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Local and tissue-scale forces drive oriented junction growth during tissue extension

Nature Cell Biology volume 17, pages 1247–1258 (2015)Cite this article

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Abstract

Convergence–extension is a widespread morphogenetic process driven by polarized cell intercalation. In the Drosophila germ band, epithelial intercalation comprises loss of junctions between anterior–posterior neighbours followed by growth of new junctions between dorsal–ventral neighbours. Much is known about how active stresses drive polarized junction shrinkage. However, it is unclear how tissue convergence–extension emerges from local junction remodelling and what the specific role, if any, of junction growth is. Here we report that tissue convergence and extension correlate mostly with new junction growth. Simulations and in vivo mechanical perturbations reveal that junction growth is due to local polarized stresses driven by medial actomyosin contractions. Moreover, we find that tissue-scale pulling forces at the boundary with the invaginating posterior midgut actively participate in tissue extension by orienting junction growth. Thus, tissue extension is akin to a polarized fluid flow that requires parallel and concerted local and tissue-scale forces to drive junction growth and cell–cell displacement.

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Figure 1: Tissue extension is scale-independent and correlates with junction growth.
Figure 2: Junction growth is driven by active stresses and can be uncoupled from tissue extension.
Figure 3: Junction growth does not correlate with E-cad density changes.
Figure 4: Medial MyoII correlates with extensions of new junctions.
Figure 5: Nano-dissections confirm the pivotal role of medial MyoII in extensions of new junctions.
Figure 6: Extrinsic forces exerted by PMG invagination contribute to tissue extension.
Figure 7: Forces during tissue extension revealed by linear laser cuts.
Figure 8: Pulling forces from PMG invagination orient junction growth.

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Acknowledgements

We thank J.-M. Philippe (IBDM, France) for designing and preparing the dsRNA probes used in this study and C. Chardès for development and assistance with the ablation set up. We thank R. Levayer, M. Rao and members of the Lecuit and Lenne groups for stimulating and useful discussions during the course of this project. We thank B. Aigouy for help with cell segmentation and tracking. This work was supported by the ANR Archiplast, the ERC (Biomecamorph no. 323027) and by the FRM (Fondation Recherche Médicale, Equipe Labelisée). C.C. was supported by a Human Frontier Science Program Long-Term Fellowship and by a post-doctoral fellowship from the FRM. We acknowledge the France-BioImaging infrastructure supported by the Agence Nationale de la Recherche (ANR-10-INSB-04-01, call ‘Investissements d’Avenir’). M.R. was supported by a PhD fellowship by the Region PACA and Amplitudes Systèmes at the time when he first discovered the infrared laser-based cell cauterization process and then by the cofunded EMBO-Marie Curie and by the Human Frontier Science Program Long-Term fellowships when he further developed the technique.

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Authors and Affiliations

  1. Aix Marseille Université, CNRS, IBDM UMR7288 13009 Marseille, France,

    Claudio Collinet, Pierre-François Lenne & Thomas Lecuit

  2. EMBL Heidelberg, Meyerhofstrasse 1 69117 Heidelberg, Germany,

    Matteo Rauzi

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  1. Claudio Collinet
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Contributions

C.C. and T.L. designed the project and planned the experiments with feedback from P.-F.L. C.C. performed all the experiments and quantifications. C.C., P.-F.L. and T.L. analysed and discussed the results. M.R. developed the laser-induced tissue cauterization. P.-F.L. performed the simulation. C.C. and T.L. wrote the paper, and the other authors made comments.

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Correspondence to Claudio Collinet or Thomas Lecuit.

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Integrated supplementary information

Supplementary Figure 2 Measurements of tissue extension.

(a) Graphical illustration of the different proportionality with the number of ‘intercalation units’ of the 2 measurements of tissue extension used in this study. The final length of a stripe of cells extending by cell intercalation is the sum of a variable length and an invariable length. While the variable length scales proportionally with the number of intercalation units, the invariable length does not. Since the distance between the centroids of the 2 outer cells of the extending stripe directly measures only the variable length it also scales proportionally with the number of intercalation units. On the contrary, since the width of the stripe (A–P width) measures also the invariable distance, it does not scale proportionally to the number of intercalation units. (b) Relative length over time (L/L0) of AP-stripes of tissue comprising different number of intercalation units measured by their A–P width (top) and by the centroid distance of the tip cells (bottom). N = 37, 31, 30, 27 and 7 stripes of 1, 3, 5, 7, 10 intercalation units respectively in one embryo. (c) Graphical representation of the different influence of cell shape changes on the 2 measurements of tissue extension employed in this study. Cell intercalation might take place without extension. The lack of extension is captured by the measurement of the A–P width but only partially by the measurement of the distance between centroids. (d,e) Example of cell intercalation without extension (1 ‘intercalation unit’). In d: still images of the intercalating cells. The extension of the ‘intercalation unit’ is measured by the dashed line (A–P width) and by the distance between the centroids (yellow arrows). In e: traces of length of the A–P width (red), of the centroid distance (blue) and of the new junction length (black). Mean ± s.e.m. are shown in b.

Supplementary Figure 3 Characterization of tissue fencing by laser cauterization.

(a) Map of laser-tissue interactions at different power densities and exposure durations (adapted from ref. 21). The grey circles give only a rough estimate of the associated laser parameters. The red circle gives a rough estimate of the conditions used to generate tissue fences in Drosophila embryos. (b) Graphical representation of the generation of tissue fences in Drosophila embryos. The near-infrared (NIR) laser is focused on an apical region of the blastoderm epithelium inducing localized thermal interactions with the tissue (darker red zone). This, in turn, generates tissue cauterization and adherence to the overlaying vitelline membrane (bold red line) and localized tissue damage (lighter red zone). (c) Tissue fences in Drosophila embryos. Top: top view of a tissue fence with transmitted light. The black arrow indicates the cauterization line and the dashed line the region of adherence to the vitelline membrane. Middle and bottom: top and lateral views of the same fence with fluorescence. The top view is a maximal projection of the top 3 μm. The white arrow indicates the cauterization line and the dashed line the region of adherence to the vitelline membrane. (d,e) Snapshot at time 0 (d) and maximum intensity projection over time (e) of a Drosophila embryo following tissue cauterization. In yellow the lines used to generate kymographs in f–h. Asterisks label fluorescent immobile particles in the glue, the white arrows indicate E-cad labeled junctions in immobilized cells and the dashed line the region of adherence to the vitelline membrane. (f–h) Kymographs along the lines indicated in d. Immobile glue particles and junctions in immobilized cells are indicated. In c images are representative of all (more than 10) cauterization experiments performed and in e of 8 embryos with posterior tissue fences.

Supplementary Figure 4 Tissue fences do not affect MyoII dynamics nor polarity in the surrounding tissue.

(a) Maximum Z-projections of MyoII in a WT embryo and in an embryo with a posterior tissue fence. The yellow arrows indicate the enrichment of MyoII at vertical junctions, the white arrows indicate MyoII clusters in the medial-apical region and the green arrows indicate supra-cellular cables of MyoII. (b,c) Close-ups on cell intercalation in regions distant (b) and proximal (c) to the tissue fence. Yellow arrows indicate the enrichment of MyoII at vertical junctions and the white arrows indicate MyoII clusters in the medial-apical region. (d–f) Quantifications of MyoII planar polarity at junctions (d), radial cell polarity (e) and of the average period of medial MyoII pulses. N indicates junctions for planar polarity (d) and cells for radial cell polarity (e) from 4 WT and 6 embryos with posterior cauterizations. In (f) N = 124 pulses in 45 cells from 2 WT embryos and 152 pulses in 50 cells from 2 embryos with posterior cauterizations. The asterisk indicates a p = 0.019 and NS a p = 0.7619 at a Mann–Whitney test. In a,b images are representative of 4 WT and 6 embryos with posterior cauterizations. Mean ± s.d. are shown in d,e.

Supplementary Figure 5 E-cad levels are low at when new cell–cell contacts form.

(a) Top: illustration of the regions where E-cad density was measured at new junctions (yellow box) and the corresponding 4 transverse junctions (white boxes). Bottom: intensity ratio plotted in b and c. (b) Time evolution of E-cad density at new junctions and their transverse junction. Traces of length are in the top graphs (black and grey curves), traces of E-cad mean intensity in the new junction (blue curves) and of the average E-cad mean intensity over the 4 neighbouring junctions (grey curves) are in the middle graphs and the intensity ratios as defined in a (red curves) are in the bottom graphs. (c) Average mean intensity ratio for 45 new junctions from 3 WT embryos. (d) Stimulated emission depletion (STED) images of new junctions shortly after T1. The white arrows indicate newly formed junctions with low levels of E-cad and poor apposition of the facing membranes. (e) Graphical representation of a new junction shortly after neighbor exchange. Note the poor apposition of facing membranes and the asymmetric distribution of E-cad clusters at the vertices. In b data are representative of 45 new junctions from 3 WT embryos and in d the images are representative of 3 WT embryos. Mean ± s.d. are shown in c.

Supplementary Figure 6 E-cad turnover and mobility at new junctions are similar to those at mature junctions.

(a,b) Examples of FRAP of single E-cad clusters (a) or of larger regions (2 μm) of junctions (b) during GBE. Right panels are corresponding kymographs showing the evolution over time of the fluorescence intensity. The red bars denote the region of FRAP and the white arrows indicate the FRAP events in the kymographs. (c,d) Average recovery curves with single exponential fits of new (red curves) and mature junctions (blue curves) following FRAP of single cluster (c) or larger regions (d). N is as e,f. (e,f) Measurements of the mobile fractions and half time of recovery (Thalf) from individual FRAP experiments targeting single cluster (e) or larger regions (f). Box plots superimposed to all data points. N = 68 cluster and 30 2 μm-regions at mature junctions and 35 clusters and 12 2 μm-regions at new junctions. The data are pooled from 11–14 embryos for single cluster FRAP and 5 embryos for FRAP of larger regions. NS indicate p > 0.1879 at a Mann–Whitney test. Red crosses label outliers. In a and b data are representative of 11–14 and 5 embryos respectively. Mean ± s.d. are shown in c,d.

Supplementary Figure 7 E-cadherin recruitment at new junctions does not precede steps of extension.

(a) Traces of length (raw measurement in black and smoothened curve in red) and of the integrated intensity of E-cadherin (raw measurements in pink and smoothened curve in magenta) of a new extending junction. Asterisks indicate discrete steps of extension. (b) Average cross-correlation functions of the extension rates and rates of change of the integrated intensity for new extending junctions labeled with E-cad::GFP or with the membrane marker Gap43::mCherry. In a data are representative of 32 junctions from 3 embryos. Mean ± s.e.m. are shown in b.

Supplementary Figure 8 Pulling forces at the posterior of the germ band do not depend on cell intercalation nor mesoderm invagination.

(a–c) Top: stills of GBE posterior to a DV fence in an embryo where PMG invagination (fog RNAi) (a), cell intercalation (eve/runtdouble RNAi) (b) or mesoderm invagination (twist/snail double RNAi) (c) are affected. The white arrows indicate cells elongated along the AP axis. The dashed line indicates the border of the tissue tear. Bottom: graphical representation of the respective observed effects. While cell elongation and tissue tearing is abolished in fog RNAi injected embryos it persists in eve/runt double RNAi and twist/snail double RNAi injected embryos. (d) Stills of ectodermal cells in an embryo injected with dsRNAs targeting eve and runt. Cells aligned along the same raw along the AP axis are labeled with the same color (white, yellow and cyan). White and cyan labeled cells do not intercalate between yellow labeled cells over 20 min indicating a defect in cell intercalation. (e) Ventral views of an embryo injected with dsRNAs targeting twist and snail. The dashed line indicates the region of the prospective mesoderm that fails to invaginate over more than 45 min after the onset of gastrulation. Images in a,b,c,d,e are representative of 6 (4 independent experiments), 3 (3 independent experiments), 7 (4 independent experiments), 3 (3 independent experiments) and 3 embryos (2 independent experiments) respectively.

Supplementary Figure 9 Procedure for the quantification of the average tissue tension by PIV.

(a) Schematic representation of a line cut experiment. Black vertical lines represent individual time frame and the yellow lightening the line ablation event. The time interval between consecutive frames is 500 ms. The frame just before the ablation event sets the time 0 and is cross-correlated using PIV to the frame at −2 s (pre-cut PIV) and +2 s (post-cut PIV). (b) Overlays of the pre-cut frame (magenta) and the frame at −2 s or at +2 s (green) are shown on the left and on the right respectively. The respective vector velocity fields (white arrows) are superimposed on the merge images. The line cut is indicated in yellow. The scaling of the white arrows is not the same between the left and right image. The magnitude of the velocity vectors is plotted in the heatmaps in b′. (c) Left: The velocity vector field resulting from the subtraction of the pre-cut PIV from the post-cut PIV is superimposed to a merge image of the pre-cut frame (in magenta) and the frame at +2 s (in green). The two regions where the velocity components orthogonal to the cut line are averaged to obtain the final v0mean value are shown in yellow. Right: Heatmap of the resulting velocity vector.

Supplementary Table 1 Sequences of the primer used to synthesize dsRNAs.
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Supplementary information

Supplementary Information

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Simulations of GBE with free and immobile boundaries.

Left: Simulations with free posterior boundary and isotropic tension relaxation (top) or ‘active’ new junctions (bottom). Right: Simulations with immobile boundaries and isotropic tension relaxation (top) or ‘active’ new junctions (bottom). Edges with high initial tension are labeled in red and new edges are labeled in blue. Green edges are exchange of neighbors involving black edges. Boundaries are labeled in light blue. (MOV 1815 kb)

Example of an immobile DV fence. Yellow dots label immobilized cells.

Labeling E-cad::GFP. Scalebar = 10 μm. (MOV 1135 kb)

GBE in a WT embryo (top), in an embryo with a posterior DV fence (middle) and in an embryo from tor−/− females (bottom).

In each case two cells labeled by the white and yellow dots have been tracked and their traces drawn. Labeling E-cad::GFP. Scalebar = 10 μm. (MOV 3521 kb)

Maximum Z-projection of Ecad::GFP during extension of new junction in a WT embryo.

Scalebar = 5 μm. (MOV 1831 kb)

GBE in a WT (water injection) embryo (left) and an embryo injected with the ROCK inhibitor H-1152 (right).

Labeling: E-cad::GFP (top) and MRLC::mCherry (bottom). Scalebar = 10 μm. (MOV 21759 kb)

Maximum Z-projection of MRLC::mCherry (green) and Ecad::GFP (magenta) during extension of new junction in a WT embryo.

White arrows indicate medial MyoII pulses pulling on transverse junctions in the A and P cells. Scalebar = 5 μm. (MOV 1042 kb)

Example of nano-ablations of MyoII (labeled with MRLC::GFP) clusters approaching a new junction in a D cell (magenta arrowhead) and in an A cells (yellow arrowhead).

The white arrows indicate the vertices of the new junction. Scalebar = 5 μm. (MOV 2157 kb)

Example of nano-ablations of junctional MyoII (labeled with MRLC::GFP) at a transverse junction neighboring two new junctions (labeled by yellow arrows).

The white arrowhead indicates the ablation. Scalebar = 5 μm. (MOV 1387 kb)

Simulations of GBE with ‘active’ new junctions and free (top) or actively moving posterior boundary (bottom).

Edges with high initial tension are labeled in red and new edges are labeled in blue. Green edges are exchange of neighbors involving black edges. Boundaries are labeled in light blue. (MOV 926 kb)

GBE posterior to a DV fence in a WT embryo.

Labeling E-cad::GFP. Scalebar = 10 μm. (MOV 2774 kb)

GBE posterior to a DV fence in an embryo from tor−/− females.

Labeling E-cad::GFP. Scalebar = 10 μm. (MOV 2590 kb)

GBE posterior to a DV fence in an embryo injected with dsRNAs against fog.

Labeling E-cad::GFP. Scalebar = 10 μm. (MOV 3906 kb)

GBE posterior to a DV fence in an embryo injected with dsRNAs against eve and runt.

Labeling E-cad:GFP. Scalebar = 10 μm. (MOV 3197 kb)

GBE posterior to a DV fence in an embryo injected with dsRNAs against twist and snail.

Labeling E-cad::GFP. Scalebar = 10 μm. (MOV 5914 kb)

Examples of an horizontal and a vertical line cut in WT embryos.

The yellow line indicates the cut. Labeling E-cad::GFP. Scalebar = 5 μm. (MOV 1252 kb)

Large views of MyoII (medial and junctional) in a WT embryo and in a embryo with a posterior DV fence.

Labeling MRLC::GFP, maximum Z-projections. Scalebar = 10 μm. (MOV 20493 kb)

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Collinet, C., Rauzi, M., Lenne, PF. et al. Local and tissue-scale forces drive oriented junction growth during tissue extension. Nat Cell Biol 17, 1247–1258 (2015). https://doi.org/10.1038/ncb3226

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