Neurosphere formation potential resides not in the caudal cell mass, but in the secondary neural tube.

The caudal cell mass (CCM) is known as the main player in secondary neurulation, forming the secondary neural tube (2NT). This suggests that the CCM may have the character of neural progenitor cells. The neural potential of the CCM and the 2NT (CCM + 2NT) was assessed by in vitro culture of neurospheres during Hamburger and Hamilton stages (HH) of secondary neurulation (HH16 to HH32). We also analyzed the neural potential of the developing central nervous system (CNS) by comparing the neurosphere culture from the brain, upper / lower spinal cord, and CCM + 2NT from various HH stages. The CCM + 2NT was capable of forming neurospheres. Of the various HH stages, the greatest number of neurospheres from CCM + 2NT were cultured at HH28. Because the 2NT is most prominent at HH28, we hypothesized that the 2NT, rather than the CCM, had the main potential to produce neurospheres. When the neurospheres were cultured separately from the CCM and the 2NT, 2NT made significantly more neurospheres. When comparing different parts of the CNS, at HH16, the greatest number of neurospheres was formed from the brain. At HH32, it was the CCM + 2NT. The region with the greatest number of neurospheres progressed in a cephalo-caudal direction during development. This study showed that neurospheres can be cultured from CCM + 2NT, and the main player in neurosphere formation was the 2NT. The present study has also revealed cephalo-caudal trend in the neural potential of developing CNS.