perfusable-vascular-network-with-tissue-model-microfluidic

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Kyushu University

Limb development has been widely studied in developmental biology. Two types of theories, one based on positional information and another based on spontaneous pattern formation, are utilized to understand this process. However, for historical reasons, these two concepts are regarded as mutually exclusive. By considering the interaction between these two mechanisms with quantitative morphological data, we can experimentally explore how the vertebrate limb skeletal pattern is established from a new direction.

Turing and Wolpert work together during limb development.

[What we can see through mathematical modeling of biological pattern formation].

Vertebrate airway has a tree-like-branched structure. This structure is generated by repeated tip splitting, which is called branching morphogenesis. Although this phenomenon is extensively studied in developmental biology, the mechanism of the pattern formation is not well understood. Conversely, there are many tree-like structures in purely physical or chemical systems, and their pattern formation mechanisms are well-understood using mathematical models. Recent studies correlate these biological observations and mathematical models to understand lung branching morphogenesis. These models use slightly different mechanisms. In this article, we will review recent progress in modelling lung branching morphogenesis, and future directions to experimentally verify the models.

Models of lung branching morphogenesis.

Plant leaf epidermal cells exhibit a jigsaw puzzle-like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo.

A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells.

Organ culture systems are used to elucidate the mechanisms of pattern formation in developmental biology. Various organ culture techniques have been used, but the lack of microcirculation in such cultures impedes the long-term maintenance of larger tissues. Recent advances in microfluidic devices now enable us to utilize self-organized perfusable capillary networks in organ cultures. In this review, we will overview past approaches to organ culture and current technical advances in microfluidic devices, and discuss possible applications of microfluidics towards the study of developmental biology.

Tissue culture on a chip: Developmental biology applications of self-organized capillary networks in microfluidic devices.

Cranial sutures are narrow mesenchymal tissues that connect skull bones to each other. Given that they serve as growth centers in the skull, these undifferentiated tissues play crucial roles in skull development. Cranial sutures are also of clinical importance, because the premature fusion of skull bones results in a pathological condition called craniosynostosis. In newborns, skull sutures are wide and straight; during adolescence, they become thinner and start winding to form an interdigitating pattern. From a functional aspect, as the degree of interdigitation becomes larger, the strength of the connection between bones increases. However, the mechanisms underlying the maintenance of mesenchymal narrow bands or formation of interdigitation remain poorly understood. In the present study, we presented a new mathematical model that can reproduce the suture width maintenance and interdigitation formation. We can predict the width of the mesenchyme bands and wavelengths of suture interdigitations from the model.

A new mathematical model for pattern formation by cranial sutures.

Notch-mediated lateral inhibition regulates binary cell fate choice, resulting in salt and pepper patterns during various developmental processes. However, how Notch signaling behaves in combination with other signaling systems remains elusive. The wave of differentiation in the Drosophila visual center or "proneural wave" accompanies Notch activity that is propagated without the formation of a salt and pepper pattern, implying that Notch does not form a feedback loop of lateral inhibition during this process. However, mathematical modeling and genetic analysis clearly showed that Notch-mediated lateral inhibition is implemented within the proneural wave. Because partial reduction in EGF signaling causes the formation of the salt and pepper pattern, it is most likely that EGF diffusion cancels salt and pepper pattern formation in silico and in vivo. Moreover, the combination of Notch-mediated lateral inhibition and EGF-mediated reaction diffusion enables a function of Notch signaling that regulates propagation of the wave of differentiation.

Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion.

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis.

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized β-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.

Reelin transiently promotes N-cadherin-dependent neuronal adhesion during mouse cortical development.

Vasculogenesis is the earliest process in development for spontaneous formation of a primitive capillary network from endothelial progenitor cells. When human umbilical vein endothelial cells (HUVECs) are cultured on Matrigel, they spontaneously form a network structure which is widely used as an in vitro model of vasculogenesis. Previous studies indicated that chemotaxis or gel deformation was involved in spontaneous pattern formation. In our study, we analyzed the mechanism of vascular pattern formation using a different system, meshwork formation by HUVECs embedded in fibrin gels. Unlike the others, this experimental system resulted in a perfusable endothelial network in vitro. We quantitatively observed the dynamics of endothelial cell protrusion and developed a mathematical model for one-dimensional dynamics. We then extended the one-dimensional model to two-dimensions. The model showed that random searching by endothelial cells was sufficient to generate the observed network structure in fibrin gels.

Mathematical modeling for meshwork formation of endothelial cells in fibrin gels.

This study demonstrates computational simulations of multicellular deformation coupled with chemical patterning in the three-dimensional (3D) space. To address these aspects, we proposes a novel mathematical model, where a reaction-diffusion system is discretely expressed at a single cell level and combined with a 3D vertex model. To investigate complex phenomena emerging from the coupling of patterning and deformation, as an example, we employed an activator-inhibitor system and converted the activator concentration of individual cells into their growth rate. Despite the simplicity of the model, by growing a monolayer cell vesicle, the coupling system provided rich morphological dynamics such as undulation, tubulation, and branching. Interestingly, the morphological variety depends on the difference in time scales between patterning and deformation, and can be partially understood by the intrinsic hysteresis in the activator-inhibitor system with domain growth. Importantly, the model can be applied to 3D multicellular dynamics that couple the reaction-diffusion patterning with various cell behaviors, such as deformation, rearrangement, division, apoptosis, differentiation, and proliferation. Thus, the results demonstrate the significant advantage of the proposed model as well as the biophysical importance of exploring spatiotemporal dynamics of the coupling phenomena of patterning and deformation in 3D space.

Article	Year
Turing and Wolpert work together during limb development. Science signaling\| PubMed ID: 23572146	2013
[What we can see through mathematical modeling of biological pattern formation]. Fukuoka igaku zasshi = Hukuoka acta medica\| PubMed ID: 24199451	2013
Models of lung branching morphogenesis. Journal of biochemistry\| PubMed ID: 25556243	2015
A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells. PLoS computational biology\| PubMed ID: 27054467	2016
Tissue culture on a chip: Developmental biology applications of self-organized capillary networks in microfluidic devices. Development, growth & differentiation\| PubMed ID: 27272910	2016
A new mathematical model for pattern formation by cranial sutures. Journal of theoretical biology\| PubMed ID: 27519950	2016
Notch-mediated lateral inhibition regulates proneural wave propagation when combined with EGF-mediated reaction diffusion. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27535937	2016
Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells. Plant & cell physiology\| PubMed ID: 28011873	2017
Reelin transiently promotes N-cadherin-dependent neuronal adhesion during mouse cortical development. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 28174271	2017
Mathematical modeling for meshwork formation of endothelial cells in fibrin gels. Journal of theoretical biology\| PubMed ID: 28648563	2017
Combining Turing and 3D vertex models reproduces autonomous multicellular morphogenesis with undulation, tubulation, and branching. Scientific reports\| PubMed ID: 29402913	2018

Takashi Miura

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