Differential cellular stiffness across tissues that contribute to Xenopus neural tube closure.

During the formation of the neural tube, the primordium of the vertebrate central nervous system, the actomyosin activity of cells in different regions drives neural plate bending. However, how the stiffness of the neural plate and surrounding tissues is regulated and mechanically influences neural plate bending has not been elucidated. Here, we used atomic force microscopy to reveal the relationship between the stiffness of the neural plate and the mesoderm during Xenopus neural tube formation. Measurements with intact embryos revealed that the stiffness of the neural plate was consistently higher compared with the non-neural ectoderm and that it increased in an actomyosin activity-dependent manner during neural plate bending. Interestingly, measurements of isolated tissue explants also revealed that the relationship between the stiffness of the apical and basal sides of the neural plate was reversed during bending and that the stiffness of the mesoderm was lower than that of the basal side of the neural plate. The experimental elevation of mesoderm stiffness delayed neural plate bending, suggesting that low mesoderm stiffness mechanically supports neural tube closure. This study provides an example of mechanical interactions between tissues during large-scale morphogenetic movements.

Distinct intracellular Ca2+ dynamics regulate apical constriction and differentially contribute to neural tube closure.

Early in the development of the central nervous system, progenitor cells undergo a shape change, called apical constriction, that triggers the neural plate to form a tubular structure. How apical constriction in the neural plate is controlled and how it contributes to tissue morphogenesis are not fully understood. In this study, we show that intracellular calcium ions (Ca2+) are required for Xenopus neural tube formation and that there are two types of Ca2+-concentration changes, a single-cell and a multicellular wave-like fluctuation, in the developing neural plate. Quantitative imaging analyses revealed that transient increases in Ca2+ concentration induced cortical F-actin remodeling, apical constriction and accelerations of the closing movement of the neural plate. We also show that extracellular ATP and N-cadherin (cdh2) participate in the Ca2+-induced apical constriction. Furthermore, our mathematical model suggests that the effect of Ca2+ fluctuations on tissue morphogenesis is independent of fluctuation frequency and that fluctuations affecting individual cells are more efficient than those at the multicellular level. We propose that distinct Ca2+ signaling patterns differentially modulate apical constriction for efficient epithelial folding and that this mechanism has a broad range of physiological outcomes.

Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis.

A peptide hormone, root meristem growth factor (RGF), regulates root meristem development through the PLETHORA (PLT) stem cell transcription factor pathway, but it remains to be uncovered how extracellular RGF signals are transduced to the nucleus. Here we identified, using a combination of a custom-made receptor kinase (RK) expression library and exhaustive photoaffinity labeling, three leucine-rich repeat RKs (LRR-RKs) that directly interact with RGF peptides in Arabidopsis These three LRR-RKs, which we named RGFR1, RGFR2, and RGFR3, are expressed in root tissues including the proximal meristem, the elongation zone, and the differentiation zone. The triple rgfr mutant was insensitive to externally applied RGF peptide and displayed a short root phenotype accompanied by a considerable decrease in meristematic cell number. In addition, PLT1 and PLT2 protein gradients, observed as a gradual gradient decreasing toward the elongation zone from the stem cell area in wild type, steeply declined at the root tip in the triple mutant. Because RGF peptides have been shown to create a diffusion-based concentration gradient extending from the stem cell area, our results strongly suggest that RGFRs mediate the transformation of an RGF peptide gradient into a PLT protein gradient in the proximal meristem, thereby acting as key regulators of root meristem development.

Force-dependent remodeling of cytoplasmic ZO-1 condensates contributes to cell-cell adhesion through enhancing tight junctions.

The physiological importance of biomolecular condensates is widely recognized, but how it is controlled in time and space during development is largely unknown. Here, we show that a tight junction protein ZO-1 forms cytoplasmic condensates in the trophectoderm (TE) of the mouse embryo before E4.0. These disappear via dissolution, and ZO-1 accumulates at the cell junction as the blastocyst cavity grows and internal pressure on TE cells increases. In contrast, this dissolution was less evident in TE cells attached to the inner cell mass because they receive weaker tensile forces. Furthermore, analyses using MDCK cells demonstrated that the ZO-1 condensates are generated and maintained by liquid-liquid phase separation. Our study also highlights that the dynamics of these condensates depends on the physical environment via an interaction between ZO-1 and F-actin. We propose that the force-dependent regulation of ZO-1 condensation contributes to the establishment of robust cell-cell adhesion during early development.

Differential Cellular Stiffness Contributes to Tissue Elongation on an Expanding Surface.

Pattern formation and morphogenesis of cell populations is essential for successful embryogenesis. Steinberg proposed the differential adhesion hypothesis, and differences in cell-cell adhesion and interfacial tension have proven to be critical for cell sorting. Standard theoretical models such as the vertex model consider not only cell-cell adhesion/tension but also area elasticity of apical cell surfaces and viscous friction forces. However, the potential contributions of the latter two parameters to pattern formation and morphogenesis remain to be determined. In this theoretical study, we analyzed the effect of both area elasticity and the coefficient of friction on pattern formation and morphogenesis. We assumed the presence of two cell populations, one population of which is surrounded by the other. Both populations were placed on the surface of a uniformly expanding environment analogous to growing embryos, in which friction forces are exerted between cell populations and their expanding environment. When the area elasticity or friction coefficient in the cell cluster was increased relative to that of the surrounding cell population, the cell cluster was elongated. In comparison with experimental observations, elongation of the notochord in mice is consistent with the hypothesis based on the difference in area elasticity but not the difference in friction coefficient. Because area elasticity is an index of cellular stiffness, we propose that differential cellular stiffness may contribute to tissue elongation within an expanding environment.

Mechanical Stress Regulates Epithelial Tissue Integrity and Stiffness through the FGFR/Erk2 Signaling Pathway during Embryogenesis.

Physical forces generated by tissue-tissue interactions are a critical component of embryogenesis, aiding the formation of organs in a coordinated manner. In this study, using Xenopus laevis embryos and phosphoproteome analyses, we uncover the rapid activation of the mitogen-activated protein (MAP) kinase Erk2 upon stimulation with centrifugal, compression, or stretching force. We demonstrate that Erk2 induces the remodeling of cytoskeletal proteins, including F-actin, an embryonic cadherin C-cadherin, and the tight junction protein ZO-1. We show these force-dependent changes to be prerequisites for the enhancement of cellular junctions and tissue stiffening during early embryogenesis. Furthermore, Erk2 activation is FGFR1 dependent while not requiring fibroblast growth factor (FGF) ligands, suggesting that cell/tissue deformation triggers receptor activation in the absence of ligands. These findings establish previously unrecognized functions for mechanical forces in embryogenesis and reveal its underlying force-induced signaling pathways.

Screening and identification of a non-peptide antagonist for the peptide hormone receptor in Arabidopsis.

Intercellular signaling mediated by peptide hormones and membrane-localized receptor kinases plays crucial roles in plant developmental processes. Because of their diverse functions, agonistic or antagonistic modulation of peptide signaling holds enormous promise for agricultural applications. Here we established a high-throughput screening system using a bead-immobilized receptor kinase and fluorescent-labeled peptide ligand to identify small molecules that bind peptide hormone receptors in competition with natural ligands. We used the Arabidopsis CLE9-BAM1 ligand-receptor pair to screen a library of ≈30,000 chemicals and identified NPD12704 as an antagonist for BAM1. NPD12704 also inhibited CLV3 binding to BAM1 but only minimally interfered with CLV3 binding to CLV1, the closest homolog of BAM1, demonstrating preferential receptor specificity. Treatment of clv1-101 mutant seedlings with NPD12704 enhanced the enlarged shoot apical meristem phenotype. Our results provide a technological framework enabling high-throughput identification of small non-peptide chemicals that specifically control receptor kinase-mediated peptide hormone signaling in plants.

Mechanical roles of apical constriction, cell elongation, and cell migration during neural tube formation in Xenopus.

Neural tube closure is an important and necessary process during the development of the central nervous system. The formation of the neural tube structure from a flat sheet of neural epithelium requires several cell morphogenetic events and tissue dynamics to account for the mechanics of tissue deformation. Cell elongation changes cuboidal cells into columnar cells, and apical constriction then causes them to adopt apically narrow, wedge-like shapes. In addition, the neural plate in Xenopus is stratified, and the non-neural cells in the deep layer (deep cells) pull the overlying superficial cells, eventually bringing the two layers of cells to the midline. Thus, neural tube closure appears to be a complex event in which these three physical events are considered to play key mechanical roles. To test whether these three physical events are mechanically sufficient to drive neural tube formation, we employed a three-dimensional vertex model and used it to simulate the process of neural tube closure. The results suggest that apical constriction cued the bending of the neural plate by pursing the circumference of the apical surface of the neural cells. Neural cell elongation in concert with apical constriction further narrowed the apical surface of the cells and drove the rapid folding of the neural plate, but was insufficient for complete neural tube closure. Migration of the deep cells provided the additional tissue deformation necessary for closure. To validate the model, apical constriction and cell elongation were inhibited in Xenopus laevis embryos. The resulting cell and tissue shapes resembled the corresponding simulation results.