ARTICLE IN PRESS 1 2 How the embryonic chick brain twists 3 4 1,2 3,4 1 1,5 1 5 rsif.royalsocietypublishing.org Zi Chen , Qiaohang Guo , Eric Dai , Nickolas Forsch and Larry A. Taber 6 1Department of Biomedical Engineering, Washington University, St Louis, MO 63130, USA 7 2Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA 8 3School of Materials Science and Engineering, Fujian University of Technology, Fuzhou 350108, 9 People’s Republic of China 10 Research 4Fujian Provincial Key Laboratory of Advanced Materials Processing and Application, Fuzhou 350108, 11 People’s Republic of China 12 5 Cite this article: Chen Z, Guo Q, Dai E, Forsch Department of Bioengineering, University of California at San Diego, La Jolla, CA 92093, USA 13 ZC, 0000-0001-5927-0249 14 N, Taber LA. 2016 How the embryonic chick 15 brain twists. J. R. Soc. Interface 20160395. During early development, the tubular embryonic chick brain undergoes a 16 http://dx.doi.org/10.1098/rsif.2016.0395 combination of progressive ventral bending and rightward torsion, one of 17 the earliest organ-level left–right asymmetry events in development. Exist- 18 ing evidence suggests that bending is caused by differential growth, but 19 the mechanism for the predominantly rightward torsion of the embryonic 20 Received: 19 May 2016 brain tube remains poorly understood. Here, we show through a combi- 21 Accepted: 20 October 2016 nation of in vitro experiments, a physical model of the embryonic 22 morphology and mechanics analysis that the vitelline membrane (VM) 23 exerts an external load on the brain that drives torsion. Our theoretical analy- 24 sis showed that the force is of the order of 10 micronewtons. We also 25 designed an experiment to use fluid surface tension to replace the mechan- 26 Subject Category: ical role of the VM, and the estimated magnitude of the force owing to 27 Life Sciences–Engineering interface surface tension was shown to be consistent with the above theoretical analy- 28 sis. We further discovered that the asymmetry of the looping heart 29 Subject Areas: determines the chirality of the twisted brain via physical mechanisms, 30 biomechanics, biophysics, bioengineering demonstrating the mechanical transfer of left–right asymmetry between 31 organs. Our experiments also implied that brain flexure is a necessary con- 32 Keywords: dition for torsion. Our work clarifies the mechanical origin of torsion and the 33 development of left–right asymmetry in the early embryonic brain. 34 left–right asymmetry, embryonic 35 development, torsion, biomechanics, 36 axial rotation 37 Q1 38 1. Introduction 39 Morphogenesis, the generation of biological form [1], has been studied for cen- 40 Author for correspondence: turies. In his classic book, On growth and form [2], Thompson suggests that the 41 Zi Chen form of an organism (and its changes) can be ‘explained by the interaction or 42 e-mail: [email protected] balance of forces’, calling attention to the need for understanding the math- 43 ematical and physical aspects of biological growth and morphogenesis. While 44 modern developmental biologists have mostly focused on the genetic and 45 molecular aspects of development, the search for mathematical and mechani- 46 cal principles of morphogenesis has never ceased and is currently being 47 revived [3–10]. 48 The transformation of a straight configuration to a chiral shape is ubiquitous in 49 natural and engineered systems [5,11–13]. Although the physical and molecular 50 mechanisms in morphogenesis have been studied for centuries [1–3,5–8,12,14], 51 except for a few studies [15–17], the roles of mechanical forces in early brain mor- 52 phogenesis are still poorly understood. In particular, the fundamental mechanism 53 underlying the torsion of the embryonic brain [18], one of the earliest morphogen- 54 etic events of left–right (L–R) asymmetry in the embryo, remains elusive. 55 Significant birth defects can result when the process of L–R specification is per- 56 turbed, including situs inversus (inversion of the positions of visceral organs), 57 isomerism (mirror image symmetry of bilaterally asymmetric tissues) and hetero- 58 taxia (random and independent occurrence of laterality defects in different 59 Electronic supplementary material is available tissues) [19]. 60 At Hamburger & Hamilton [20] (HH) stage 10 (after approx. 33 h of incu- online at rs.figshare.com. 61 bation), the neural tube of the chick embryo is nearly straight. The anterior 62 63 & 2016 The Author(s) Published by the Royal Society. All rights reserved. rsif20160395—26/10/16—20:13–Copy Edited by: Not Mentioned ARTICLE IN PRESS 64 (a) (b) Previous studies have suggested that bending of the brain 2 65 cranial tube could be attributed in part to differential growth [22], brain rsif.royalsocietypublishing.org 66 flexure but the biomechanical mechanism of brain torsion remains 67 brain cervical poorly understood. Interestingly, the heart almost always 68 flexure loops in the same direction (to the right side) that the brain 69 twists, leading some researchers to speculate that the looping heart 70 heart thoracic heart affects the direction of brain torsion [23,24]. Neverthe- 71 flexure less, little direct evidence exists, and the mechanical origin 72 LR spinal cord of brain torsion remains elusive. 73 Here, we use experiments in developing chick embryos 74 and a physical model to show that (i) the vitelline membrane 75 (VM) exerts a mechanical load that causes the brain tube to J. R. Soc. Interface 76 twist as it bends, (ii) the direction of the looping heart deter- 77 mines the direction of torsion, and (iii) brain flexure is a 78 vitelline necessary condition for torsion. 79 (c) membrane 80 20160395 81 brain 82 heart 83 2. Results 84 2.1. The vitelline membrane applies the compressive 85 86 load necessary for torsion 87 In a normal HH11 chick embryo (after approx. 40–45 h of 88 incubation), the embryonic brain tube has not yet begun to splanchnopleure 89 twist (figure 1a). After around 20 h of cultivation (to 90 Figure 1. Flexure and torsion in early brain development of a normal chick approx. HH stage 16), the brain tube is bent and twisted 91 embryo (dorsal view). (a) Dorsal view of nearly straight brain tube at Ham- rightward, whereas the spinal cord is still in its original orien- 92 burger and Hamilton (HH) stage 11. The white dots are virtual markers on tation (figure 1b). A thin membrane called the VM, which is 93 the neural tube for visualizing the large deformation during flexure and tor- under tension, constrains the embryo on its original dorsal 94 sion. Scale bar, 1 mm. (b) Dorsal view of same embryo 20 h later featuring side, and the splanchnopleure (SPL) membrane constrains 95 flexure and torsion in the brain tube. The embryonic brain turns rightward, so the heart on the ventral side (figure 1c). Studies have 96 that its left side lies on the substrate. (c) Transverse section of HH stage 12 shown that the SPL membrane is a primary cause of torsion 97 chick embryo obtained by optical coherence tomography; the heart is on the of the looping heart [25]. 98 embryonic right, the VM is dorsal to the embryo, whereas the SPL is ventral To determine whether the VM, an often-overlooked 99 to the embryo. L, left; R, right. Scale bar, 0.5 mm. developmental structure, plays a mechanical role in brain 100 morphogenesis, we removed this membrane before the 101 onset of torsion. More specifically, the VM was cut from 102 the anterior end of the embryo to the spinal cord at the 103 part of the neural tube expands to create the primitive brain point of the thoracic flexure (figure 2b). Notably, embryos 104 tube (figure 1a), which then undergoes shape changes that (starting from stage HH11, as shown in figure 2a; n ¼ 12, 105 involve ventral bending (flexure) and rightward twisting (tor- where n is the number of embryos tested) cultivated for 106 sion). These shape changes eventually alter the position of the 27 h to HH stage 17 without the dorsal constraint of the 107 embryo relative to the yolk [21]. VM exhibited considerably less torsion than control embryos. 108 Flexure is considered to consist of two main phases. First, For example, the torsional angle near the eyes 34 degrees in 109 the embryo bends about a transverse axis through the mid- such an embryo shown in figure 2b (see electronic sup- 110 brain (cranial flexure, figure 1b), followed by bending from plementary material, texts and figure S3 for the method of 111 the hindbrain to the spinal cord (cervical flexure). During tor- measuring the torsional angle) when compared with the tor- 112 sion, the brain tube twists rightward, so that the left side of sional angle 88 degrees in a control embryo (figure 2d). 113 the brain rests upon the yolk, whereas the posterior part of Furthermore, when we lowered the fluid level in the Petri 114 the neural tube (future spinal cord) maintains its original dish to apply surface tension to the embryo (i.e. when the 115 orientation (electronic supplementary material, movie S1). embryonic brain is exposed to the air–fluid interface it 116 Flexure and torsion occur almost concurrently in chick becomes subjected to the fluid surface tension) [26], we 117 embryos, as torsion first appears almost simultaneously found that the brain instantaneously twisted rightward 118 with the beginning of cranial flexure and continues as flexure (figure 2c and electronic supplementary material, movie S2), 119 progresses. Also noteworthy is the fact that torsion usually with degrees of torsion similar to the control embryos (e.g. 120 occurs, so that the head turns rightward.