Interpretation of morphogen gradients by a synthetic bistable circuit

ARTICLE https://doi.org/10.1038/s41467-020-19098-w OPEN Interpretation of morphogen gradients by a synthetic bistable circuit 1234567890():,; Paul K. Grant 1 ✉, Gregory Szep 1,2,9, Om Patange 3,7,8,9, Jacob Halatek 1, Valerie Coppard1, Attila Csikász-Nagy 2,4, Jim Haseloff 5, James C. W. Locke1,3,6, Neil Dalchau 1 & Andrew Phillips 1✉ During development, cells gain positional information through the interpretation of dynamic morphogen gradients. A proposed mechanism for interpreting opposing morphogen gradients is mutual inhibition of downstream transcription factors, but isolating the role of this specific motif within a natural network remains a challenge. Here, we engineer a synthetic morphogen-induced mutual inhibition circuit in E. coli populations and show that mutual inhibition alone is sufficient to produce stable domains of gene expression in response to dynamic morphogen gradients, provided the spatial average of the morphogens falls within the region of bistability at the single cell level. When we add sender devices, the resulting patterning circuit produces theoretically predicted self-organised gene expression domains in response to a single gradient. We develop computational models of our synthetic circuits parameterised to timecourse fluorescence data, providing both a theoretical and experimental framework for engineering morphogen-induced spatial patterning in cell populations. 1 Microsoft Research, 21 Station Road, Cambridge CB1 2FB, UK. 2 Randall Centre for Cell and Molecular Biophysics, King’s College London, London WC2R 2LS, UK. 3 Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, UK. 4 Faculty of Information Technology and Bionics, Pázmány Péter Catholic University, Budapest 1083, Hungary. 5 Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK. 6 Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK. 7Present address: Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA. 8Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 9These authors contributed equally: Gregory Szep, Om Patange. ✉email: ; NATURE COMMUNICATIONS | (2020)11:5545 | https://doi.org/10.1038/s41467-020-19098-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19098-w T he positional information solution to the French flag problem, in which cells compute their spatial position by responding to the concentration of a morphogen in a gradient1, provides crucial insight into how patterns of gene expression form in a developing organism. The simplest formulation of this model – concentration thresholds leading directly to gene expression states – requires a static morphogen gradient to produce a stable pattern of gene expression2,3. However, quantitative measurements in developing embryos reveal that morphogen gradients are both dynamic and transient4,5, and genetic perturbations reveal that pattern formation is robust to changes in morphogen concentration6–8. A gene regulatory network topology of mutual inhibition downstream of antiparallel morphogen gradients9–12 (Fig. 1a) has been proposed to robustly interpret dynamic gradients (Fig. 1b). However, while certain features of this topology are common to a number of developmental contexts such as the early Drosophila embryo and the vertebrate neural tube (reviewed in 3), demonstrating how this network functions and whether it is indeed sufficient remains a challenge, due to the complexities of the different biological contexts in which it operates. Recent work in synthetic biology has proven the utility of building multicellular patterning circuits both for understanding development and for learning engineering principles13–18. Here we show that the mutual inhibition motif19 is sufficient to produce stable domains of gene expression in response to dynamic and transient morphogen gradients. By taking a synthetic biology approach20–23 we have built a morphogen-induced a mutual inhibition circuit from scratch that acts in isolation in E. coli and used it to investigate the conditions under which patterning occurs. We have also added morphogen production to the core circuit to create a reaction-diffusion patterning system that responds to a single gradient by producing two domains of gene expression with a self-organized boundary. The experimental control and precise measurement afforded by a synthetic biology framework allowed us to understand the behaviour of these patterning mechanisms at a quantitative level in the context of a mathematical model parameterized against data, and to uncover general design principles for engineering multicellular systems. Results Engineering mutual exclusivity. To investigate whether a simple mutual inhibition network topology can interpret dynamic gradients, we built a synthetic Exclusive Receiver circuit (Fig. 1c), based on a previous Receiver circuit design (pR33S17524) that responds to two homoserine lactone (HSL) input signals, 3O-C6-HSL (C6) and 3O-C12-HSL (C12) with fluorescent protein outputs. We engineered mutual inhibition by introducing genes encoding TetR, expressed bicistronically with eYFP, and LacI, expressed bicistronically with eCFP. In addition, the C12-binding receiver protein LasR was expressed under the control of a LacI-repressible promoter, while the C6-binding receiver protein LuxR was expressed under the control of a TetR-repressible promoter. The Exclusive Receiver therefore consists of two signalling pathways that mutually repress each other, such that LasR, eYFP and TetR are expressed in the c Morphogen 1 source 3O-C12-HSL Morphogen 2 source PLas81 PLac (R0011) Diffusion Transient morphogen gradients LasR YFP TetR CFP LacI Signal transduction Ptet (R0040) Repression Transcription Transcription factor 1 factor 2 PLux76 LuxR Stable boundary 3O-C6-HSL YFP 600 2500 600 2500 300 0 100 0 250 100 2500 0 25,000 2.5 0 200 25 2.5 250 200 0 25,000 25 250 25 2.5 [C12] (nM) 2500 250 [C12] (nM) 25,000 400 25 300 500 25 250 25 250 2500 400 0 500 250 2.5 0 2500 [C6] (nM) 25,000 [C6] (nM) 700 25,000 Transcription factor 2 expression 25,000 2500 Transcription factor 1 expression [Morphogen 2] 700 25 [Morphogen 1] Space [C12] (nM) [C12] (nM) 25,000 2.5 Receiver [C6] (nM) Space 25 0 20 [C12] (nM) 250 2.5 40 0 25,000 250 2500 0 25 60 0 50 2.5 80 25 2.5 100 0 100 250 250 2.5 2500 2500 2500 150 25,000 120 25,000 200 25 140 2.5 250 250 Merge 25,000 [C6] (nM) 300 2500 2.5 CFP 25,000 25 d 2.5 After boundary formation [C6] (nM) Before boundary formation Exclusive receiver [C6] (nM) b [C12] (nM) Fig. 1 A synthetic gene circuit for morphogen interpretation. a Schematic representation of a developing embryo. Mutual inhibition of transcription factors (cyan and yellow) downstream (...truncated)