Fibroblast growth factor promotes the development of deep cerebellar nuclear neurons in dissociated mouse cerebellar cultures

Abstract

Neurons of the deep cerebellar nuclei and excitatory cerebellar interneurons arise from the rhombic lip of the cerebellar anlage. In contrast, Purkinje cells and inhibitory interneurons arise in the neuroepithelium of the fourth ventricle. During development, the projection neurons of the cerebellar nuclei are born first (embryo age (E)9–E12 in mouse) followed closely by the Purkinje cells (E10–E13). Cerebellar interneurons arise later and differentiate postnatally. We have examined the development of cerebellar nuclear neurons in primary cultures. Embryonic cerebella from E15 to E18 pups were cultured 21 days in vitro. Three distinct classes of large neurons were identified: those expressing calbindin, typical of Purkinje cells; those expressing neurogranin (Golgi cells); and a third class expressing parvalbumin but not calbindin, consistent with the morphology of large projection neurons of the cerebellar nuclei. These neurons also express Tbr1, a specific antigenic marker of cerebellar nuclear neurons. Birthdating by using BrdU incorporation shows that the putative DCN neurons are not born in vitro. To confirm their identity the E18 cerebellum was dissected into cerebellar nuclear-containing (ventral) and -lacking (dorsal) halves, which were then dissociated and cultured separately. Only the ventral cultures produce putative cerebellar nuclear neurons. In contrast to E15–E18 cultures, dissociated E13–E14 cerebella in vitro do not yield putative cerebellar nuclear neurons. However, E14 cultures do produce them when fibroblast growth factors are added to the medium. We conclude that FGF signaling is required for the maturation of cerebellar nuclear neurons.