System-level synchronization and subtype-specific modules govern brain-wide serotonin axon innervation

The serotonin system modulates nearly all neural circuits across development and adulthood and is a major pharmacological target for mood and anxiety disorders. Forebrain-projecting serotonin neurons are organized into projection-defined subsystems that align with molecular heterogeneity, but how these subsystems assemble during development remains unclear. We combined whole-brain developmental projection maps with single-cell transcriptomics to define the spatiotemporal and molecular logic of postnatal serotonin innervation. Using a deep-learning pipeline, we generated four-dimensional whole-brain maps of postnatal serotonin axon density and uncovered two coupled modes of regulation: region-specific trajectories of axon growth and refinement, and an early-postnatal, brain-wide synchronization of innervation. To link these dynamics to cell-intrinsic programs, we profiled single-cell transcriptomes of forebrain-projecting serotonin neurons from embryonic through postnatal stages and reconstructed developmental trajectories. We found that serotonin neuron heterogeneity was established embryonically and that the subtype taxonomy was largely conserved between mice and humans. Across serotonin subtypes, we observed a coordinated surge in wiring-molecule expression at mouse postnatal day 4 that aligned with the subsequent global innervation wave. Tracing subtypes defined by developmentally conserved markers at birth revealed complementary projection territories and engagement of distinct postnatal wiring modules that matched region-specific terminal maturation. Together, these results show that brain-wide serotonin innervation is built through embryonic subtype specification and two layers of postnatal control: system-level synchronization and subtype-specific terminal development.