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An abnormal serotonergic (5\u2010HT) system has been implicated in the etiology of ASD. In the present study, we have examined the expression and distribution of two early inducers of 5\u2010HT neurons in rat embryos, to elucidate the prenatal development of 5\u2010HT neurons after VPA exposure at embryonic day (E) 9.5. Whole\u2010embryo <jats:italic>in situ<\/jats:italic> hybridization at E11.5 showed that the expression of <jats:italic>sonic hedgehog<\/jats:italic>, one of the early inducers of 5\u2010HT neurons, was reduced around the isthmus in the VPA\u2010exposed group. Furthermore, whole\u2010mount immunohistochemistry of the hindbrain and quantitative analysis of 5\u2010HT neurons in the rostral raphe nucleus (rRN) revealed that neuronal distribution in the caudal part of the rRN was narrower at E15.5 in the VPA\u2010exposed group than in controls. Thus, the early development of 5\u2010HT neurons was altered after VPA exposure <jats:italic>in utero<\/jats:italic>. 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