking versus growing junctions (defined by their orientation within 20o of AP or DV embryonic axis, respectively; shrinking: n = 44, growing: n = 21). Statistical significance calculated by Mann–Whitney test. (H) Quantification of string lengths at stage 7 versus stage 8 (stage 7: n = 42, stage 8: n = 49). Statistical significance calculated by Mann–Whitney test. Data for graphs F–H can be found at https://doi.org/10.17863/CAM.44798. AP, anteroposterior; DE-Cad, DE-Cadherin; DV, dorsoventral; GFP, green fluorescent protein; Sdk, Sidekick; SIM, Structured Illumination Microscopy; YFP, yellow fluorescent protein.
Fig 3.
tAJs marked by Sdk-YFP are separate at rosette centres.
(A) Sdk localisation during rosette formation imaged over 15 minutes in live embryos labelled with Sdk-YFP and Gap43-mCherry. Time is indicated in seconds since start of the intercalation rosette. Each image is a maximum intensity projection over 3 z-slices spanning 1.5 μm. Movie is representative of behaviour found in all of n = 6 full rosette-like intercalation events. Scale bars = 5 μm. Close-up images of the rosette centre are shown in yellow boxes for the Sdk-YFP channel. Cartoon below illustrates the dynamics of the Sdk-YFP puncta seen in the movie. (B) Sdk-YFP string localisation at a rosette centre involving six cells, imaged by super-resolution SIM. The image is from a stage 8 embryo fixed and stained for GFP and DE-Cad. Maximum projection over 12 slices = 1.5 μm. Close-ups of the rosette centre with different projections are shown in yellow boxes to demonstrate that three distinct strings can be resolved. Cartoon shown to interpret images. Scale bars for the main SIM panels are 5 μm and for the close-ups 1 μm. DE-Cad, DE-Cadherin; Gap43, growth-associated protein 43; GFP, green fluorescent protein; Sdk, Sidekick; SIM, Structured Illumination Microscopy; tAJ, tricellular adherens junction; YFP, yellow fluorescent protein.
Fig 4.
Cell shapes are more anisotropic in sdk mutants versus the WT during GBE.
(A,B) Movie frame of ventral ectoderm at 30 mins into GBE from representative WT (A) and sdk (B) movies, labelled with E-cadherin-GFP. (C,D) Measurement of cell shape anisotropy and orientation (see also S5A–S5C Fig). Cell shape anisotropy is calculated as the log ratio of the principal axes of best-fit ellipses to tracked cell contours. An isotropic cell shape (a circle) will have a log-ratio value of 0 and a very elongated cell a value of over 1. Cell orientation is given by the cosine of the angular difference between the ellipse’s major axis and the DV embryonic axis. Negative values indicate cells that are elongated in the AP axis, positive values in the DV axis. Cell shape anisotropy and orientation measures are then multiplied together to give a composite measure (termed ‘axial shape elongation’) of how elongated cells are in the orientation of the embryonic axes (Materials and Methods). (D) Axial shape elongation measure (y-axis) for the first 30 mins of GBE (x-axis) for WT and sdk embryos. In this graph and hereafter, the ribbon’s width indicates the within-embryo confidence interval, and the dark grey shading indicates a difference (p < 0.05) (Materials and Methods). (E–F) Measures of AP and DV cell lengths in WT and sdk embryos (see also S5D and S5E Fig). Cell shape ellipses are projected onto AP and DV axes to derive a measure of cell length in each axis. (G,H) Evolution of AP-oriented and DV-oriented cell–cell interface lengths (y-axis) as a function of time in GBE (x-axis) (see also S5F and S5I Fig). Tracked cell–cell interfaces are classified as AP- or DV-oriented according to their orientation relative to the embryo axes. Data for graphs D–H can be found at https://doi.org/10.17863/CAM.44798. AP, anteroposterior; DV, dorsoventral; GBE, germband extension; GFP, green fluorescent protein; Sdk, Sidekick; WT, wild type.
Fig 5.
Apical adhesion is disrupted during polarised cell intercalation in sdk mutants.
(A) A single z-frame at the level of AJs showing a gap or tear in the cortex at a presumed rosette centre in an sdk mutant embryo. Left panel shows merge between DE-Cad-GFP and Sqh-mCherry (shortened as MyoII-mCherry) signals, right panel, DE-Cad-GFP channel only. In the bottom panel, the different cells have been coloured to highlight the apical gap in the middle. (B) Single z-frames at different positions along the apicobasal axis of an apical gap in an sdk mutant embryo (from movie shown in S6A Fig) at the level of AJs (0 μm) and 1 and 2 μm below. The gap present at the level of AJs is closed in the planes basal to the AJs. Bottom panels show colourised cells, highlighting the apical gap in the apical-most z-slice. (C) Fixed and stained sdk mutant embryo against DE-Cad and GFP (to reveal MyoII-GFP), imaged by SIM super-resolution microscopy at stage 7. Each image is a maximum intensity projection over 3 μm at the level of AJs in the ventral ectoderm. Regions bounded by yellow and blue lines show discontinuities in E-Cad signal, indicating holes in apical adhesion, and are shown below as close-ups. (D–F) Apical gaps quantifications in WT and sdk mutant movies as shown in A, B. (D) Quantification of the number of gaps found at the level of AJs, normalised to a given area (2,500 μm2) of the ectoderm. One to two regions (embryo sides) were quantified per movie: WT, n = 7, from seven embryos; sdk mutant n = 10, from eight embryos; Mann–Whitney, p-value = 0.0018. (E) Quantification of how long apical gaps persist in the tissue. (F) Quantification of how long apical gaps persist as a function of the number of cells present at the gap’s border. We detected gaps where four to seven cells and more meet. For both E and F, the number of gaps quantified was n = 92 for WT and n = 115 for sdk mutant. In D, Mann–Whitney, p-value < 0.0001. Data for graphs D–F can be found at https://doi.org/10.17863/CAM.44798. AJ, adherens junction; DE-Cad, DE-Cadherin; GFP, green fluorescent protein; MyoII, Myosin II; Sdk, Sidekick; SIM, Structured Illumination Microscopy; Sqh, spaghetti-squash; WT, wild type.
Fig 6.
Sdk is required for normal polarised cell intercalation.
(A) Graphical illustration of our measures of tissue and cell shape SRs (Materials and Methods). The cell intercalation SR is derived from these two measures. (B–D) Average SRs in the direction of extension (along AP) for five WT (blue) and five sdk mutant (red) embryos for the first 30 minutes of GBE. Total tissue SR (B), cell shape SR (C), and cell intercalation SR (D). Units are in pp per minute. (E) Diagram of a T1 transition leading to a loss of neighbours 1 and 3 along AP and a gain of neighbours 2 and 4 along DV. (F,G) Analysis of the number and orientation of T1 transitions averaged for five WT (blue) and five sdk mutant (red) embryos for the first 30 minutes of GBE (see also S8D and S8E Fig). (F) Orientation of all T1 transitions relative to the AP embryonic axis. Orientation is given by the angle of cell interfaces relative to AP, 5 minutes before a T1 swap (Kolmogorov–Smirnov test, N = 1,786 for WT and 1,890 for sdk mutant, D = 0.1115, p < 0.0001). (G) Cumulative proportion of T1 swaps contributing to axis extension in AP (called productive T1 swaps; see Materials and Methods) for the first 30 minutes of GBE and expressed as a pp of DV-oriented interfaces tracked at each time point. Data for graphs B–G can be found at https://doi.org/10.17863/CAM.44798. AP, anteroposterior; DV, dorsoventral; GBE, germband extension; pp, proportion; Sdk, Sidekick; SR, strain rate; WT, wild type.
Fig 7.
Vertex models of the sdk mutant phenotype.
(A) Cell rearrangement (T1 transition) is usually implemented in vertex models as follows: edges with length below a threshold are removed, and a new edge is created between previously non-neighbouring cells. (B–C) Alternative implementation of cell rearrangement used in this paper. (B) Shortening junctions merge to form a four-way vertex and a protorosette, which has a probability, p4, of resolving at every time step. (C) Formation of rosettes around higher-order vertices (formed of five cells, as shown here, or more) due to the shortening of junctions connected to four-way vertices. Edges connected to the shortening junction are merged into the existing vertex, which now has a probability, p5+, of resolving at every time step. (D) Initial configuration for each simulation. The tissue is a tiling of 14 × 20 regular hexagons with periodic boundary conditions. All cells are bestowed one of four stripe identities, {S1, S2, S2, S4}, representing identities within parasegments, as in [49] (see S9A Fig for an illustration). (E) Wild-type simulation of Drosophila GBE in the presence of a posterior pulling force, implementing cell rearrangements as outlined in B and C with and
. Model parameters used were (Λ, Γ) = (0.05, 0.04). (F) Tissue strain rate in the A–P (extension) direction for wild-type tissues with parameters used in E. Solid line and shading represent mean and 95% confidence intervals from five independent simulations. (G) Simulation of GBE in a tissue in which T1 swaps are less likely to resolve, with
and
. All other parameters are kept equivalent to wild-type simulation. (H) Tissue strain rate in the A–P (extension) direction for tissues with parameters used in G compared to strain rate of wild-type tissue in E. Solid line and shading represent mean and 95% confidence intervals from five independent simulations. (I) Simulation of GBE in a tissue in which T1 swaps are less likely to resolve, with
and
, as in G, and additionally in which the shear modulus of the tissue (in the absence of actomyosin cables) has been reduced by setting Γ = 0.01. All other parameters are kept equivalent to wild-type simulation. (J) Tissue strain rate in the A–P (extension) direction for tissues with parameters used in I compared to strain rate of wild-type tissue in E. Solid line and shading represent mean and 95% confidence intervals from five independent simulations. As shown in key, for B, C, E, G, and I, cell colouring indicates the vertex rank of a cell, defined as the maximum number of cells sharing one of its vertices (note that darker blue is for rosettes of rank 5 and above). Further details about models and simulations can be found in S1 Text. Data for graphs F, H, and J can be found at https://doi.org/10.17863/CAM.44798. A–P, anterior–posterior; GBE, germband extension; Sdk, Sidekick.