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.

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.

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.

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 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 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.

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.

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 tln1^d4 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 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 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.

Extended Data Fig. 7. Generation and characterization of itgb1b:Itgb1b^ΔNPxY-sfGFP mutant knock-in line and F-actin retrograde flow analysis

a, Itgb1b^ΔNPxY-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 Itgb1b^ΔNPxY-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 Itgb1b^ΔNPxY-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 Itgb1b^ΔNPxY-sfGFP at the myotendinous junction. GFP intensities are pseudo-colored as heat maps. Scale bars = 10 μm. e, Graph of Itgb1b-sfGFP and Itgb1b^ΔNPxY-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 Itgb1b^ΔNPxY-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 Video 6. Kymographs of 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.

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 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 × 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 100^th 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.

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 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 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 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. (l–o) Data correspond to the at the 80 min time point of Video 10.

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

a, Itgb1b-tdTomato-to-Itgb1b-sfGFP (left) and Itgb1b-tdTomato-to-Itgb1b^ΔNPxY-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 Itgb1b^ΔNPxY-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.

Fig. 1.. The primordium migrates on top of a basement membrane and directly under the skin

a, TEM images of the skin (s), primordium (p, purple hue), the muscle (m), and BM (white arrows). n = 1 embryo. Scale bar = 1 μm. b, Optical sections through a primordium in a 31 hpf embryo expressing LamC1-sfGFP. Scale bar = 50 μm. c, Optical section through a primordium labeled with F-tractin-mCherry in a live 32 hpf embryo expressing Cdh1-sfGFP. n = 7 embryos. Scale bar = 25 μm. A: apical, B: basal. d, schematic illustration of the environment around the primordium.

Fig. 2.. Primordium migration requires an intact basement membrane

a, Control and Ctnna1-depleted primordia (arrowheads) in 48 hpf embryos. Scale bar = 0.5 mm. Close-up of region indicated by a dashed square. b, Quantification of the migration distance for primordia shown in a. Individual data points, means and SD are indicated. **: p=0.0013 (two-tailed Mann-Whitney test). c, Speed of Ctnna1-depleted primordium cells. Solid line = median, dashed line = quartile. n = cell speeds from more than 7 primordia with each more than 100 cells). d, Left. TEM images of the ultrastructure of the BM between the skin (s) and the muscle (m) in control (n = 2) and lamC1−/− embryos (n = 1). White arrows indicate the BM. Scale bar = 2 μm. Right. Antibody staining against Collagen IV in control and lamC1−/− embryos. Scale bar = 50 μm. e, Quantification of Collagen IV filaments in control and lamC1−/− embryos. Individual data points, means and SD are indicated. **: p=0.0056 (two-tailed Mann-Whitney test). f, Strategy to express Cxcl12a in a few muscle cells in lamC1−/− embryos and siblings. g, Images of the migrating primordium in cxcl12a−/− and cxcl12a−/−; lamC1−/− 32 hpf embryos with clones in the trunk muscle that express mCherry (not shown) or Cxcl12a together with mCherry (not shown). Asterisks indicate the ear and arrowheads the primordium. Scale bar = 0.5 mm. h, Quantification of the distance migrated by the primordium in the indicated experimental conditions at 32 hpf. Individual data points, means and SD are indicated. ***: p=0.0002, ****: p<0.0001, n.s: p=0.0879 (two-tailed Mann-Whitney test). i, Principle of the Cxcl12a sensor. j, Images of the Cxcl12a sensor in primordia of cxcl12a−/− and cxcl12a−/−; lamC1−/− live embryos with clones in the muscle of the trunk that express mCherry or Cxcl12a. Scale bar = 20 μm. k, Quantification of the Cxcr4b-Kate-to-memGFP ratio in the primordia of embryos shown in j. Individual data points, means and SD are indicated. *: p=0.038, ***: p=0.0001, n.s: p=0.1349 (one way ANOVA followed by Holm-Sidak’s multiple comparison test). Note, controls are lamc1+/+ and lamc1−/+ embryos. For a, b, e, h, k, n = number of embryos.

Fig. 3.. β1-Integrin and Talin form small short-lived clusters at the basal sides of the primordium cells

a, Expression of Itgb1b-sfGFP from the endogenous locus in a 33 hpf embryo. The image is a single slice from a z-stack. The primordium is outlined by a dotted, yellow line. Scale bar = 20 μm. b, Expression of Tln1-YPet from the tln1:tln1-YPet BAC transgene in a 33 hpf embryo. The image is a single slice from a z-stack. The primordium is outlined by a dotted, yellow line. Scale bar = 20 μm. c, Schematic of blastomere transplantation experiments. d, Intensity profiles of Itgb1b-sfGFP (left) and Tln1-YPet (right) with F-tractin-mCherry along the perimeter on the basal sides of the clones shown in (e) and (f) over time. e, Images of Itgb1b-sfGFP and F-tractin-mCherry localization at the basal side of a clone in the primordium over time. Images are single slices from a time-lapse movie (Video 3). Arrowheads indicate Itgb1b-sfGFP/F-tractin-mCherry clusters. Scale bar = 10 μm. f, Images of Tln1-YPet and F-tractin-mCherry localization at the basal side of a clone in the primordium over time. Images are single slices from a time-lapse movie (Video 3). Arrowheads indicate Tln1-YPet/F-tractin-mCherry clusters. Scale bar = 10 μm.

Fig. 4.. β1-Integrin, Talin and their interaction are required for efficient primordium migration

a, Maximum-projected z-stacks of migrating primordia in wild-type and itgb1b−/− embryos temporally color-coded as indicated. Scale bar = 100 μm. b, Cumulative primordium migration distance in control and itgb1b−/− embryos. Means and SDs are indicated. **: p=0.0053 (two-tailed t-test at the end point). c, Maximum-projected z-stacks of primordia in wild-type and the itgb1b−/− embryos. Scale bar = 25 μm. d, Primordium circularity in control and itgb1b−/− embryos. Means and SDs are indicated. *: p=0.035 (two-tailed t-test at the end point). e, Crossing scheme to generate embryos with primordium-specific depletion of Itgb1b-sfGFP. f, Primordium migration in 48 hpf embryos of indicated genotypes. Primordium-specific Itgb1 depletion refers to genotype shown in e. Arrows indicate primordia, arrowheads indicate the cxcr4b:zGrad transgene marker. Scale bar = 500 μm. g, Primordium migration distance of embryos shown in (f). Individual data points, means and SD are indicated. ****: p<0.0001 (one-way ANOVA followed by Tukey’s multiple comparison test). h, Crossing scheme to generate embryos with reduced Talin activity in the primordium. i, Maximum-projected z-stacks of migrating primordia in embryos of indicated genotypes color-coded for time as indicated. Scale bar = 100 μm. j, Cumulative primordium migration distance in cxcr4b:zGrad and (MZ)tln1−/−; (Z)tln2a+/− or +/+; (MZ)tln2b−/−; cxcr4b:zGrad embryos. Means and SD are indicated. ****: p<0.0001 (two-tailed t-test at the end point). k, Amino acid alignment of indicated Integrin-β1 cytoplasmic domains, the two NPxY motives are indicated in red. l, Itgb1b-sfGFP and Itgb1b^ΔNPxY-sfGFP localization in the primordium. Images are single transverse section from z-stacks. Scale bar = 25 μm. m, Quantification of the Itgb1b-sfGFP and Itgb1b^ΔNPxY-sfGFP distribution on the membrane of primordium cells along the apicobasal axis normalized to the fluorescence intensity of membrane-tethered mCherry. *: p=0.012 at 1.3 μm (two-tailed Welch’s t-test). Individual data points (pale dots), means (dots) and SDs are indicated. n, Primordium position in itgb1b:itgb1b-sfGFP/− and the itgb1b:itgb1b^ΔNPxY-sfGFP/− 48 hpf embryos. Arrows indicate primordia. Scale bar = 0.5 mm. o, Quantification of the primordium migration distance. Individual data points, means and SD are indicated. ****: p<0.0001 (two-tailed Mann-Whitney test). For b, d, g, j, m, o, n = number of embryos.

Fig. 5.. β1-Integrin couples cell-substrate adhesion to actin flow in the primordium

a, Experimental design to assess actin flow. b, Images of F-tractin-mNeonGreen localization at the basal sides of wild-type and itgb1b−/− primordium cells (top). White arrows indicate the direction of migration. Scale bar = 2 μm. Images are single optical sections from Video 6. Kymographs of Video 6 along the dotted line indicated in top images (bottom). The dotted cyan and magenta lines indicate the rates of actin flow and protrusion, respectively. c, Quantification of the rates of actin flow (left) and actin polymerization (right) in the primordium basal cells. Individual data points (dots), means (horizontal lines) and SD (vertical lines) are indicated. ****: p<0.0001 (two-tailed Mann-Whitney test). d, Quantification of the rate of actin flow in superficial primordium cells. n.s.: p=0.3708 (two-tailed t-test). Individual data points (dots), means (horizontal lines) and SD (vertical lines) are indicated. For c, d, n = number of cells pooled from more than 5 primordia in each condition.

Fig. 6.. Traction stress measurements indicate that the primordium exerts highest stresses in its rear

a, Strategy to measure traction stresses in vivo. b, Representative force curve of BM stiffness measurement by AFM. Fit of the first 200 nm after the contact point at 0 μm to the Hertz model is indicated in cyan. Only the approach part of the force curve is shown. c, Quantification of the BM stiffness with and without Collagenase treatment. N = number of embryos, n = number of recorded approach curves. Individual data points, means, and SD are indicated ****: p<0.0001 (two-tailed Mann-Whitney test). d, Maximum-projected z-stack (Video 8). Scale bar = 25 μm. e, BM displacement by the primordium (grey) shown as a vector field along the X-, Y- and Z-axes (Video 10). The displacement vector magnitude is indicated as a color map. XY-view is shown from the basal side of the primordium. s: skin, m: muscle. Scale bar = 25 μm. f, BM displacement in the XY-plane by the primordium (grey) shown as a vector field (Video 10). Displacement field in the XY-plane is shown from the basal side of the primordium. Scale bar = 25 μm. Bottom panel is a magnification of the outlined region in top panel. Scale bars = 25 μm (top) and 5 μm (bottom), arrow = 50 μm (top) and 10 μm (bottom). g, Traction stress magnitudes on the BM indicated as color map (Video 10). Scale bar = 25 μm. h, Schematic of the approach to quantify the traction stresses and BM displacement vector direction. i, BM displacement vector angles with respect to the migration direction (0°). Mean, 25th–75th percentiles (box), and min/max (whiskers) are indicated N = number of embryos, n = number of bleached cylinders. ****: p<0.0001 and n.s.: p=0.6291. j, Pooled traction stresses exerted by the primordium on the BM. Magnitude in XY-plane (left) and along the Z-axis (right) with median (dashed line) and quartile (dotted line) are shown. *: p=0.0302, ****: p<0.0001, n.s.: p=0.2273 (two-tailed Mann-Whitney test). N = number of embryos, n = number of bleached cylinders analyzed. For c, i, j, n was used for statistical tests.

Fig. 7.. The basement membrane wrinkles around the primordium

a, F-tractin-mCherry distribution in the primordium (top) and LamC1-sfGFP around the primordium (bottom) in a 32 hpf embryo. LamC1-GFP fluorescence intensity is pseudo-colored as a heat map. The image is a maximum-projected z-stack. Scale bar = 50 μm. b, Transverse section through the F-tractin-mCherry-expressing primordium and the underlying LamC1-sfGFP-labeled BM (left top). Corresponding image showing the LamC1-sfGFP fluorescence intensity as a heat map (left bottom). Arrows indicate apposed clusters of F-tractin-mCherry and LamC1-sfGFP. Images are single sections along the YZ-plane of a z-stack. Scale bar = 10 μm. Fluorescent intensity profiles of F-tractin-mCherry and LamC1-sfGFP of image shown in left along the Y-axis (right). Arrows indicate the position of the apposed clusters of F-tractin-mCherry and LamC1-sfGFP indicated by arrows in left. c, Quantification of the LamC1-sfGFP intensity within 3 μm-wide bands around the perimeter of the primordium (left) and at a distance of 6 μm from the primordium’s perimeter (right). ****: p<0.0001 (two-tailed paired t-test). n = number of embryos.

Fig. 8.. The primordium generates larger forces in the rear

a, Myl12.1-mScarlet distribution in the primordium. Middle, Myl12.1-mScarlet in the entire primordium. Bottom, Myl12.1-mScarlet on the primordium’s basal side pseudo-colored. Scale bar = 20 μm. b, Quantification of the basal Myl12.1-mScarlet intensity at indicated positions in the primordium. Data points, means, and SD are indicated. (one-way ANOVA followed by Tukey’s multiple comparison test). c, Images of F-tractin-mNeonGreen at the basal sides of primordium cells (top). Arrows, direction of migration. Scale bar = 2 μm. Kymographs along the dotted line indicated in top images (bottom). d, Actin flow rates in basal primordium cells. n = number of cells pooled from > 5 primordia. Data points, means, and SD are indicated. (two-tailed Mann-Whitney test). e, Distribution (top) and quantification (bottom) of Itgb1b-sfGFP on the primordium cell membranes. Itgb1b-sfGFP intensity is pseudo-colored. Scale bar = 20 μm. Mean and SD are indicated. f, Fluorescence intensity ratio images of Itgb1b-tdTomato to Itgb1b-sfGFP (top left) and Itgb1b^ΔNPxY-sfGFP (bottom left) on the membrane of primordium cells. Quantification of intensity ratios (right). Ratios are shown in pseudo-color. Mean and SD are indicated. g, BM displacement around the primordium (grey) in itgb1b−/− embryos shown as a vector field along the X-, Y- and Z-axes (Video 10). Vector magnitude is indicated as a color map. XY-view is from the basal side of the primordium. Scale bar = 25 μm. h, Traction stresses on the BM indicated as a color map (Video 10). Scale bar = 25 μm. i, Pooled traction stresses exerted by the primordium on the BM in itgb1b−/− embryos. Median (thick line) and quartile (thin line) are shown. Wild-type control is the same as Fig. 6j. (two-tailed Mann-Whitney test). N = number of embryos, n = number of bleached cylinders. n was used for statistics. j, Left, staining against Paxillin (top) and phosphorylated Paxillin (bottom) protein in primordia shown as a heat map. Right, quantification of the fluorescence intensity on the primordium membrane for Paxillin (top) and phosphorylated Paxillin (bottom). Mean and SD are indicated. Scale bar = 20 μm. k, Model for primordium motility. For b, e, f, j, n = number of embryos.