Sequential addition of neuronal stem cell temporal cohorts generates a feed-forward circuit in the Drosophila larval nerve cord.

How circuits self-assemble starting from neuronal stem cells is a fundamental question in developmental neurobiology. Here, we addressed how neurons from different stem cell lineages wire with each other to form a specific circuit motif. In Drosophila larvae, we combined developmental genetics (twin-spot mosaic analysis with a repressible cell marker, multi-color flip out, permanent labeling) with circuit analysis (calcium imaging, connectomics, network science). For many lineages, neuronal progeny are organized into subunits called temporal cohorts. Temporal cohorts are subsets of neurons born within a tight time window that have shared circuit-level function. We find sharp transitions in patterns of input connectivity at temporal cohort boundaries. In addition, we identify a feed-forward circuit that encodes the onset of vibration stimuli. This feed-forward circuit is assembled by preferential connectivity between temporal cohorts from different lineages. Connectivity does not follow the often-cited early-to-early, late-to-late model. Instead, the circuit is formed by sequential addition of temporal cohorts from different lineages, with circuit output neurons born before circuit input neurons. Further, we generate new tools for the fly community. Our data raise the possibility that sequential addition of neurons (with outputs oldest and inputs youngest) could be one fundamental strategy for assembling feed-forward circuits.