Insights into temporal and spatial dynamics of short association fiber formation in the human fetal brain.

Short association fibers (SAFs) form the local scaffold of cortical connectivity and support early functional specialization. However, their development before birth remains largely unknown. Using fetal diffusion magnetic resonance imaging from the Developing Human Connectome Project and histology from the Zagreb Collection of human brains, we present an in utero reconstruction of SAF pathways in the human brain. We tracked their volumetric and microstructural developmental trajectories in 243 fetuses spanning a critical period of connectome formation. We found that SAFs emerge before sulcal folding, initially as flat, loosely arranged pathways along the subplate-white matter interface, and later reorganize into coherent U-shaped bundles. Their maturation followed a sensorimotor-to-association gradient, paralleling cortical development. Nonlinear, bundle-specific trajectories captured multiphasic maturation, with subplate dynamics preceding increases in axonal coherence and early myelination. These findings reveal how the brain's local wiring is established during prenatal development and provide insight into the origins of individual variability.