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Fig. S1. Dorsal tissues may be under a small but finite pre-strain within the embryo. (A) A representative stage 16 embryo immediately before microsurgery. (B) A dorsal isolate from the embryo in A within 60 seconds of the first image. The epithelium of the dorsal isolate contracts ∼10% along the anterior-posterior axis after microsurgery (compare A′ with B′). (C) Wound healing over the next 10 minutes does not significantly change the strain but does ‘round’ the corners of the isolate. (D) Pre-strain in the anterior-posterior and mediolateral direction and area dilation in the dorsal epithelium from five embryos-isolate pairs. (E) Nine-minute uniaxial compression tests. Five examples of 9-minute stress-relaxation curves. Dorsal isolates exhibit typical viscoelastic properties with an immediate viscous response and a long-term steady-state elastic response to applied strain. Tissues nearly reach residual stiffness (steady-state elastic modulus) within 3 minutes. Arrows show examples of transient active-force generation by the tissues which may represent either a contraction event, wound healing, or elongation forces. (F) Five repetitions of the 3-minute uniaxial compression tests. 20% strain is applied to the same dorsal isolate, held for 3 minutes, and followed immediately by 1-minute zero strain rest before the next test. The elastic modulus is shown for four examples of stress-relaxation curves in response to multiple compression tests. Three isolates are measured in the fourth repetition and two in the fifth repetition, owing to the isolates slipping out from the test apparatus. Dorsal isolates increased their stiffness with repeated cycles, but still remain viscoelastic. (G) One-minute uniaxial compression test with increasing strain. A 10% strain is initially applied to a single dorsal isolate, held for 1 minute, and the strain increased 5% every minute in next 4 minutes. A representative trace of the time-dependent elastic modulus show that the dorsal isolate remain viscoelastic under different strains. A strain-stress diagram of five different dorsal isolates reveal that dorsal isolates become stiffer with increasing strain.
Fig. S2. Standard Linear Solid Model representation of a dorsal isolate in unconfined uniaxial compression exhibiting creep and stress relaxation. The first step in quantifying the viscous and elastic properties of these tissues is to determine the time-(t)-dependent Young’s modulus, E(t). The Young’s modulus is defined by the fundamental relationship between stress (σ) and strain (ε): E=σ / ε. Strain is a dimensionless measure of deformation and stress is a measure of force applied per unit area. For the special case of unconstrained compression the modulus, stress and strain can be represented by single numbers. Strain is the deformation of the material along the direction of compression: ε=L(0)-L(t)/L(0). Stress is the axial force applied to the tissue divided by the cross-sectional area perpendicular to the direction of the applied force: σ=F/A. Each of the values of F, A, L(t) and L(0) can be measured during an unconfined compression test and E(t) can be represented by a viscoelastic model-material consisting of a network of springs and dashpots. (A) The Standard Linear Solid Model (SLS), consisting of a spring (k1) in parallel to a spring (k2) and dashpot (η) in series, captures both the creep and stress-relaxation behaviors exhibited by the dorsal isolate. The springs are ideal Hookean springs and the dashpot is an idealized Newtonian fluid. The form of the Young’s modulus of this network is: E(t)=k1 + k2 * exp(-t/τ); where τ=η/k2. Nonlinear regression fitted parameters k1, k2 and η to real data (B, broken line) reproduces the general form of the time-dependent Young’s modulus (E(t); B, unbroken line) measured from a real dorsal isolate. The time-dependent force, F(t), is determined from the displacement of the fiber optic from the center-null position on the quadrant detector. The time-varying axial length of the tissue, L(t), is determined from the visual time-lapse recording during the 180-second time-course of the compression testing. In general we use the final length L (180 seconds) for this term as it does not vary more than 3% over the course of compression (data not shown). The cross-sectional area, A(t), cannot be measured during the course of the experiment but is instead determined at the conclusion of the compression test. The area at the end of each compression test, A(180s), is measured from the cross-sectional area of each dorsal isolate after fixation (Davidson, 2007). The resulting time dependent modulus is: E(t)=(F(t) * L0)/(A(180)*(L(0)−L(180).
Fig. S3. Superposition model of dorsal tissues: composite analysis of stiffness. (A) Tissues within the dorsal isolate can be broken down into discrete structural elements: notochord, paraxial-medial mesoderm, paraxial-lateral mesoderm, neural plate ectoderm and archenteron roof-plate endoderm. (B) The relative area contributions of these five tissues and their contributions to midline (N), paraxial-medial (M) and paraxial-lateral (L) tissues are measured from transverse confocal optical sections from rhodamine-dextran-labeled cells (red) and fibronectin fibril-labeled tissue boundaries (green). The gray line overlay indicates the boundaries between these regions. The schematic on the right highlights the discrete structural elements present in this confocal section.
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