Semaphorin 3A repulsion directs the caudal projection of pioneer longitudinal axons in the developing chicken brain

The medial longitudinal fasciculus (MLF) is the first axon tract to develop in the ventral vertebrate brain. It originates in the diencephalon and projects caudally into the spinal cord, pioneering the path for later developing axons. Previous anatomical and expression analyses in the chicken suggested Semaphorin 3 A (Sema3A) as the candidate to repel the amniote MLF from the forebrain. However, studies in the zebrafish implicated a distantly related semaphorin with a role in axon fasciculation, not guidance. Thus, the mechanism accounting for the caudal projection of the MLF remains unclear. Here we show that misexpression of Sema3A or grafting of Sema3A-expressing cells into the path of the MLF diverts the axons or blocks their outgrowth in chicken embryos. In vitro, Sema3A exposure resulted in the collapse of MLF growth cones. A dominant-negative approach or siRNA to interfere with the function of the Sema3A receptor Neuropilin1 allowed MLF axons to project rostrally. Together, this suggests that Sema3a repulsion directs the caudal extension of the MLF to pioneer the ventral longitudinal tract.