Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, β2 laminins, and collagen α(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (α1/2) promote clustering of synaptic vesicles as nerve terminals form. β2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (α3–6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.
