Optogenetic Modulation of FGF-Secreting Organizer Cells Alters Trajectory Bias and Pattern Encoding in the Limb-Bud Progress Zone during Gradient Conditioning
July 17
When a localized chemoattractant is experimentally displaced from its endogenous fibroblast growth factor (FGF) source in the developing limb bud, mesenchymal progenitors adopt one of two stereotyped migration modes. A subpopulation navigates toward the FGF-rich organizer region itself (source-directed trajectory), whereas another converges on the displaced chemoattractant focus (signal-directed trajectory). The emergence of the signal-directed mode, but not the source-directed mode, requires intact FGF signaling within the progress zone (PZ). We previously reported that chemoattractant-evoked calcium transients in the PZ display distinct spatiotemporal profiles in signal- versus source-directed cells. However, a causal link among FGF secretion, PZ activity, and adoption of the signal-directed strategy has not been demonstrated.
Using Fgf8::CreERT2 embryos maintained in ex vivo limb-bud culture, we expressed either an optogenetic silencer or activator specifically in apical ectodermal ridge (AER) FGF-secreting cells and monitored mesenchymal migration together with two-photon calcium imaging of the PZ. Transient photoinhibition of AER cells precisely at the moment of chemoattractant presentation prevented the emergence of signal-directed trajectories while leaving source-directed migration unchanged. Conversely, continuous photostimulation of the same organizer cells did not accelerate acquisition of the signal-directed mode; yet termination of stimulation halted further adoption of this trajectory bias.
Both photoinhibition and photostimulation rapidly reconfigured activity in a subset of PZ mesenchymal cells, leading to progressive alterations in chemoattractant- and source-related calcium dynamics across successive conditioning epochs. These data support the view that signal-directed and source-directed migration are products of two separable developmental learning processes—one reliant on organizer-derived FGF and one independent of it—and that the impact of AER-FGF signaling on signal-directed migration is mediated through dynamic modulation of the limb-bud progress zone.