Mechanical properties during convergent extension
Multiscale analysis of architecture, cell size and the cell cortex reveals cortical F-actin density and composition are major contributors to mechanical properties during convergent extension.
Shawky JH, Balakrishnan UL, Stuckenholz C, Davidson LA.
Development 2018 145: dev161281 doi: 10.1242/dev.161281 Published 5 October 2018.
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The large-scale movements that construct complex three-dimensional tissues during development are governed by universal physical principles. Fine-grained control of both mechanical properties and force production is crucial to the successful placement of tissues and shaping of organs. Embryos of the frog Xenopus laevis provide a dramatic example of these physical processes, as dorsal tissues increase in Young''s modulus by six-fold to 80 Pascal over 8 h as germ layers and the central nervous system are formed. These physical changes coincide with emergence of complex anatomical structures, rounds of cell division, and cytoskeletal remodeling. To understand the contribution of these diverse structures, we adopt the cellular solids model to relate bulk stiffness of a solid foam to the unit size of individual cells, their microstructural organization, and their material properties. Our results indicate that large-scale tissue architecture and cell size are not likely to influence the bulk mechanical properties of early embryonic or progenitor tissues but that F-actin cortical density and composition of the F-actin cortex play major roles in regulating the physical mechanics of embryonic multicellular tissues.
Fig. 1. Multiscale contributors to tissue mechanical properties. (A) Structural elements at the tissue, cell and molecular scale may contribute to bulk tissue mechanical properties. Germ layers in the dorsal axis are depicted in different colors: ectoderm (blue), mesoderm (red) and endoderm (yellow). (B) Time-dependent Young’s modulus [E(t)] of dorsal tissues measured by uniaxial stress relaxation. Dorsal tissues from Xenopus laevis embryos are microsurgically isolated and loaded into the nanoNewton force measurement device (nNFMD). Tissues are compressed to a fixed strain (ε) and the compressive force is measured using a calibrated force transducer. Modulus is calculated from strain, force and the cross-sectional area measured after fixation (Zhou et al., 2009). (C) Residual elastic modulus [E(180)] determined from testing shows that dorsal tissues stiffen ∼150% between stages 14 and 21. Two clutches were tested (number of explants in each set indicated in parentheses below the plot). ***P<0.001 by Mann–Whitney U test. Error bars represent s.d. Note: explants were treated with 0.5% DMSO
Fig. 2. Young’s modulus depends on stage, not architecture. (A) Transverse sections of stage 14 and stage 21 dorsal tissues stained for F-actin (phallacidin). Note the large-scale tissue architecture change between stages. A, anterior; D, dorsal; Ec, ectoderm; En, endoderm; Ep, prospective epidermis; M, mesoderm; Ne, neural ectoderm; No, notochord; P, posterior; V, ventral. The flat neural plate (Ec; stage 14) bends, folds and internalizes to form the neural tube (Ne). (B) Cell shapes in PSM tissues in dorsal isolates mosaically injected with the F-actin reporter Lifeact-eGFP. By stage 21, mesoderm cells lengthen dorso-ventrally (as revealed by the transverse aspect ratio), reflecting tissue-level mesoderm thickening (***P<0.001; Mann–Whitney U test; n=cells, explants). Arrowheads indicate filopodia-like protrusions from the lateral surface of the cells. Schematics depict the morphological changes in cell shape in the PSM during neurulation. (C) Schematic of tissue architecture disruption. Tissues were isolated at neurula stage, dissociated, and re-aggregated into ‘scrambled’ tissues. Mechanical properties and extracellular matrix organization within scrambled tissues were compared with native tissues. The re-aggregation process is detailed in the inset beneath the summary schematics. Dissociated cells were loaded into custom-made chambers and centrifuged to generate single ∼0.8×1 mm elliptical sheets of tissue that were dissected into regular dorsal isolate-shaped blocks. (D) Fibronectin and β-catenin staining reveal loss of bulk architecture, stereotypic fibronectin organization and cell shape in scrambled tissues. (E) Cross-explant mixing occurs within ECM encapsulated clusters as seen in scrambled tissues made from half rhodamine dextran (red)-injected embryos and half FITC-dextran (green)-injected embryos. (F) En face z-slices of dorsal isolate and scrambled tissues stained for the somite marker 12/101 and F-actin (phallacidin). Dashed white lines indicate optical sections at three points along the dorsal axis from anterior (a) to posterior (c). Cells within aged scrambled tissues (to the equivalent of stage 18) express 12/101, indicating differentiation to somitic tissue. (G) En face z-slices of dorsal isolate and scrambled tissues stained for F-actin, laminin and fibrillin. Insets reveal high levels of fibrillin between notochord cells (yellow arrow). Aged scrambled tissues (to the equivalent of stage 18) reveal de novo synthesis of laminin and fibrillin. (H) Scrambled tissues (hashed bars) have the same Young’s modulus as native control tissues (unhashed bars) in early and late neurula tissues (stage 14: 27.9 Pa versus 33.8 Pa; twoway ANOVA, P=0.371; stage 21: 85.5 Pa versus 85.1 Pa; two-way ANOVA, P=0.971). Each cluster represents one experiment and n value (beneath the bars) represents number of explants tested per group. Error bars represent s.d. Xenopus stage schematics adapted from Nieuwkoop and Faber (1967). n.s., not significant. Scale bars: 100 μm.
Fig. 3. Testing predictions of the cellular solids model: cell size correlates with stiffness. (A) To generate large cells in tissue explants, we arrested the cell cycle using a combination of cell cycle inhibitors, hydroxyurea and aphidicolin (HUA). Tissues were microsurgically isolated at stage 14 and mechanically tested. (B-E′) Representative images of control (B-E) and HUA-treated (B′-E′) tissues stained for nuclei (Yo-Pro-1), fibronectin (4H2) and F-actin (phallacidin). (F,G) Tissues treated with HUA show reduced nuclei/volume (***P=0.01; Mann–Whitney U test; F) and a 25% decrease in Young’s modulus (two-way ANOVA, P=0.04; G). Dashed red line indicates CSM prediction. (H) To generate small cells in tissue explants, we induced a cell division in the PSM by inhibiting a developmentally regulated cell cycle inhibitor, Wee2, using anti-sense morpholino knockdown. Tissues were microsurgically isolated at stage 18 and mechanically tested. (I) Depletion of Wee2 in whole embryos compared with control morpholino (CMO). Note reduced convergence of neural folds at stage 18 and shortened axis at stage 24. (J) Increased nuclear density within Wee2-depleted mesoderm tissue (morpholino co-injected with rhodamine dextran; n=8; **P<0.002, Student’s t-test). (K) Transverse sections of stage 18 unilaterally Wee2-depleted tissues stained for nuclei (DAPI) and fibronectin (Fn1). Note increased nuclear density within Wee2-depleted tissues. (L) Normalized F-actin is unperturbed in Wee2-depleted tissues compared with control morpholino. (M) Transverse sections of stage 18 unilaterally Wee2-depleted tissues stained for F-actin (phallacidin). (N) Tissues depleted of Wee2 show 12% increase in Young’s modulus (two-way ANOVA, P=0.038). Young’s modulus is normalized to control morpholino (CMO)-injected tissues. Dashed red line indicates CSM prediction. Each cluster represents one experiment and n value (beneath the bars in F,G,N) represents number of explants tested per group. Error bars represent s.d. Xenopus stage schematics adapted from Nieuwkoop and Faber (1967). n.s., not significant. Scale bars: 100 μm.
Adapted with permission from The Company of Biologists on behalf of Development: Shawky et al. (2018). Multiscale analysis of architecture, cell size and the cell cortex reveals cortical F-actin density and composition are major contributors to mechanical properties during convergent extension. Development 2018 145: dev161281 doi: 10.1242/dev.161281 Published 5 October 2018. Copyright (2018).Last Updated: 2018-11-19