Single-molecule observation of Hedgehog gradient formation, and a new model to think about how molecules diffuse through natural tissues

Single-molecule observation of Hedgehog gradient formation, and a new model to think about how molecules diffuse through natural tissues

Dr Gavin Schlissel; Whitehead Institute & Massachusetts Institute of Technology

Gavin is a postdoctoral fellow with Pulin Li at Whitehead & MIT. His work uses synthetic biology and single-molecule microscopy to understand how morphogens diffuse through the extracellular matrix. His work to date has focused on Sonic Hedgehog signaling in mice, but the lessons are generalizable to a range of developmental or immunological signaling molecules across organisms. Gavin trained at UC Berkeley with Jasper Rine, where he studied the mechanism of chromatin replication to understand the molecular basis of epigenetic memory. When he isn’t in lab, Gavin can usually be found cooking, hiking or sailing all over the American Northeast.

Animals use a small number of morphogens to pattern and maintain tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. This question is particularly controversial in the case of lipid-modified morphogens like Hedgehog, where there is intense debate about whether hydrophobic Hedgehog molecules diffuse extracellularly, or whether they are delivered from sender cells to receiver cells via direct cell-cell contact. We used single-molecule imaging in cultured mouse cells and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion, and we developed a new model to explain how morphogen gradient lengthscale can be controlled by modulating transition dynamics between discrete biochemical forms of Sonic Hedgehog. Under our model, cell-cell gaps create topological diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. By catalytically regulating interconversion of Hedgehog complexes, SCUBE allows Hedgehog molecules to jump between cells. Notably, this mechanism of gradient regulation uncouples the Hedgehog gradient lengthscale in distinct tissues, allowing evolution to explore variation in Hedgehog patterns without interfering with conserved Hedgehog functions. Our work motivates a fresh assessment of the evolvability of morphogen gradients, and provides mechanistic clarity about Hedgehog diffusion dynamics. We anticipate that our understanding of morphogen diffusion will generalize to all other signaling molecules that promiscuously bind receptors or co-receptors during gradient formation, including developmental morphogens, immunological cytokines and chemokines, or synthetic proteins designed to mimic natural signaling molecules.

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