Supplementary Figure 1

(a) Confocal projection of 20s limb bud visualising pHH3 (red), Tcf/Lef::H2B-Venus (green), and DAPI (blue). Shown are multiple images stitched together. (b) xy sections of limb field in embryos treated with water control (left) or 100 μM NSC23766 (right) visualising actin (red) and DAPI (blue). (c–e) xy sections at the DV boundary of initiating limb bud ectoderm visualising Myosin IIB (c), Myosin IIA (d), phospho-Myosin Light Chain-1 (e) (green) and DAPI (blue). (f) Composite confocal projection of 17-somite stage lateral plate visualising DAPI used to measure dimensions of pre-limb lateral plate ectoderm for simulation. (g) Scanning electron micrograph of a custom 10 μm spherical AFM probe. (h) Two-paired Prony series fitting of limb bud viscoelastic properties. (i) Illustration of finite element model of initiating limb bud. Red arrow indicates direction of mesodermal growth, yellow arrowhead indicates fixed support. (j) Percentage of pHH3-positive cells relative to total cells in control versus PEG-injected embryos (n = 3 18–21 som. embryos; p = 0.9871 (Student’s t-test)). (k) xy section away from DV boundary of pre-AER 23 som. limb bud ectoderm visualising Myosin IIB (green) and Actin (red). (l) xy section of the apical plane of 19 som. limb bud visualising actin (red) and myosin (green). (m) Model of 22 som. pre-AER limb bud. Black arrow indicates mesodermal growth, yellow arrowhead indicates fixed support. (n) Model of AER progenitor intercalation at DV boundary. Black arrows indicate direction of ectodermal tension (modelled as pulling force). (o,p) Stresses are linearly proportional to the pulling force applied at the DV midline; (o) 2 pN, (p) 1 pN. (q,s,u) Distance between two vertices attached to cut interface for (q) distal AP versus proximal AP, p = 0.0142, (s) lateral plate AP versus lateral plate DV, p = 0.1381, (u) intact AP versus post-cut AP interfaces, p = 0.0475. (r,t,v) Peak retraction velocities of ablated (r) distal AP versus proximal AP, (t) lateral plate AP versus lateral plate DV, (v) AP versus post-cut AP interfaces interfaces ((r) p = 0.0023, (t) p = 0.1544, (v) p = 0.0488; (q–v) n ∼ 15 ablations for each of 4 embryos). ((q,r) Student’s t-test with Holm’s correction). Scale bars indicate 5 μm (g), 10 μm (c–e,k,l), 20 μm (a,b), 100 μm (f). Error bars indicate SEM.