Glass, Mox, and FoxQ2 co-staining with serotonin. FoxQ2 mRNA is detected throughout the thickened neurogenic ectoderm at the animal pole of prisms (72 hours), but in 96 and 120 hour plutei there was no hybridization detectable. Tyramide amplification produces small foci of fluorescence in the cytoplasm of the cells that hybridize probe. There is diffuse background fluorescence throughout the remainder of the embryo. (a to c) In 72 hour prisms that have strong hybridization of the FoxQ2 probe to the neurogenic ectoderm, the anti-serotonin immunoreactive cells were localized outside the FoxQ2 region. (d to f) Single confocal optical section clearly shows serotonergic cells are FoxQ2 negative (white arrow). Mox mRNA was detected in the neurogenic ectoderm of prism and pluteus larvae. (g to l) In prism larvae, all of the serotonergic neurons were Mox positive. There are also some cells that are not immunoreactive with anti-serotonin, and they hybridize the Mox probe (not shown). (m to o) In plutei, neurons that are weakly immunoreactive with anti-serotonin hybridize with the Mox probe (yellow arrow). However, Mox mRNA was not detected in the neurons that strongly expressed serotonin. As the serotonergic neurons continue to differentiate during these stages, this may indicate that Mox is only expressed early in neurogenesis (asterisks in panels m to o). These preparations have relatively high background. (p to r) Glass mRNA appears not to co-localize with anti-serotonin immunoreactive cells in 72 hours prisms (white and yellow arrowheads).