Summary Developmental variation in brain-wiring contributes to behavioural individuality 1,2 . However, how and when individualized wiring diagrams emerge and become stable during development remains largely unknown. Here, we explored axon targeting dynamics in individual brains using live-imaging of a developing Drosophila visual circuit and discovered that targeting choice is an algorithmic multi-step growth process with variable outcomes. Using optogenetics, we found that temporally restricted Notch lateral-inhibition defines a subset of neurons with a probabilistic potential to innervate distal targets. Next, axons from Notch OFF neurons amplify into long actin-rich multi-fibre structures necessary for distal growth. A subset of these Notch OFF neurons create distal targeting axons by stabilizing microtubule growth in one of their actin fibres. Amplified axons without tubulin-stabilized fibres retract, resulting in the stochastic selection of a different number of distal targeting axons in each brain. Pharmacological microtubule destabilization suffices to inhibit this targeting. We observed a similar axonal amplification-stabilization process in the developing chick spinal cord, suggesting a conserved mechanism. Finally, early microtubule patterns predict the adult brain-wiring of an individual in a target-independent manner prior to synapse formation 3,4 . Thus, we show that a temporal succession of genetically encoded stochastic processes explains the emergence of individual wiring variation. One-Sentence Summary The temporal succession of stochastic developmental processes explains the emergence of individual wiring variation.

Probabilistic axon targeting dynamics lead to individualized brain wiring