How the embryonic chick brain twists

Abstract

During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left-right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic morphology and mechanics analysis that the vitelline membrane (VM) exerts an external load on the brain that drives torsion. Our theoretical analysis showed that the force is of the order of 10 micronewtons. We also designed an experiment to use fluid surface tension to replace the mechanical role of the VM, and the estimated magnitude of the force owing to surface tension was shown to be consistent with the above theoretical analysis. We further discovered that the asymmetry of the looping heart determines the chirality of the twisted brain via physical mechanisms, demonstrating the mechanical transfer of left-right asymmetry between organs. Our experiments also implied that brain flexure is a necessary condition for torsion. Our work clarifies the mechanical origin of torsion and the development of left-right asymmetry in the early embryonic brain.

Keywords: axial rotation; biomechanics; embryonic development; left–right asymmetry; torsion.

Figure 1.

Flexure and torsion in early brain development of a normal chick embryo (dorsal view). (a) Dorsal view of nearly straight brain tube at Hamburger and Hamilton (HH) stage 11. The white dots are virtual markers on the neural tube for visualizing the large deformation during flexure and torsion. L, left; R, right. Scale bar, 1 mm. (b) Dorsal view of same embryo 20 h later featuring flexure and torsion in the brain tube. The embryonic brain turns rightward, so that its left side lies on the substrate. (c) Transverse section of HH stage 12 chick embryo obtained by optical coherence tomography; the heart is on the embryonic right, the VM is dorsal to the embryo, whereas the SPL is ventral to the embryo. Scale bar, 0.5 mm.

Figure 2.

Effects of external forces on brain torsion (dorsal view). (a) Embryo with a nearly straight brain tube and VM removed at HH stage 11. (b) Same embryo 27 h later showing a relatively small amount of rightward torsion. (c) Same embryo under surface tension immediately underwent significant additional brain torsion (see electronic supplementary movie). (d) Control embryo with normal brain torsion at a comparable stage. (e) Embryo with VM removed at HH stage 11, shown after approximately 20 h of cultivation under fluid surface tension. Brain torsion is normal, suggesting that the fluid surface tension replaced the mechanical forces formerly supplied by the VM. Scale bar, 1 mm.

Figure 3.

Effects of heart-looping direction and brain flexure on torsion (ventral view). (a) HH stage 12 embryo with heart looped normally to the right side. (b) Same embryo with heart pushed to the left side. Surface tension is immediately applied to keep the heart on the left. (c) Same embryo 20 h later; the brain tube twists leftward. (d) HH stage 13 embryo with the head rotated partially rightward. (e) Same embryo with heart pushed to the left side, and surface tension immediately applied. (f) Same embryo 18 h later. The brain turns right as in a normal embryo. (g) Chick embryo at HH stage 11 with eyelash implanted in the brain tube to suppress flexure. (h) Same embryo 18 h later shows little brain torsion. (i) Normal embryo at a comparable stage to the manipulated embryos in (b) and (h). (j) Chick embryo at HH stage 10+ with an eyelash implanted in the brain tube (not reaching the first somite). (k) Same embryo 20 h later exhibiting comparable amount of torsion to control. (l) Control embryo (an eyelash was inserted in the brain tube at HH stage 10+ and immediately removed) with normal rightward torsion. Scale bar, 1 mm.

Figure 4.

Physical model of embryonic brain torsion. (a) Simplified geometry of a chick embryo at HH stage 14–17 in computer-aided design (Comsol MultiPhysics). (b) Silicone elastomer cast physical model of simplified geometry. (c) Dorsal view of model with heart on right side before downwards force application by coverslip. (d) Dorsal view of model following downwards force application by coverslip, with brain beginning to twist rightwards. (e) Model after further force application, with brain twisted more drastically rightwards. Fiducial markers (marks and pins), used to visualize torsion, are also shown. Scale bars, (b–d) 1 cm.