Synthetic mammalian pattern formation driven by differential diffusivity of Nodal and Lefty

Abstract

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...

Using Synthetic Biology to Engineer Spatial Patterns

“…The Weiss and the Ebisuya labs have recently published self-organizing systems that are reminiscent of a canonical Turing system but lack some of its distinctive features. [78,79] As opposed to the classical conception of the Turing mechanism, which depicted a very limited parameter space allowing for patterning, recent theoretical studies suggest that Turing patterns may not actually be so demanding, especially in network designs with more than two nodes. [68,74,82] Therefore, future attempts to engineer biological Turing systems may not focus on two-node networks with high parameter sensitivity, but will presumably explore the recently proposed more complex and robust network topologies.…”

Synthetic tissue engineering: Programming multicellular self-organization by designing customized cell-cell communication

“…Morphogens diffuse and form a graded concentration in developing embryos, which is interpreted by cells as positional information guiding cell differentiation spatially. In the synthetic biology field, soluble cell-cell signaling has been mainly engineered with bacterial quorum sensing systems [ 43 – 46 ], but synthetic mammalian soluble signaling system will be a powerful tool to build and test pattern formation circuits and provide a new way for spatial control of cell fates [ 47 , 48 ]. Developing the molecular toolkits for cell-cell signaling channels and morphogenetic effector genes will expand the synthetic biology platform to engineer more complex developmental programs ( Fig.…”

Programming cell growth into different cluster shapes using diffusible signals

“…Recent advances in genetic engineering technologies have made it possible to encode desired sets of rules within the genetic programs of cells, and these have been used to create distinct spatial patterns [1][2][3][4][5]. For example, the use of a synthetic notch receptor system to encode changes in expression levels of cadherin molecules (in 'receiver cells' engineered with receptors that trigger downstream cellular responses when activated) upon contact with another cell type ('sender cells' engineered with ligands on cell surface) led to self-organization of clusters with distinct spatial arrangements of different cell types [3,6].…”

“…In addition to juxtacrine signaling i.e. signaling through direct cell-cell contact (ligand/receptor systems), cells can also be engineered to communicate via diffusible signals [4][5][6]. In particular, a graded pattern of signaling activity was obtained by culturing engineered * yipeiguo@g.harvard.edu Hedgehog-responding cells next to engineered Hedgehogsecreting cells [4], and Turing-like patterns were generated by reconstituting an activator-inhibitor circuit of two diffusible molecules [5].…”