Our latest: We developed a chemo-optogenetic system for precise spatiotemporal control of morphogen production. Using dual light + small molecule control of Sonic Hedgehog production, we recapitulated neural tube patterning in vitro & measured spread of Shh 🧵 www.sciencedirect.com/science/arti...
The challenge: Most existing optogenetic systems suffer from either weak activation OR leaky expression in the dark To get around this we fused the light-responsive transcription factor GAVPO to a small-molecule responsive degron domain (DD-GAVPO) Best of both worlds: chemo-optogenetics
The dual-control system exhibits minimal leakiness while allowing robust gene induction By titrating Shld-1 concentration, we can precisely tune activation levels - something challenging to achieve with light alone It's a dimmer switch, not just a toggle
We applied DD-GAVPO to control Sonic hedgehog (Shh) production in a stem cell model of neural tube development Spatially restricted light illumination recreated long-range Shh gradients that faithfully recapitulated neural tube patterning
Key finding #1: Shh has an extracellular half-life ~1h. Turnover much faster than downstream gene expression dynamics, indicating morphogen gradient is continually renewed during patterning Suggests Shh's effective diffusion rate: ~0.125 μm²/s, remarkably similar to Dpp/Wingless in Drosophila
Key finding #2: Cholesterol-modified Shh showed normal patterning, while non-cholesterol modified Shh (NShh) extended signalling range 3-fold Membrane-tethered Shh-CD4 abolished long-range signalling
Similarly, the Shh interacting protein Scube2 increased Shh range by ~50%, confirming its role as a "gradient expander" in mammalian systems (previously shown in zebrafish) The system provides a quantitative readout for morphogen modulator activity
Key finding #3: Dynamic xpts revealed progenitor identity depends on both concentration & duration of Shh More ventral markers (Nkx2.2) appear later but domain sizes remain constant This suggests progressive fate assignment reflects gene regulatory dynamics, not gradient dynamics
Is this why having a floor plate is important? (as opposed to only rely on the notochord -who will eventually disappear/turn into something else- to secrete the ligand)(naïve question)
Bottom line: DD-GAVPO represents a powerful tool for precise gene control. We used it to generate quantitative measurements of a mammalian morphogen but it is versatile and could be used in a wide range of applications All credit to @dbenzinger.bsky.social who conceived & implemented the project.
@katwrog.bsky.social if you haven't seen this...
very cool!!
Waste of time posting this without an explanation. Why is it important, in terms most can understand. Otherwise it’s just fluffing. I’m a scientist. It no wonder nobody listens.
its a great vention
Very neat and elegant ! Congratulations @dbenzinger.bsky.social @jamesbriscoe.bsky.social and to all authors👏😀!!
Have you tried to modulate the extracellular space size or geometry and/or the cell volume (e.g. through osmotic perturbation) to see how this would affect SSH effective diffusion coefficient?
Yeah, interesting questions. On the list, but easier to propose than to do....
Thanks, James! I agree that these are very difficult experiments, but perhaps they could be carried out using an in vitro system like yours, where cell volume and osmolarity could be controlled 'pharmacologically'...
A few years ago, we introduced a new theoretical framework that treats tissues as biphasic poroelastic media. Through simulations, we demonstrated that the effective diffusion coefficient of morphogens essentially depends on:
i- The size and geometry of extracellular space; ii- Interactions of morphogens with cells through binding-unbinding or endo-/exocytosis; iii- Mechano-osmotic effects associated with cell volume regulation and intercellular fluid flows.