11Pattern formation is fundamental for embryonic development. Although synthetic 12 biologists have created several patterns, a synthetic mammalian reaction-diffusion pattern 13 has yet to be realized. TGF-β family proteins Nodal and Lefty have been proposed to 14 meet the conditions for reaction-diffusion patterning: Nodal is a short-range activator that 15 enhances the expression of Nodal and Lefty whereas Lefty acts as a long-range inhibitor 16 against Nodal. However, the pattern forming possibility of the Nodal-Lefty signaling has 17 never been directly tested, and the underlying mechanisms of differential diffusivity of 18 Nodal and Lefty remain unclear. Here, through a combination of synthetic biology and 19 theoretical modeling, we show that a reconstituted minimal network of the Nodal-Lefty 20 signaling spontaneously gives rise to a pattern in mammalian cell culture. Surprisingly, 21 extracellular Nodal was confined underneath the cells as small clusters, resulting in a 22 narrow distribution range compared with Lefty. We further found that the finger 1 domain 23 of the Nodal protein is responsible for its short-range distribution. By transplanting the 24 finger 1 domain of Nodal into Lefty, we converted the originally long-range distribution 25 of Lefty to a short-range one, successfully preventing the pattern formation. These results 26 indicate that the differences in the localization and domain structures between Nodal and 27 Lefty, combined with the activator-inhibitor topology, are sufficient for reaction-diffusion 28 pattern formation in mammalian cells. 29 30 Main 31One of the goals of synthetic biology is creating a synthetic tissue to understand natural 32 developmental mechanisms 1-3 , to explore the origin of multicellularity 4 and to engineer a 33 programmable tissue for therapeutic purposes 5,6 . The first step towards a synthetic tissue 34 is controlling pattern formation, which enables to place different types of cells properly 35 in a tissue. Several synthetic cellular patterns have been reported previously: Ring patterns were created in genetically engineered bacteria that can sense the concentrations 1 of small molecules 7,8 . In mammalian cells, 2D and 3D patterns were created based on 2 engineered cell sorting mechanisms 9,10 . However, there is another pattern formation 3 mechanism that has not been artificially created in mammalian cells despite its 4 importance: the reaction-diffusion (RD) patterning system. 5The concept of a self-organizing RD system was first proposed by Alan Turing 6 as a chemical system of interacting and diffusible molecules giving rise to various stable 7 patterns, such as spots and stripes [11][12][13][14] . Recent studies have suggested that RD system 8 underlies a number of developmental patterning phenomena, including digit formation in 9 the limb 15,16 , pigmentation on the skin 17 , the formation of hair follicles and feather buds 10 on the skin 18,19 , branching morphogenesis in the lung 20 and rugae formation in the palate 21 . 11In the field of sy...

A synthetic mammalian reaction-diffusion pattern has yet to be created, and Nodal-Lefty signaling has been proposed to meet conditions for pattern formation: Nodal is a short-range activator whereas Lefty is a long-range inhibitor. However, this pattern forming possibility has never been directly tested, and the underlying mechanisms of differential diffusivity of Nodal and Lefty remain unclear. Here, through a combination of synthetic and theoretical approaches, we show that a reconstituted Nodal-Lefty network in mammalian cells spontaneously gives rise to a pattern. Surprisingly, extracellular Nodal is confined underneath the cells, resulting in a narrow distribution compared with Lefty. The short-range distribution requires the finger 1 domain of Nodal, and transplantation of the finger 1 domain into Lefty shortens the distribution of Lefty, successfully preventing pattern formation. These results indicate that the differences in localization and domain structures between Nodal and Lefty, combined with the activator-inhibitor topology, are sufficient for reaction-diffusion patterning.

Phenotypic diversification of cells is crucial for developmental and regenerative processes in multicellular organisms. The diversification concept is described as the motion of marbles rolling down Waddington’s landscape, in which the number of stable states changes as development proceeds. In contrast to this simple concept, the complexity of natural biomolecular processes prevents comprehension of their design principles. We have constructed, in Escherichia coli , a synthetic circuit with just four genes, which programs cells to autonomously diversify as the motion on the landscape through cell–cell communication. The circuit design was based on the combination of a bistable toggle switch with an intercellular signaling system. The cells with the circuit diversified into two distinct cell states, “high” and “low,” in vivo and in silico, when all of the cells started from the low state. The synthetic diversification was affected by not only the shape of the landscape determined by the circuit design, which includes the synthesis rate of the signaling molecule, but also the number of cells in the experiments. This cell-number dependency is reminiscent of the “community effect”: The fates of developing cells are determined by their number. Our synthetic circuit could be a model system for studying diversification and differentiation in higher organisms. Prospectively, further integrations of our circuit with different cellular functions will provide unique tools for directing cell fates on the population level in tissue engineering.

Control of the cell-type ratio in multistable systems requires wide-range control of the initial states of cells. Here, using a synthetic circuit in E. coli, we describe the use of a simple gene-overexpression system combined with a bistable toggle switch, for the purposes of enabling the wide-range control of cellular states and thus generating arbitrary cell-type ratios. Theoretically, overexpression induction temporarily alters the bistable system to a monostable system, in which the location of the single steady state of cells can be manipulated over a wide range by regulating the overexpression levels. This induced cellular state becomes the initial state of the basal bistable system upon overexpression cessation, which restores the original bistable system. We experimentally demonstrated that the overexpression induced a monomodal cell distribution, and subsequent overexpression withdrawal generated a bimodal distribution. Furthermore, as designed theoretically, regulating the overexpression levels by adjusting the concentrations of small molecules generated arbitrary cell-type ratios.

Among the various biological devices developed and characterized in synthetic biology, light-sensing biological devices can serve as an input-output system owing to their light modulation property. The well-characterized devices in living systems are useful for modulating cellular sensing and transducing information. In this study, we examined short pulse responsiveness of a light-sensing two-component system (TCS), Cph8-OmpR, which was generated by replacing the sensor domain of the EnvZ-OmpR osmoregulatory system with the light sensor Cph1. We varied the input pulse width of the Cph8-OmpR system and found that an input width of <1 s was sufficient to alter the accumulation of a reporter gene upregulated by Cph8 phosphorylation of OmpR. Based on this result and the mathematical model showing that the timescale for the upstream Cph8-activity transition was much faster than that of downstream gene expression, we evaluated the merit of a TCS with such an unbalanced cascade. Our mathematical simulation of a cascade TCS suggests that high-frequency noise arising from fast transitions in kinase activity was attenuated throughout the cascade reaction. In terms of noise attenuation, these results can contribute to analyze biological cascade systems with the balance of reaction rates in each process.