Figure 1

Figure 1

Design of Small Molecule Screen in two phases. (A) Schematic depiction of primary screening and secondary screening procedure. We selected 32 small molecule inhibitors that target chromatin regulators. Zebrafish larvae were exposed to the small molecule inhibitors during distinct phases of DA and NA neuron development ranging from 24 to 72 hpf. We screened all compounds at different concentration to find the optimal dosage for each small molecule inhibitor. During the primary screen we evaluated the effects of all selected small molecule inhibitors on DA and NA neurons by whole mount in situ hybridization (WISH) analysis of tyrosine hydroxylase expression. Small molecules that caused a dose-dependent change in DA and NA marker expression were considered a screening hit. For these compounds, we performed a secondary screen to evaluate DA and NA neurogenesis steps affected. (B) Schematic drawings of the distribution of DA and NA neurons (top, taken from Mahler et al., 2010), as well as of Isl1 expressing cranial motor neurons in the larval zebrafish brain at 96 hpf. DA neurons (blue, color intensity encodes dorsal to ventral position of DA groups as indicated) analyzed during the screening procedure reside in the olfactory bulb, subpallium (telencephalon, Tc), retina, pretectum (PrT) and ventral diencephalon (DC1–6) (Holzschuh et al., 2001; Rink and Wullimann, 2002). NA neurons (red) reside within the locus coeruleus (LC) and medulla oblongata (MO) area of the hindbrain. The analyzed Isl1 expressing cranial motor neurons reside within the telencephalon (Tc), the midbrain (MN II/IV) and the hindbrain (MN Va, Vp and MN VII) (Higashijima et al., 2000). AC, amacrine cells; DA, dopaminergic; DC, diencephalic; LC, locus coeruleus; MN, motor neuron cluster; MO, medulla oblongata; NA, noradrenergic; Tc, telencephalon.