Sequential and independent probabilistic events regulate differential axon targeting during development in Drosophila...

Variation in brain wiring contributes to non-heritable behavioral individuality. How and when these individualized wiring patterns emerge and stabilize during development remains unexplored. In this study, we investigated the axon targeting dynamics of Drosophila visual projecting neurons called DCNs/LC14s, using four-dimensional live-imaging, mathematical modeling and experimental validation. We found that alternative axon targeting choices are driven by a sequence of two independent genetically encoded stochastic processes. Early Notch lateral inhibition segregates DCNs into Notch^ON proximally targeting axons and Notch^OFF axons that adopt a bi-potential transitory state. Subsequently, probabilistic accumulation of stable microtubules in a fraction of Notch^OFF axons leads to distal target innervation, whereas the rest retract to adopt a Notch^ON target choice. The sequential wiring decisions result in the stochastic selection of different numbers of distally targeting axons in each individual. In summary, this work provides a conceptual and mechanistic framework for the emergence of individually variable, yet robust, circuit diagrams during development.