Neurogenesis and neurite outgrowth in the spinal cord of chicken embryos and in primary cultures of spinal neurons following knockdown of Class III beta tubulin with antisense morpholinos.

Microtubules are the primary cytoskeletal constituent of extending neurites. We used antisense morpholinos to knock down expression of neuron-specific Class III beta tubulin in the right half of the neural tube of chicken embryos in ovo. There was a significant (p < 0.01) reduction in the number of Class III beta tubulin immunostained interneurons 24 h following electroporation of the morpholinos when compared with the contralateral side of the neural tube. However, neural crest-derived sensory neurons labeled with the fluorescently tagged morpholinos developed distinct processes. Moreover, there was no significant difference in the number of interneurons labeled on either side of the neural tube with a second marker of developing neurons, anti-microtubule associated protein (MAP) 1b. Neural tubes were also excised and dissociated following antisense or control morpholino electroporation. The resulting neurons were cultured for 48 h and immunostained with anti-Class III beta tubulin and anti-MAP 1b. Neurons that had taken up the antisense morpholino had significantly shorter neurites (p < 0.01) than neurons from the same neural tubes that did not; they also had significantly shorter neurites (p < 0.05) than labeled neurons from neural tubes electroporated with a control morpholino. Thus, normal expression of Class III beta tubulin may not be necessary for neurogenesis in the early avian spinal cord in situ, but is required for neurite outgrowth in vitro.