Clonally related, Notch-differentiated spinal neurons integrate into distinct circuits.

Shared lineage has diverse effects on patterns of neuronal connectivity. In mammalian cortex, excitatory sister neurons assemble into shared microcircuits. In Drosophila , in contrast, sister neurons with different levels of Notch expression (Notch ON /Notch OFF ) develop distinct identities and diverge into separate circuits. Notch-differentiated sister neurons have been observed in vertebrate spinal cord and cerebellum, but whether they integrate into shared or distinct circuits remains unknown. Here, we evaluate how sister V2a (Notch OFF )/V2b (Notch ON ) neurons in the zebrafish integrate into spinal circuits. Using an in vivo labeling approach, we identified pairs of sister V2a/b neurons born from individual Vsx1+ progenitors and observed that they have somata in close proximity to each other and similar axonal trajectories. However, paired whole-cell electrophysiology and optogenetics revealed that sister V2a/b neurons receive input from distinct presynaptic sources, do not communicate with each other, and connect to largely distinct targets. These results resemble the divergent connectivity in Drosophila and represent the first evidence of Notch-differentiated circuit integration in a vertebrate system.