Control of spatio-temporal patterning via cell growth in a multicellular synthetic gene circuit

Creators: Santorelli, Marco¹; Bhamidipati, Pranav S.^{2, 1}; Courte, Josquin¹; Swedlund, Benjamin¹; Jain, Naisargee¹; Poon, Kyle¹; Schildknecht, Dominik²; Kavanagh, Andriu^{1, 3}; MacKrell, Victoria A.¹; Sondkar, Trusha¹; Malaguti, Mattias⁴; Quadrato, Giorgia¹; Lowell, Sally⁴; Thomson, Matt²; Morsut, Leonardo¹

1. University of Southern California
2. California Institute of Technology
3. California State University, Northridge
4. University of Edinburgh

Abstract

A major goal in synthetic development is to build gene regulatory circuits that control patterning. In natural development, an interplay between mechanical and chemical communication shapes the dynamics of multicellular gene regulatory circuits. For synthetic circuits, how non-genetic properties of the growth environment impact circuit behavior remains poorly explored. Here, we first describe an occurrence of mechano-chemical coupling in synthetic Notch (synNotch) patterning circuits: high cell density decreases synNotch-gated gene expression in different cellular systems in vitro. We then construct, both in vitro and in silico, a synNotch-based signal propagation circuit whose outcome can be regulated by cell density. Spatial and temporal patterning outcomes of this circuit can be predicted and controlled via modulation of cell proliferation, initial cell density, and/or spatial distribution of cell density. Our work demonstrates that synthetic patterning circuit outcome can be controlled via cellular growth, providing a means for programming multicellular circuit patterning outcomes.

Copyright and License

© 2024, The Author(s). This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Acknowledgement

We thank D. Carra and Z. Shappell from the Morsut lab for sharing the L929 mCherry Senders and tagBFP Receivers line; the Flow Cytometry Facility and Optical Imaging Facility of the Eli and Edith Broad CIRM Center; M. Johnson for her critical review of this manuscript and valuable feedback; A. Subramanian for critical scientific discussions; J. Bois and M. Elowitz for sharing teaching materials for mathematical modeling; S.O. for coding advice; and the Morsut and Thomson labs for their scientific input and support. The authors acknowledge their family and friends that support them always and in particular for the times of this work that took place during the COVID-19 pandemic. This work was partially funded by the Human Frontier Science Program (HFSP) Organisation, funding M.S. with Long Term Fellowship (LT000469/2019-L); the CIRM-Bridges internship fellowship (A.K. and K.P.); a Belgian American Educational Foundation (BAEF) postdoctoral fellowship (B.S.); the National Institute of General Medicine of the NIH award number R35 GM138256 (L.M.); the National Science Foundation award number CBET-2034495 RECODE and CBET-2145528 Faculty Early Career Development Program (L.M.); grant 2023-332386 from the Chan Zuckerberg Initiative Donor Advised Fund, CZI DAF, an advised fund of the Silicon Valley Community Foundation (L.M., M.T.); the Heritage Medical Research Institute (M.T.); and the David and Lucile Packard Foundation (M.T.). S.L. is supported by a Wellcome Trust Senior Fellowship [220298]. M.M. is supported by a University of Edinburgh School of Biological Sciences new staff start-up award. Figures 1B and 3A, E are created with BioRender.com.

Contributions

These authors contributed equally: Marco Santorelli, Pranav S. Bhamidipati, Josquin Courte.

L.M. and M.T. directed the research and acquired funding; M.S. discovered the phenomenon of density dependent signaling and initiated the experimental branch, with help from A.K., V.A.M., and T.S.; G.Q. and L.M. co-mentored M.S. for his fellowship acquisition; P.S.B. led the computational branch, and together with M.S. developed the material for the first submission; J.C. led the revision work, focusing on investigating the mechanisms of density dependence; B.S., N.J., and K.P. supported the experimental work in revision; D.S. supported the computational work; M.M. and S.L. provided the mES cells material and directions.

Conflict of Interest

L.M. is an inventor on a synNotch patent for applications in cancer cell therapy licensed to Gilead. The remaining authors declare no competing interests.