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Published May 4, 2018 | Supplemental Material + Accepted Version
Journal Article Open

Morphogen gradient reconstitution reveals Hedgehog pathway design principles


In developing tissues, cells estimate their spatial position by sensing graded concentrations of diffusible signaling proteins called morphogens. Morphogen-sensing pathways exhibit diverse molecular architectures, whose roles in controlling patterning dynamics and precision have been unclear. In this work, combining cell-based in vitro gradient reconstitution, genetic rewiring, and mathematical modeling, we systematically analyzed the distinctive architectural features of the Sonic Hedgehog pathway. We found that the combination of double-negative regulatory logic and negative feedback through the PTCH receptor accelerates gradient formation and improves robustness to variation in the morphogen production rate compared with alternative designs. The ability to isolate morphogen patterning from concurrent developmental processes and to compare the patterning behaviors of alternative, rewired pathway architectures offers a powerful way to understand and engineer multicellular patterning.

Additional Information

© 2018 American Association for the Advancement of Science. Received 14 June 2017; accepted 22 March 2018; Published online 5 April 2018. We thank J. Briscoe and A. McMahon for DNA constructs and helpful discussion, Y. Antebi for data analysis programs and discussion of modeling, J. Bois for advice on model simulation, Z. Singer for cloning ERT2-GAL4, F. Tan for suggesting the rocker experiment, and the Elowitz laboratory for discussion. We also thank U. Alon, N. Barkai, P. Beachy, A. Eldar, J. Garcia-Ojalvo, L. Goentoro, E. Hui, P. Jordan, B. Shilo, B. Shraiman, and D. Sprinzak for discussion and feedback. Funding: The work was funded by the Howard Hughes Medical Institute (M.B.E.), American Cancer Society Postdoc Fellowship 127270-PF-15-032-01-DDC (P.L.), NICHD (Eunice Kennedy Shriver National Institute of Child Health and Human Development) Pathway to Independence Career Award K99HD087532 (P.L.), NIH Ruth Kirschstein National Research Service Award F32 AR067103 (J.S.M.), Institute for Collaborative Biotechnologies contract W911NF-09-D-0001 through the U.S. Army Research Office, and BBSRC (UK Biotechnology and Biological Sciences Research Council)–NSF award 1546197. The RNA-sequencing work was supported by the Millard and Muriel Jacobs Genetics and Genomics Laboratory at the California Institute of Technology. Author contributions: P.L. and M.B.E. conceived the project and designed the experiments. P.L., S.W., and V.V. performed the experiments. P.L., J.S.M., and S.W. analyzed the experimental data. J.S.M., P.L., and S.C. developed the mathematical models. P.L., J.S.M., and M.B.E. wrote the paper. Competing interests: The authors declare no competing interests. Data and materials availability: Original data, Matlab code, DNA constructs, and cell lines are available upon request.

Attached Files

Accepted Version - nihms-1016389.pdf

Supplemental Material - aao0645_Li_SM.pdf

Supplemental Material - aao0645s1.mp4

Supplemental Material - aao0645s2.mp4

Supplemental Material - aao0645s3.mp4

Supplemental Material - aao0645s4.mp4

Supplemental Material - aao0645s5.mp4

Supplemental Material - aao0645s6.mp4


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August 19, 2023
October 17, 2023