Fated Path of a Neuron: Neural Circuit Assembly is Regulated by a Temporally Associated Guidance Receptor Code

Functional nervous systems require that neurons wire together during development to form neuronal circuits. To establish circuits, neurons from different developmental origins must grow to the same space ithin the central nervous system to build specific connections. However, it was unknown how spatial pathfinding and circuit wiring are coordinated across synaptic partners. Investigations at the tissue and single neuron levels identified multiple classes of ligands and receptors, or guidance cues, that regulate neuronal growth and pathfinding. For example, along the medial-lateral axis, the repellent ligand Slit and its three Roundabout (Robo) receptors regulate neuronal morphology and midline crossing. Therefore, we investigated the extent to which a code of guidance receptors is shared across neuronal partners to coordinate circuit wiring. We first determine that medial-lateral guidance receptors fra, unc-5, robo2, and robo3 are differentially expressed with robo2 and robo3 patterns associated with developmental birth order. While robo1 is ubiquitously expressed across the Drosophila melanogaster ventral nerve cord (VNC). With this pattern in mind, I assayed a well-characterized neural lineage that establishes connections with two distinct somatosensory circuits. Neuroblast 3-3 (NB 3-3) produces two cohorts of Even-skipped (+) Lateral (EL) sensory processing interneurons depending on the birth order of the neuron. Early-born ELs receive input from Chordotonal (CHO) sensory neurons, and late-born ELs receive input from Dorsal Bipolar Dendrite (DBD) sensory neurons. Previous work established that CHO sensory neurons express and require robo 3 to position their axon terminals (Zlatic et al. 2003). We show that the early-born ELs express robo 3 like their CHO inputs, while late-born ELs are robo 3 negative, similar to their DBD inputs. Therefore, robo 3 is differentially expressed at the level of neural lineages to potentially coordinate unique circuit wiring outcomes. We find, through both a genetic knockout MARCM assay and an early-born EL specific knockdown, that Robo 3 is necessary for early-born EL morphology, specifically their dendritic arborization. Additionally, we show that Robo 3 is necessary for the sensory processing function of early-born ELs. Finally, we demonstrate that robo 3 differential expression across EL interneurons and the VNC is regulated by early but not late temporal transcription factor expression. Our findings reveal that neuronal partners share a guidance receptor code for circuit wiring. Altogether, our research establishes a new mechanism for neuronal circuit wiring in which guidance receptors are differentially regulated during development to coordinate the wiring of synaptic partners and functional circuits.