Abstract
Development of multicellular organisms depends on the proper establishment of signaling information in space and time. Secreted molecules called morphogens form concentration gradients in space and provide positional information to differentiating cells within the organism. Although the key molecular components of morphogen pathways have been identified, how the architectures and key parameters of morphogen pathways control the properties of signaling gradients, such as their size, speed, and robustness to perturbations, remains challenging to study in developing embryos. Reconstituting morphogen gradients in cell culture provides an alternative approach to address this question. Here we describe the methodology for reconstituting Sonic Hedgehog (SHH) signaling gradients in mouse fibroblast cells. The protocol includes the design of morphogen sending and receiving cell lines, the setup of radial and linear gradients, the quantitative time-lapse imaging, and the data analysis. Similar approaches could potentially be applied to other cell–cell communication pathways.
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Acknowledgments
We thank the Michael Elowitz lab where the method was initially developed. We also thank Yaron Antebi for providing the single-cell segmentation/tracking program. This work was funded by NIH Pathway to Independence Career Award 1R00HD087532 and Mathers Foundation MF-1905-00336.
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Kim, J.S., Pineda, M., Li, P. (2021). Reconstitution of Morphogen Signaling Gradients in Cultured Cells. In: Ebrahimkhani, M.R., Hislop, J. (eds) Programmed Morphogenesis. Methods in Molecular Biology, vol 2258. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1174-6_4
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DOI: https://doi.org/10.1007/978-1-0716-1174-6_4
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