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Rear traction forces drive adherent tissue migration in vivo

Abstract

During animal embryogenesis, homeostasis and disease, tissues push and pull on their surroundings to move forward. Although the force-generating machinery is known, it is unknown how tissues exert physical stresses on their substrate to generate motion in vivo. Here, we identify the force transmission machinery, the substrate and the stresses that a tissue, the zebrafish posterior lateral line primordium, generates during its migration. We find that the primordium couples actin flow through integrins to the basement membrane for forward movement. Talin- and integrin-mediated coupling is required for efficient migration, and its loss is partially compensated for by increased actin flow. Using Embryogram, an approach to measure stresses in vivo, we show that the rear of the primordium exerts higher stresses than the front, which suggests that this tissue pushes itself forward with its back. This unexpected strategy probably also underlies the motion of other tissues in animals.

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Fig. 1: The primordium migrates on top of the BM and directly under the skin.
Fig. 2: Primordium migration requires an intact BM.
Fig. 3: Integrin-β1 and talin form small short-lived clusters at the basal sides of the primordium cells.
Fig. 4: Integrin-β1, talin and their interaction are required for efficient primordium migration.
Fig. 5: Integrin-β1 couples cell–substrate adhesion to actin flow in the primordium.
Fig. 6: Traction stress measurements indicate that the primordium exerts the highest stresses in its rear.
Fig. 7: The BM wrinkles around the primordium.
Fig. 8: The primordium generates larger forces in the rear.

Data availability

Previously published genome assemblies as either GRCz10 or GRCz11 that were used here for the design of gRNA constructs are available for tln1, tln2a, tln2b, itgb1a and itgb1b under accession codes ENSDARG00000100729, ENSDARG00000017901, ENSDARG00000110973, ENSDARG00000071863 and ENSDARG00000104484, respectively. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The code for Embryogram software has been deposited at Zenodo (https://zenodo.org/record/5762146#.Ya5X0y-B1QJ)29. The codes for image analysis using ImageJ and R are provided as a zip file.

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Acknowledgements

We thank R. Lehmann, L. Christiaen, D. Rifkin, M. Schober, J. Torres-Vázquez, W. Qian, P. Vagni, S. Lau and T. Colak-Champollion for critical comments; T. Gerson, T. Colak-Champollion and A. Feitzinger for reagents; T. Gerson, J. Proietti and S. Pirani for excellent fish care; N. Paknejad for advice on AFM; M. Cammer and Y. Deng for advice on microscopy; A. Liang, C. Petzold and K. Dancel-Manning for consultation and assistance with TEM work; and A. Ferrari and N. Chala for AFM consultation. The use of the NYULH DART Microscopy Laboratory (P30CA016087) and the Memorial Sloan Kettering Molecular Cytology Core Facility (P30 CA008748) is gratefully acknowledged. For providing the zebrafish knockout allele lamC1sa9866, we thank the Zebrafish International Resource Center. For providing the cdh1:cdh1-tdTomato line, we thank M. Cronan and D. Tobin. This work was supported by NIH grant NS102322 (H.K.), by an NYSTEM fellowship C322560GG (N.Y.), by an American Heart Association fellowship 20PRE35180164 (N.Y.), in part through the NYU IT High Performance Computing resources, services, and staff expertise, the NSF CAREER award 1652515 (D.N.), the NSF grants IIS-1320635 (D.N.), OAC-1835712 (D.N.), OIA-1937043 (D.N.), CHS-1908767 (D.N.), CHS-1901091 (D.N.), a gift from Adobe Research (D.N.), a gift from nTopology (D.N.), and a gift from Advanced Micro Devices (D.N.).

Author information

Authors and Affiliations

Authors

Contributions

N.Y., D.P. and H.K. conceptualized the study and designed the experiments. N.Y. performed all the zebrafish experiments with support from H.K., except for the AFM measurements, which were performed by B.W. with samples prepared by N.Y. The Embryogram software was developed by Z.Z., T.S. and D.P. (with inputs from N.Y. and H.K.). N.Y. analysed most of the data with help from Z.Z. and T.S. for the traction stress analysis and from Z.Z. and B.W. for the AFM data analysis. N.Y. and H.K. wrote the main manuscript (with input from Z.Z., T.S. and D.P.). Z.Z., T.S. and D.P. wrote Supplementary Note 1 (with input from N.Y. and H.K.). All authors approved of and contributed to the final version of the manuscript.

Corresponding authors

Correspondence to Daniele Panozzo or Holger Knaut.

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Nature Cell Biology thanks Anna Huttenlocher, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Ultrastructure of the basement membrane along the migratory route of the primordium and the characterization of the TgBAC(lamC1:lamC1-sfGFP) line.

a, Overview of the primordium. Dotted lines indicate the location of the cross-sections shown in (b-e). b-e, TEM images of cross-sections at the level of the most recently deposited neuromast (b), at the level of the primordium’s rear (c), at the level of the primordium’s front (d), and in front of the primordium (e). Scale bars = 10 μm. b’-e’, Magnification of area outlined by a dotted line in (b-e). Scale bars = 1 μm. The skin (s), primordium (p, purple hue), the muscle (m), and the BM (arrows) are indicated. n = 1 embryo. f, Schematic of the TgBAC(lamC1:lamC1-sfGFP) transgene. g, Image of the expression of LamC1-sfGFP from TgBAC(lamC1:lamC1-sfGFP) transgene in a 28 hpf embryo. The image is a sum-projected z-stack. Scale bar = 0.5 mm. h, The TgBAC(lamC1:lamC1-sfGFP) transgene partly rescues the lamC1 mutant phenotype. Crosses from lamC1-/+; lamC1:lamC1-sfGFP to lamC1-/+ fish resulted in embryos with three different phenotypes shown on the left. Quantification of the phenotypic categories from these crosses for non-transgenic embryos and embryos expressing LamC1-sfGFP are shown on the right. Note that the mild phenotype correlates with the presence of LamC1-sfGFP and the severe phenotype represents the lamC1 mutant phenotype. Scale bars = 0.5 mm. i, Quantification of the primordium migration in the presence of different copy numbers of the TgBAC(lamC1:lamC1-sfGFP) transgene. Data points, means, and SD are indicated. n.s.: p = 0.6514 (non-transgenic vs. lamC1:lamC1-sfGFP/+), p = 0.7842 (non-transgenic vs. lamC1:lamC1-sfGFP/lamC1:lamC1-sfGFP) (two-tailed Mann-Whitney test). j, Cross-section along apical-basal axis of a primordium (dotted line in top panel) of embryos expressing LamC1-sfGFP (BM) and the Cdh1-tdTomato (skin). The LamC1-sfGFP signal is enhanced to saturated levels (bottom panel). Scale bars = 25 μm. k, Images of slices from a z-stack of 32 hpf TgBAC(cxcr4b:EGFP-CaaX) embryos stained for Fibronectin and GFP. Orthogonal views are shown. l, Images of slices from a z-stack of 32 hpf TgBAC(cxcr4b:EGFP-CaaX) embryos stained for Chondroitin sulfate and GFP. Orthogonal views are shown. For h, i, n= number of embryos.

Source data

Extended Data Fig. 2 Depletion of Ctnna1-Citrine by zGrad and characterization of the lamC1 mutants.

a, Principle of zGrad-mediated protein degradation. b, Left: 8 hpf embryos injected with sfGFP-ZF1 mRNA and mCherry-ZF1 mRNA with or without co-injected zGrad mRNA. Middle: 8 hpf embryos injected with YPet-ZF1 mRNA and mCherry-ZF1 mRNA with or without co-injected zGrad mRNA. Right: 8 hpf embryos injected with mNeonGreen-ZF1 mRNA and mCherry-ZF1 mRNA with or without co-injected zGrad mRNA. n ≥ 20 embryos. Scale bar: 1 mm. c, Single confocal slices of primordia in prim:mem-mCherry; ctnna1:ctnna1-citrine control (left) and prim:mem-mCherry; ctnna1:ctnna1-citrine; cxcr4b:zGrad 32 hpf embryos (right). Lower panels show the Ctnna1-Citrine fluorescence as a heat map. Scale bar = 20 μm. d, Quantification of the Ctnna1-Citrine fluorescence intensity in control and zGrad-expressing embryos at 32 hpf. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed Welch’s t-test). e, Expression of cxcl12a in control (wild-type or lamc1-/+) and lamC1 mutant 30 hpf embryos. Bracket indicates the location of interrupted cxcl12a expression domain. Scale bar = 0.5 mm. f, mCherry-expressing clones in muscle of 26 hpf control (wild-type or lamC1-/+) and lamC1 mutant embryos also transgenic for cldnB:lyn2GFP. Arrowheads indicate the position of primordium. Scale bar = 0.5 mm. g, Quantification of the distance from the ear to the first somite in the indicated genotypes at 26 hpf. Data points, means, and SD are indicated. n.s.: p=0.5516 (two-tailed Mann-Whitney test). h, Images of the primordium in wild-type and cxcl12a-/- 32 hpf embryos with clones in the trunk muscle that express Cxcl12a together with mCherry (not shown) (Left). Asterisks indicate the ear and arrowheads the primordium. Scale bar = 0.5 mm. Quantification of the distance migrated by the primordium in the indicated experimental conditions at 32 hpf (Right). Data points, means, and SD are indicated. ****: p<0.0001 (One-way ANOVA followed by Tukey’s multiple comparison test). i, Cross-sectional images of the Cxcl12a sensor in primordia of cxcl12a-/- and cxcl12a-/-; lamC1-/- embryos with clones in the muscle of the trunk that express mCherry or Cxcl12a. Quantification shown in Fig. 2k. Scale bar = 20 μm. For d, e, g, h, n = number of embryos.

Source data

Extended Data Fig. 3 β-integrin and talin expression analysis.

a, Expression analysis of β-integrins in the migrating primordium by in situ hybridization on 32 hpf embryos. Note that itgb8 could not be amplified from embryonic cDNA. Arrows indicate expression in the primordium. Scale bar = 0.5 mm. b, Schematics of the itgb1b locus, the itgb1b targeting cassette, and the modified itgb1b locus. c, Itgb1b-sfGFP expression in a 28 hpf itgb1b:itgb1b-sfGFP embryo. The image is a sum-projected z-stack. Scale bar = 0.5 mm. d, in situ hybridization against the three zebrafish talin genes on 32 hpf embryos. Arrow indicates enriched talin expression in the primordium. Scale bar = 0.5 mm. e, Schematic of the TgBAC(tln1:tln1-YPet) transgene and its protein product. f, Tln1-YPet expression in a 28 hpf tln1:tln1-YPet embryo. The image is a sum-projected z-stack. Scale bar = 0.5 mm.

Extended Data Fig. 4 Integrin-β1b and Talin1 dynamics in cells of the primordium.

a, Localization of Itgb1b-sfGFP and F-tractin-mCherry at the apical side of superficial cells in the primordium. The images are single optical slices. Arrowheads indicate Itgb1b-sfGFP clustering. Scale bar = 10 μm. b, Localization of Itgb1b-sfGFP (top) and Tln1-YPet (bottom) with membrane-mCherry at the basal sides of cells in clones in the primordium imaged over time taken from Supplementary Video 3. The images are single optical slices. Arrowheads indicate Itgb1b-sfGFP and Tln1-YPet clustering. Scale bar = 10 μm. c, Intensity profiles of Itgb1b-sfGFP (left) and Tln1-YPet (right) together with membrane-tethered mCherry along the contours of clones at indicated times taken from Supplementary Video 3. Arrows indicate Itgb1b-sfGFP and Tln1-YPet clusters that do not coincide with membrane-tethered mCherry clustering. Representative profile of 5 or more imaged cells. d, Montage of 10 consecutive images of the basal sides of the clones. The images are single transverse sections from a time lapse video. Scale bar = 10 μm. e, Quantification of co-localization of Itgb1b-sfGFP and Tln1-YPet with F-tractin-mCherry and membrane tethered mCherry. Li’s ICQ co-localization indices of 0.5 and −0.5 indicate perfectly co-localized and perfectly anti-co-localized signals, respectively. n = number of cells. Data points, means, and SD are indicated. Three data points were analyzed from the same embryo. **: p=0.0015 (two-tailed t-test). f, Images from time-lapse video after photo-bleaching of Itgb1b-sfGFP at the myotendinous junction of embryos treated with DMSO or 50 μM Rockout. GFP intensities are pseudo-colored as a heat map. Scale bars = 10 μm. g, Graph of Itgb1b-sfGFP fluorescence intensity over time before and after photo-bleaching in embryos treated with DMSO or 50 μM Rockout. The fluorescence intensities are normalized to the minimal intensities after photo-bleaching. Dots indicate mean intensities and error bars are SD. n = number of experiments, N = number of embryos. h, Plot of the percent recovery of Itgb1b-sfGFP fluorescence intensity at 28 sec after photo-bleaching in embryos treated with DMSO or 50 μM Rockout. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed Welch’s t-test). n = number of experiments (used for statistical test), N = number of embryos. i, Images from time-lapse video after photo-bleaching of Itgb1b-sfGFP in the primordium (left) and at the myotendinous junction (right). Fluorescence intensities are pseudo-colored as a heat map. Scale bars = 10 μm. j, Graph of Itgb1b-sfGFP fluorescence intensity over time before and after photo-bleaching in the primordium and at the myotendinous junction. The fluorescence intensities are normalized to the minimal intensities after photo-bleaching. Dots indicate mean intensities and error bars are SD. n = number of experiments, N = number of embryos. k, Plot of the percent recovery of Itgb1b-sfGFP fluorescence intensity at 27 sec after photo-bleaching in the primordium and at the myotendinous junction. n = number of experiments (used for statistical test), N = number of embryos. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed Welch’s t-test). l, Experimental design to culture primordium cells. m, Antibody staining against Itgb1b-GFP and F-tractin-mCherry on cultured primordium cells. Arrowheads indicate actin stress fibers (m’) with Itgb1b-GFP clusters (arrows in m”) in the cell center and in protrusions (m”’). Scale bars = 20 μm (m-m”) and 1 μm (m”’). n, Antibody staining against Tln1-YPet and F-tractin-mCherry on cultured primordium cells. Arrowheads indicate actin stress fibers (n’) with Tln1-YPet clusters (arrows in n”) in the cell center and in protrusions (n”’). Scale bars = 20 μm (n-n”) and 1 μm (n”’). Images are max-projected z-stacks. Close-ups (right panels) are magnifications of the regions indicated by dotted squares in the middle panels.

Source data

Extended Data Fig. 5 β1-integrin mutational analysis.

a, Schematic of the itgb1a and itgb1b alleles. d and i denote deletion and insertion, respectively. b, Primordium migration and morphology defects in embryos with different levels of integrin-β1 activity at 48 hpf. M and Z denote maternal and zygotic mutants, respectively. Arrows indicate the position of the primordium. Scale bar = 0.5 mm. c, Quantification of the primordium migration distance (left), the body length (middle), and primordium migration distance normalized to body length (right) in 48 hpf itgb1 mutant embryos. n = number of embryos. Data points, means, and SD are indicated. ****: p<0.0001 (one-way ANOVA followed by Tukey’s multiple comparison test, left plot), **: p=0.0026 (two-tailed Welch’s t-test, middle plot), and ***: p=0.0002 (two-tailed t-test, right plot). d, Overall morphology of control (wild-type or itgb1b-/+) and itgb1b mutant embryos at 24 hpf. Scale bar = 0.5 mm. e, Primordium migration in control (wild-type or itgb1b-/+) and itgb1b mutant embryos at 54 hpf. Scale bar = 0.5 mm. The arrows indicate the position of the primordium and the arrowheads indicate the position of the tip of the tail. f, Quantification of primordium migration in control (wild-type or itgb1b-/+) and itgb1b mutant embryos at 54 hpf. Data points, means, and SD are indicated. n = number of embryos. ****: p<0.0001 (two-tailed Welch’s t-test). g, prim:mem-mCherry; itgb1b:itgb1b-sfGFP control (left) and prim:mem-mCherry; itgb1b:itgb1b-sfGFP; cxcr4b:zGrad 33 hpf embryos (right). Lower panels show the Itgb1b-sfGFP fluorescence as a heat map. Scale bar = 25 μm. Images are single confocal slices from a z-stack. h, Quantification of the Itgb1b-sfGFP fluorescence intensity in control and zGrad-expressing embryos at 33 hpf. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed Welch’s t-test). n = number of embryos.

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Extended Data Fig. 6 Generation and characterization of talin mutant and analysis of primordium migration in embryos or primordia with depleted Talin activity.

a, Schematic of the tln1, tln2a and tln2b mutant alleles. The sequence around the deletions (d) and insertions (i) are shown. The start codons are indicated for tln1d4 and tln2ai23, and the premature stop codon for tln2ai23. b, Primordium migration distance in 48 hpf embryos with different levels of talin activity. Scale bar = 0.5 mm. c, Quantification of the primordium migration distance (left), the body length (middle), and primordium migration distance normalized to body length (right) in tln mutants at 48 hpf. Data points, means, and SD are indicated. ****: p<0.0001 (one-way ANOVA with Tukey’s multiple comparisons test, left plot), ***: p=0.0002 (two-tailed Welch’s t-test, middle plot). n = number of embryos. d, Crosses to generate embryos with depleted Talin activity. e, in situ hybridization against cxcl12a mRNA on 28 hpf wild-type (top) and Talin-depleted (bottom) embryos injected with zGrad mRNA. Scale bar = 0.5 mm. f, Quantification of the percentage of control and Talin-depleted embryos with perturbed cxcl12a expression along the horizontal myoseptum in 28 hpf embryos. n = number of embryos. g, Quantification of the body length in control and Talin-depleted 28 hpf embryos. n = number of embryos. Data points, means, and SD are indicated. **: p=0.0063, ****: p<0.0001, n.s.:p=0.1610 (two-tailed Mann-Whitney test). h, prim:mem-mCherry; tln1:tln1-YPet control (left) and prim:mem-mCherry; tln1:tln1-YPet; cxcr4b:zGrad 33 hpf embryos (right). Lower panels show the Tln1-YPet fluorescence as a heat map. Scale bar = 25 μm. Images are single confocal slices from a z-stack. i, Quantification of the Tln1-YPet fluorescence intensity in control and zGrad-expressing embryos at 33 hpf. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed t-test). n = number of embryos. j, Experimental strategy to generate embryos with Talin-depleted clones in the primordium. k, Migration of wild-type primordia with clones of control cells (top) and Talin-depleted cells (bottom). Images are maximum-projected z-stacks from Supplementary Video 5. The dotted lines indicate the location of primordium tip. Scale bar = 20 μm. l, Kymographs of migrating chimeric primordia shown in (k) and Supplementary Video 5. m, Quantification of the cumulative migration distance of primordia with clones of control cells and Talin-depleted cells. Dots are means, error bars are SD. *: p=0.03131, p=0.03046 and p=0.04856 (45, 50 and 55 min in the graph) (two-tailed t-test). n = number of embryos.

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Extended Data Fig. 7 Generation and characterization of itgb1b:Itgb1bΔNPxY-sfGFP mutant knock-in line and F-actin retrograde flow analysis.

a, Itgb1bΔNPxY-sfGFP expression in a 28 hpf itgb1bΔNPxY-sfGFP embryo. Image is sum-projected z-stacks. Scale bars = 0.5 mm. b, Distribution of Itgb1b-sfGFP and Itgb1bΔNPxY-sfGFP in muscle of 33 hpf embryos. Images are single z-slices through muscle at the myotendinous junction imaged and scaled identically. The GFP intensity is pseudo-colored as a heat map. Scale bars = 20 μm. c, Quantification of the Itgb1b-sfGFP and Itgb1bΔNPxY-sfGFP fluorescence intensities at the myotendinous junction (MTJ). Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed Mann-Whitney test). n = number of experiments (used for statistical analysis), N = number of embryos. d, Images from time-lapse video after photo-bleaching of Itgb1b-sfGFP and Itgb1bΔNPxY-sfGFP at the myotendinous junction. GFP intensities are pseudo-colored as heat maps. Scale bars = 10 μm. e, Graph of Itgb1b-sfGFP and Itgb1bΔNPxY-sfGFP fluorescence intensities over time before and after photo-bleaching. The GFP fluorescence intensities are normalized to the minimal intensities after photo-bleaching. Dots indicate mean intensities and error bars are SD. n = number of experiments, N = number of embryos. f, Plot of the percent recovery of Itgb1b-sfGFP and Itgb1bΔNPxY-sfGFP fluorescence intensities at 1 min after photo-bleaching shown in e. n = number of experiments (used for statistical analysis), N = number of embryos. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed t-test). g, Images of F-tractin-mNeonGreen localization at the apical sides of wild-type and itgb1b-/- primordium superficial cells (top). White arrows indicate the direction of migration. Scale bar = 2 μm. Images are single optical sections from Supplementary Video 6. Kymographs of Supplementary Video 6 along the dotted line indicated in top images (bottom). The dotted cyan line indicates the rate of actin flow. h, Protrusion rates in wild-type and itgb1b mutant primordium basal cells. Data points, means, and SD are indicated. n.s.: p=0.3167 (two-tailed t-test). n = number of cells. i, Plot of the protrusion rate versus the actin flow rate in individual primordium basal cells. n = number of cells.

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Extended Data Fig. 8 LamC1-sfGFP mobility, Embryogram workflow, and basement membrane stiffness measurements.

a, Optical sections along the indicated planes of a z-stack of the primordium and the BM labeled with LamC1-sfGFP from Supplementary Video 7. LamC1-sfGFP was bleached in front of the primordium in a hexagonal pattern. Scale bar = 50 μm. b, Hexagonal bleach pattern on LamC1-sfGFP-labeled BM underneath the migrating primordium (left). Dotted line indicates location of intensity profile shown on right. Scale bar = 5 μm. The image is a maximum-projected z-stack. c, FRAP analysis of LamC1-sfGFP and extracellular mCherry in heat-shocked hsp70l:sec-mCherry; lamC1:lamC1-sfGFP embryos. Images from the time course are shown on the left and quantification of fluorescence recovery is shown on the right. Scale bar = 10 μm, error bars = SD, n = measurements from N embryos, dots = means, n was used for statistical analysis. d, Extended FRAP analysis of LamC1-sfGFP over 50 min in lamC1:lamC1-sfGFP embryos. Images from time course are shown on the left and quantification of fluorescence recovery is shown on the right. Scale bar = 10 μm, error bars = SD, dots = means, n = measurements from N embryos. n was used for statistical analysis. e, Image of Embryogram application user interface. f, In Embryogram, candidate locations for the bleached markers are identified by a grid search (1), clustered in the XY-plane (2), and then along the Z-axis (3). We match these candidates with a regular hexagonal grid using the iterative closest point algorithm (4). Markers are tracked in subsequent frames using optical flow and numerical optimization. The user can manually offset rigid body motions caused by the movement of the microscope, sample movement or sample growth (6). The displacement of each dot is calculated using the mesh for the first time frame as the relaxed reference (7). To perform finite element analysis (FEA), the user constructs a volumetric tetrahedral mesh above, below or both (8) and inputs the Young’s modulus and the Poisson ratio of the material. The results of the FEA can be exported and visualized in other software packages such as ParaView (9). For detail see Supplementary Note 1. g, 28 hpf embryos with labeled skin and BM before and after surgical skin removal. Images are maximum-projected z-stacks. Scale bar = 100 μm. h, Deskinned and collagenase-treated lamC1:lamC1-sfGFP; cdh1:cdh1-TagRFP embryo. Image is a maximum-projected z-stack. Scale bar = 50 μm. i, TEM-image of the BM underneath the primordium. The semi-transparent yellow line traces the BM and the black lines indicate the thickness of the BM. Scale bar = 1 μm. j, Bright-field image of a deskinned embryo tail with the cantilever during an AFM measurement (left). A grid of 8×8 squares (20 μm by x 20 μm) on the BM was probed for its stiffness (square in left image) and the resultant stiffness map is shown on the right. Scale bar = 1 mm. k, Representative force curves showing the approach (red) and retraction (blue) curves for a deskinned embryo (top) and a collagenase-treated deskinned embryo (bottom). Cross-hairs indicate contact point position and force. Red dots on the approach curves indicate the first 200 nm from the contact point. The fit to the baseline and the Hertz model is indicated by a dotted black line. l, Analysis of the effect of repeated probing of the same area by AFM. The left image is a montage of the stiffness values obtained for the same location after measurements 1 to 100. The order of the measurements is indicated by the arrows. The force curves for the first and 100th measurements are shown on the right. The fit to the Hertz model is indicated in cyan. m, Quantification of the stiffness of the BM of deskinned embryos when fitting the first 500 nm after the contract point to the Hertz model. Data points, mean and SD are shown. Values for the fit of the first 200 nm to the Hertz model are shown for comparison. ****: p<0.0001 (two-tailed Mann-Whitney test). n, Representative force curves that meet (left) and do not meet (right) the indicated quality criteria.

Source data

Extended Data Fig. 9 Distribution of stresses under the skin, under the primordium, and in the absence of the primordium.

a, Images of LamC1-sfGFP (left) and basal skin cell membranes (middle) from Supplementary Video 9. The LamC1-sfGFP intensity is pseudo-colored as a heat map. The area outlined by a dotted line was analyzed using Embryogram to calculate the traction stresses (right) pseudo-colored as temperature map (right). The arrowhead indicates a spot of transient accumulation of LamC1-sfGFP. Images are maximum-projected z-stacks. Scale bar = 5 μm. b, Quiver plots of the BM displacement at 0 min in the XY- and XZ-planes. The XZ-plane quiver plot shows a subset of the vector field outlined by the orange rectangle. The magnitude of the vectors was increased by a factor of 3 for visualization purposes. Scale bar = 5 μm. c, Image of cell membrane at −1 min with arrows indicating the direction of movement from time point −1 min to 0 min as determined by PIV. Vector magnitudes are magnified three-times. Scale bar = 5 μm. d, Quantification of traction stresses. Traction stresses at the three vertices closest to a given wrinkle were averaged. Individual data points are shown. Individiual data points are indicated. **: p=0.0027 (−1 min vs. 0 min), p=0.0043 (0 min vs. 2 min) and n.s.: p=0.1479 (0 min vs. 1 min), p=0.7341 (−1 min vs. 2 min) (two-tailed paired t-test). n = number of measured cells, N = number of embryos, n was used for statistical analysis. e, Quantification of LamC1-sfGFP accumulation during BM wrinkling. Intensity profiles were obtained from a line plot across the BM wrinkle at 0 min indicated by the arrowhead in a, and from line plots at the same location of the images at the time points −1 min and 1 min. Intensities were normalized to the mean intensities at time point −1 min. Mean and SD are shown. n = number of measurements, N = number of embryos. f, Deformation of the BM before, during, and after primordium (magenta) migration. Images are from Supplementary Video 8. The white arrow indicates the direction of migration. Scale bar = 5 μm. g, Quantification of the displacement of bleached marks (yellow circles 1–4 in f) relative to control bleached marks (cyan circles in f). h, Quiver plot of the stresses in the direction of primordium migration. The magnitude of the stress vectors is color-coded. i, Distribution of the tensile and shear stresses around the migrating primordium outlined by a dotted line. The value of each unique component of the stress tensor is colored as a temperature map. The X and Y direction are indicated. The Z direction is orthogonal to the image plane. j, Experimental design of the stress analysis with blocked primordium migration. k, Images of a heat-shocked control embryo at 0 min and 80 min of Supplementary Video 8. The dotted line indicates the region used for the analysis. Images are maximum-projected z-stacks. Scale bar = 50 μm. l, Quiver plot of the displacement vectors shown along the Z, Y and X axes. The magnitude of the displacement vectors is color coded. Scale bar = 10 μm. m, Quiver plot of the displacement vectors projected in the XY-plane. The magnitude of the vectors was increased twofold. Scale bar = 10 μm. n, Distribution of the traction stress magnitudes color-coded using a temperature map. Scale bar = 10 μm. o, Quiver plot of the stresses in the direction of horizontal myoseptum. The magnitude of the stress vectors is color-coded. Scale bar = 10 μm. (lo) Data correspond to the at the 80 min time point of Supplementary Video 10.

Source data

Extended Data Fig. 10 The primordium is a continuously migrating tissue.

a, Itgb1b-tdTomato-to-Itgb1b-sfGFP (left) and Itgb1b-tdTomato-to-Itgb1bΔNPxY-sfGFP (right) ratio images in trunk muscle cells. Images are single optical slices from z-stacks. Ratios are color-coded as indicated. Scale bar = 25 μm. b, Quantification of ratios Itgb1b-tdTomato to Itgb1b-sfGFP and Itgb1b-tdTomato to Itgb1bΔNPxY-sfGFP at the myotendinous junction and lateral sides of muscle cells. Data points, means, and SD are indicated. ****: p<0.0001 (two-tailed Mann-Whitney test). n = number of measurements at indicated locations, N = number of embryos, n was used for statistical analysis. c, Image of Cxcr4b-EGFP and membrane-tethered Kate2 expressed from the Cxcl12a sensor in the primordium. Image is a maximum-projection of a z-stack. Scale bar = 25 μm. d, Quantification of the Cxcr4b-EGFP/Kate2 ratio across the primordium. Mean (black line) and SD (gray lines) are shown. n = number of embryos. e, Illustrations and predictions for two models of tissue migration. f, Quantification of junction length (left) and the cumulative migration distance over time for three primordia. g, Trajectories of individual primordium cells (left) and frequency plots for angles between any two given cell velocity vectors (right). h, Localization of Cadherin-2-mCherry and membrane-tethered EGFP in the primordium (left). Cdh2-mCherry fluorescence intensity pseudo-colored as a temperature map (middle) and on the primordium’s rear at higher magnification (right). Images are single confocal slice from the z-stack. Scale bars = 25 μm (left) and 10 μm (right). i, Images of slices from a z-stack of 32 hpf TgBAC(cxcr4b:EGFP-CaaX) embryos stained for F-actin (left) or phospho-MLC (right) and GFP. Orthogonal views are shown.

Source data

Supplementary information

Supplementary Information

Supplementary Note 1 and Supplementary Table 1.

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Supplementary Video 1

Migration of wild-type and Ctnna1-depleted primordia. Time lapse of the nuclei of migrating primordia in ctnna1:ctnna1-citrine/ctnna1:ctnna1-citrine; cxcr4b:h2a-mCherry (top) and ctnna1:ctnna1-citrine/ctnna1:ctnna1-citrine; cxcr4b:h2a-mCherry; cxcr4b:zGrad embryos (bottom). Time stamp in minutes. Each time frame is a sum-projected z-stack. The video starts at 36 h.p.f. Scale bar, 50 μm.

Supplementary Video 2

Z-stacks of Itgb1b–sfGFP and Tln1–YPet protein localization. Z-stack of the primordium from lateral (apical side of primordium) to medial (basal side of primordium) in wild-type embryos transgenic for itgb1b:itgb1b-sfGFP; prim:mem-mCherry (top) and TgBAC(tln1:tln1-YPet); prim:mem-mCherry (bottom). Scale bar, 50 μm, z-slice, 1.01 μm.

Supplementary Video 3

Time lapses of the dynamics of Itgb1b–sfGFP and Tln1–YPet proteins with the cell membrane or F-actin in a clone of cells in the primordium. Time lapse of cell clones expressing Itgb1b–sfGFP and mem-mCherry (top left), Tln1–YPet and mem-mCherry (bottom left), Itgb1b–sfGFP and F-tractin–mCherry (top right) and Tln1–YPet and F-tractin–mCherry (bottom right) in unlabelled primordia of 32 h.p.f. chimeric embryos. The Itgb1b–sfGFP and Tln1–YPet fluorescence intensities with the mem-mCherry or F-tractin–mCherry fluorescence intensity (left), the Itgb1b–sfGFP and Tln1–YPet fluorescence intensities only (middle), and the mem-mCherry fluorescence intensity or F-actin–mCherry only (right) are shown. Scale bar, 5 μm. Time stamp in seconds. Each time frame is a single slice at the basal side of the cells from a z-stack.

Supplementary Video 4

Time lapses of migrating primordia in wild-type and itgb1b mutant embryos. (Top) Time lapse of migrating primordia in wild-type (top) and itgb1b mutant embryos (lower top) labelled with the prim:mem-mCherry transgene. Scale bars, 50 μm. Each time frame is a sum-projected z-stack. The video starts at 34 h.p.f. (Bottom) High temporally resolved time lapse of primordium migration in wild-type (upper bottom) and itgb1b mutant embryos (bottom) labelled with the cldnB:lyn2GFP transgene. Scale bars, 30 μm. Time stamp in minutes. Each time frame is a maximum-projected z-stack. The video starts at 32 h.p.f.

Supplementary Video 5

Time lapses of migrating primordia with reduced talin activity. (Top) Time lapse of migrating primordia in cxcr4b:zGrad control (top) and (MZ)tln1–/–; (Z)tln2a+/–; (MZ)tln2b–/–; cxcr4b:zGrad embryos (lower top). The primordia are labelled with the prim:mem-mCherry transgene. Scale bar, 100 μm. Time stamp in minutes. Each time frame is a sum-projected z-stack. The time lapses start at 32 h.p.f. (Bottom) Time lapse of wild-type primordia labelled with the cldnB:lyn2GFP transgene (green) containing wild-type (upper bottom) and talin-depleted cells (bottom) labelled with the prim:mem-mCherry transgene (magenta) at the tip of the primordium. Scale bar, 20 μm. Time stamp in minutes. Each time frame is a maximum-projected z-stack. The videos start at 36 h.p.f.

Supplementary Video 6

Time lapses of F-actin dynamics in single wild-type and itgb1b mutant primordium basal cells and superficial cells. Time lapse of F-actin dynamics in single basal cells (top) and superficial cells (bottom) expressing the F-actin marker F-tractin–mNG from the injected cxcr4b:F-tractin-mNeonGreen BAC construct in wild-type primordia (left) and itgb1b mutant primordia (right). Scale bar, 2 μm. Time stamp in seconds. Each time frame is a single slice at the basal side of the cell from an z-stack. Images were thresholded using the Huang algorithm in Fiji to extract pixels with high signal. The videos start at 33–36 h.p.f.

Supplementary Video 7

Z-stack of LamC1–sfGFP-labelled BM with bleach marks. Z-stack of a 32 h.p.f. lamC1:lamC1-sfGFP; prim:mem-mCherry embryo in which LamC1–sfGFP was bleached in a hexagonal pattern to optically mark the BM in front of the migrating primordium. Optical section thickness is 0.4 μm. Scale bar, 50 μm.

Supplementary Video 8

Time lapses of primordia with the BM marked by a bleached pattern in wild-type, Cxcl12a overexpressing, itgb1b mutant embryos. Time Lapse of lamC1:lamC1-sfGFP; prim:mem-mCherry (top), lamC1:lamC1-sfGFP; prim:mem-mCherry; hsp70:cxcl12a (middle) and itgb1b–/–; lamC1:lamC1-sfGFP; prim:mem-mCherry (bottom) embryos with bleach marks in front of the primordium at the first time point. Montage of orthogonal views of the z-stack are shown along the cross-hair for each time point (xy, xz and yz planes). Note that the view of the xy plane is a maximum-projection of the z-stack while the views of the xz and yz planes are single slices through the z-stack. Ubiquitous Cxcl12a expression was induced by a heat shock to block primordium migration (middle). Scale bar, 25 μm. Time stamp in minutes.

Supplementary Video 9

Time lapse of BM wrinkling and traction stresses. Montage of time lapses showing the membrane of the skin cells labelled by mem-mCherry expressed from injected lyn2mCherry mRNA and the LamC1–sfGFP-labelled BM bleach pattern (TgBAC(lamC1:lamC1-sfGFP), left), the LamC1–sfGFP signal intensities as a fire map (middle left), mem-mCherry signal intensities as a greyscale (middle right), and the magnitude of the traction stresses as a temperature map (right). Note that the first time frame of the time lapse serves as the reference for the undeformed BM. Therefore, no stresses can be calculated for the −2 min time frame. Images are maximum-projected z-stack. Scale bar, 5 μm. Time stamp in minutes.

Supplementary Video 10

BM deformation and stresses in control, wild-type and itgb1b mutant embryos. Montage of time lapses showing the displacement vector field (left), the displacement vector field projected onto the xy plane (middle left), the traction stress magnitudes (middle right) and the stress vectors calculated in the direction of migration (right) together with the migrating primordium (top), in the control condition with blocked primordium migration (middle) and in itgb1b mutant embryos (bottom). For the displacement vector field the orthogonal views of the xz (left, bottom) and yz planes are also shown (left, left). Note that the magnitude of the displacement vectors are increased twofold for visibility, the magnitude of the traction stresses are indicated as a temperature map, the magnitude of the stress vectors calculated in the direction of migration are indicated as a colour code, and the xy planes are shown from the basal side of the primordium. Supplementary Video 8 shows the original time lapses that were used for this analysis. Scale bar, 25 μm.

Supplementary Software 1

ImageJ-based and R-based codes used for image analysis.

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Yamaguchi, N., Zhang, Z., Schneider, T. et al. Rear traction forces drive adherent tissue migration in vivo. Nat Cell Biol 24, 194–204 (2022). https://doi.org/10.1038/s41556-022-00844-9

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