S. Okuda1,2,3,*, N. Takata1,†, Y. Hasegawa1,‡, M. Kawada1, Y. Inoue3, T. Adachi3, Y. Sasai3,§ and M. Eiraku1,3,*
1RIKEN Center for Developmental Biology, Kobe 650-0047, Japan.
2PRESTO, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan.
3Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan.
↵*Corresponding author. Email: eiraku@infront.kyoto-u.ac.jp (M.E.); satoru.okuda@riken.jp (S.O.)
↵† Present address: Center for Vascular and Developmental Biology, Feinberg Cardiovascular Research Institute, Northwestern University Chicago, IL 60611, USA.
↵‡ Present address: Department of Neurology, Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA 19104, USA.
↵§ Deceased.
Science Advances 21 Nov 2018:
Vol. 4, no. 11, eaau1354
DOI: 10.1126/sciadv.aau1354
Organogenesis is a self-organizing process of multiple cells in three-dimensional (3D) space, where macroscopic tissue deformations are robustly regulated by multicellular autonomy. It is clear that this robust regulation requires cells to sense and modulate 3D tissue formation across different scales, but its underlying mechanisms are still unclear. To address this question, we developed a versatile computational model of 3D multicellular dynamics at single-cell resolution and combined it with the 3D culture system of pluripotent stem cell–derived optic-cup organoid. The complementary approach enabled quantitative prediction of morphogenesis and its corresponding verification and elucidated that the macroscopic 3D tissue deformation is fed back to individual cellular force generations via mechanosensing. We hereby conclude that mechanical force plays a key role as a feedback regulator to establish the robustness of organogenesis.
INTRODUCTION
During organogenesis, morphogens dynamically organize spatial patterns of cell differentiation in three-dimensional (3D) tissues (1, 2). According to the pattern, individual cells generate characteristic mechanical forces to form the entire organ structure in 3D space (3–5). Many molecules have been identified as key signaling factors that regulate each step of patterning and force generation. However, these molecular signals are not enough to explain the entire regulatory mechanism of morphogenesis. In particular, it is still unclear how individual cells sense and modulate the entire 3D tissue formation across different scales. Previous studies have revealed cellular mechanosensing mechanisms (6–8), which may also be involved in the cross-scale regulatory mechanism of 3D tissue formation. Therefore, in this study, we focus on the mechanical aspect of morphogenesis and reveal the role of mechanical force in regulating 3D tissue formation across different scales.
Recent progress in the stem cell field has enabled us to form various 3D tissues in vitro (9, 10). For instance, we have reported a culture system of pluripotent stem cell–derived optic-cup organoids, which well recapitulates a typical process seen in vivo (11, 12); on the basis of the Wnt antagonism, the distal part of optic vesicle (OV) differentiates into neural retina (NR), whereas the adjacent part becomes retinal pigment epithelium (RPE). According to the differentiation pattern, the NR invaginates into the surrounding RPE in the apically convex direction. Subsequently, a hinge structure is formed along the boundary between the inner NR and outer RPE to generate a cup-like tissue shape. From a mechanical point of view, this stepwise process proceeds autonomously without external forces from the surroundings such as lens placode and periocular mesenchyme.
To explain this self-organizing process, we have previously found several key candidates of driving force and suggested a relaxation-expansion model (11) that explains the mechanism of optic-cup formation through four sequential phases (fig. S1A). In phase 1, semispherical OV autonomously generates the differentiation pattern composed of distal NR and the surrounding RPE. In phase 2, the distal NR decreases its stiffness according to the reduction of apical myosin accumulation. In phase 3, the boundary between NR and RPE causes apical constriction, by which the NR is passively invaginated. In phase 4, the NR causes rapid proliferation and facilitates the NR invagination by itself. Although this model is consistent with previous experimental findings, our further mechanical analyses have prompted the investigation of more elaborate mechanisms.
In the present study, we elucidate a mechanical force that is fed back from macroscopic 3D tissue deformation to individual cellular force generation during optic-cup morphogenesis. On the basis of previous mathematical models (13–19), we developed a versatile 3D vertex model that adequately describes general 3D multicellular dynamics at single-cell resolution. The in vitro culture of optic-cup formation enables us to observe and perturb specific cell behaviors in 3D living tissues, whereas the in silico recapitulation enables us to predict its mechanisms comprehensively (15, 16, 20, 21). By combining the in vitro and in silico approaches, we found key cell behaviors required for the NR invagination and the subsequent hinge formation along the NR-RPE boundary and elucidate the key role of mechanical force in the self-organizing optic-cup formation.
RESULTS
Quantitative simulations predict key mechanisms in optic-cup morphogenesis
To elucidate mechanisms of 3D tissue morphogenesis, we attempted to combine in vitro and in silico systems. The optic-cup formation is a complex 3D deformation process to form several characteristic structures such as the apically convex, thicker NR, the adjacent, thinner RPE, and the sharply wedged hinge structure at the NR-RPE boundary (Fig. 1, A to D). The optic-cup formation follows various single-cell behaviors in 3D space, such as cellular contraction, extension, stiffening, softening, adhesion, growth, rearrangement, division, and apoptosis (11). In particular, cells at the NR-RPE boundary form an anisotropic hinge shape (Fig. 1E and movie S1). These cell behaviors dynamically change according to the differentiation state of individual cells from OV to NR and RPE in a dorsoventrally asymmetric manner (2, 12, 22).
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To elucidate mechanisms of 3D tissue morphogenesis, we attempted to combine in vitro and in silico systems. The optic-cup formation is a complex 3D deformation process to form several characteristic structures such as the apically convex, thicker NR, the adjacent, thinner RPE, and the sharply wedged hinge structure at the NR-RPE boundary (Fig. 1, A to D). The optic-cup formation follows various single-cell behaviors in 3D space, such as cellular contraction, extension, stiffening, softening, adhesion, growth, rearrangement, division, and apoptosis (11). In particular, cells at the NR-RPE boundary form an anisotropic hinge shape (Fig. 1E and movie S1). These cell behaviors dynamically change according to the differentiation state of individual cells from OV to NR and RPE in a dorsoventrally asymmetric manner (2, 12, 22).
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