Zhou J et al. (2015), Force production and mechanical accommodation d...

XB-ART-50600

Development 2015 Feb 15;1424:692-701. doi: 10.1242/dev.116533.

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Force production and mechanical accommodation during convergent extension.

Zhou J , Pal S , Maiti S .

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Forces generated within the embryo during convergent extension (CE) must overcome mechanical resistance to push the head away from the rear. As mechanical resistance increases more than eightfold during CE and can vary twofold from individual to individual, we have proposed that developmental programs must include mechanical accommodation in order to maintain robust morphogenesis. To test this idea and investigate the processes that generate forces within early embryos, we developed a novel gel-based sensor to report force production as a tissue changes shape; we find that the mean stress produced by CE is 5.0±1.6 Pascal (Pa). Experiments with the gel-based force sensor resulted in three findings. (1) Force production and mechanical resistance can be coupled through myosin contractility. The coupling of these processes can be hidden unless affected tissues are challenged by physical constraints. (2) CE is mechanically adaptive; dorsal tissues can increase force production up to threefold to overcome a stiffer microenvironment. These findings demonstrate that mechanical accommodation can ensure robust morphogenetic movements against environmental and genetic variation that might otherwise perturb development and growth. (3) Force production is distributed between neural and mesodermal tissues in the dorsal isolate, and the notochord, a central structure involved in patterning vertebrate morphogenesis, is not required for force production during late gastrulation and early neurulation. Our findings suggest that genetic factors that coordinately alter force production and mechanical resistance are common during morphogenesis, and that their cryptic roles can be revealed when tissues are challenged by controlled biophysical constraints.

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Species referenced: Xenopus laevis
Genes referenced: fn1 grap2 myod1

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	Fig. 1. Gel force sensor. (A) Schematic of gel force sensor shows a dorsal isolate embedded in agarose gel with fluorescent beads. A confocal optical stack is collected near the mid-plane of the tissue to detect the gel deformation. (B) When tissues converge in mediolateral (ML) direction and extend along the anterio-posterior (AP) axis, they compress the gel at the AP ends of the isolate. (C) Flow chart of the procedure for computing elongation forces. (D) As tissue extends, the immediately surrounding gel (black) is deformed and the deformation tracked (blue) by embedded fluorescent beads at time=0 (red) and after 4â€…h (green). (E) The beads closest to the ends of the tissue along the AP axis (dashed box in D) are the most displaced.
	Fig. 2. Rate of stress production by a dorsal isolate. (A) Bead positions at end of dorsal isolate before deformation (time=0âh), and 2 and 4âh later. The dorsal isolate (asterisk) is outlined (white line). (Aâ²) Bead displacements between 0 (red) and 4âh (cyan) shown in overlay of the region in the dashed box in A. Dark-blue lines connect beads at 0 and 4âh. (B) von Mises stress calculated in the gel shown in A at 0, 2 and 4âh. (C) Maximal bead displacements in the AP direction over time. (D) Increasing maximal stress (Ïmax) of the dorsal isolate. (E) Profile of stress across the AP face of the isolate calculated at 2 and 4âh. The profile is approximately centered on the dorsal midline of the isolate.
	Fig. 3. Mapping forces of convergent extension using the gel force sensor. (A) An elongating dorsal isolate is positioned in agarose gel, with the ML axis and AP axis aligned with the plane of the confocal section. (B) Dorsal isolate is embedded in gel (dorsal view) at time=0. (Bâ€²) When the isolate extends, it deforms the agarose gel at both its anterior and posterior ends. As the isolate converges, it loses contact with the gel along its ML sides (arrows). (C) Beads within the gel are visible in the â€˜null forceâ€™ (red) and â€˜force-loadedâ€™ (green) states. Note: some green beads (asterisk) â€˜appearâ€™ at 4â€…h. These are not due to deformation but become visible because they are no longer obscured by converging tissues. (D) Gel displacements are calculated by image registration of the two images in C and drawn as arrows. (E) Stress distribution computed using an FE model shows that the posterior end produces a higher, more axially focused pattern of stress than that of the anterior end (F). (G) Dorsal isolates positioned with their DV and AP axes aligned with the plane of the confocal sections. (H) Stress produced by both the tissue elongation forces in AP axis and dorsal ventral thickening forces in DV axis. (I) Gel displacement in AP axis caused by tissue elongation is focused along the anterior and posterior axis. (J) Gel displacement in DV axis caused by tissue thickening is located at dorsal and ventral sides. There is no gel displacement along the DV direction at anterior and posterior ends caused by either tissue elongation or thickening. (K) Dorsal isolates positioned with their DV and ML axes aligned with the plane of the confocal section. (L) Stress produced by tissue thickening forces at a point midway down the AP axis. The arrow lengths in D, H and K indicate a tenfold distance of the actual gel displacement. See supplementary material Fig.Â S7 for contour maps of displacements.
	Fig. 4. Elongation stress production but not unconstrained deformation is regulated by myosin II contractility. (A) Dorsal isolates elongate more in DMSO and 40â€…ÂµM Y27632 without mechanical constraint than they elongate after being embedded in agarose gel. (B) Tissues elongate at same rates in DMSO or Y27632 without mechanical constraint and elongate much less in Y27632 when embedded in gel. (C,D) Stress field surrounding isolates cultured in DMSO (C) or Y27632 (D) over 4â€…h. (E) Stress distribution along the anterior and posterior ends of the DMSO and Y27632-treated isolates. (F,G) Both the maximum stress (F) and mean stress (G) of the extending isolates in DMSO were significantly greater than those of isolates in Y27632. Significance of stress measurements among multiple clutches were calculated using two-way ANOVA (P<0.05; P<0.01; **P<0.005). Error bars in B, F and G indicate s.d.
	Fig. 5. Dorsal isolates in stiffer gel produced greater force: experiment and model. (A-C) Stress maps of dorsal isolates embedded in 30-Pa gel (A), in 200-Pa gel (B) and in 500-Pa gel (C). (D) Stress distribution along the midline axis of dorsal isolates embedded in gels with different elastic modulus. (E,F) Both the maximum stress (E) and mean stress (F) of the dorsal tissues in a 30-Pa gel are significantly less than those of tissues in 200-Pa or 500-Pa gels. Significance of stress measurements among multiple clutches were calculated using a two-way ANOVA (P<0.05; P<0.01; **P<0.005). Error bars in E,F indicate s.d. (G) Elongation stress simulated in a sample dorsal isolate. (H) FE grid surrounding the isolate. Higher densities of the elements were used within the isolate and near its surface. (I-K) Stress fields produced from constant ML stress in a 30-Pa dorsal isolate within a 30-Pa elastic gel (I), a 200-Pa elastic gel (J) and a 500-Pa elastic gel (K). (L) The stress profiles across the face of the simulated elongating isolate show increasing stress in stiffer gels.
	Fig. 6. Contribution of the notochord to force production. (A) Mock-operated control dorsal isolates were split axially and then re-combined. (B) Notochord was microsurgically removed along notochord-paraxial mesoderm boundary to produce notochord-less dorsal isolates. (Aâ€²) Representative average projections and transverse maximum projections of confocal sections of stained fibronectin fibrils of mock control and notochord-less dorsal isolates (Bâ€²) show that the mock control dorsal isolate contains neural ectoderm (ne), notochord (n), paraxial mesoderm (s) and endoderm (e), whereas the microsurgically prepared notochord-less dorsal isolate lacks a notochord (posterior, p; anterior, a). (C) Stress distribution along the midline axis of mock control and notochord-less dorsal isolates. (D,E) Both the maximum stress (D) and mean stress (E) of the mock control and notochord-less dorsal isolates are not significantly different. Significance of stress measurements among multiple clutches were calculated using two-way ANOVA (P<0.05; P<0.01; **P<0.005). Error bars in D,E indicate s.d.
	Fig. 7. Posterior axial tissues and neural plate are additional sources of force production. (A-C) The dorsal isolate can be bisected into a posterior and anterior half for measurement of stress production. Anterior and posterior dorsal isolate halves extend equally well (see supplementary material Fig.Â S10 and MovieÂ 3). The anterior half of the dorsal isolate produces lower normalized maximum stress (B) and normalized mean stress (C) than the full-sized dorsal isolate. Posterior halves typically produce less stress than the full-sized dorsal isolate, but this difference is not statistically significant. (D) Two neural plates can be recombined to produce a single neural sandwich (NS). Neural sandwiches extend as well as dorsal isolates (see supplementary material Fig.Â S11 and MovieÂ 4). (E-H) Normalized maximum (E,G) and mean stress (F,H) production in neural sandwiches is variable but no significant differences were detected after 1â€…h of elongation (three clutches) or after 2â€…h (two clutches). Significance of stress measurements among two clutches were calculated using two-way ANOVA (*P<0.05). Error bars indicate s.d.

References [+] :

Balgude, Agarose gel stiffness determines rate of DRG neurite extension in 3D cultures. 2001, Pubmed