Structured and target-specific development of cortico-cortical connectivity in the mouse visual cortex

The mammalian cortex exhibits highly stereotyped long-range connectivity, yet the developmental principles specifying precise cortico-cortical projection patterns remain poorly defined. Two dominant models propose that target specificity arises either from exuberant outgrowth followed by pruning, or through initially directed axonal targeting. To resolve this, we systematically mapped the postnatal development of V1 cortico-cortical projection neurons (CCPNs) to eleven higher visual areas (HVAs) in mice using retrograde, anterograde, and single-cell tracing methods. V1→HVA connectivity develops via spatiotemporally staggered axon extension and pruning, aligned with target position along the medial-lateral axis, while reciprocal HVA→V1 feedback is refined concurrently. Multiplexed retrograde tracing and MAPseq-based single-cell profiling revealed that individual V1 neurons initialize and retain specific projection motifs with limited variation, arguing against global exuberance and selective inter-areal pruning. Instead, distinct V1 CCPN subtypes establish selective projections early, followed by local, target-dependent refinement, providing an anatomical framework for how precise long-range cortical networks emerge.