Hh-responsive progenitors of the hypothalamus give rise to dopaminergic, serotonergic, and GABAergic neurons. A, Native (green) Kaede expression in Hh-responsive hypothalamic cells at 9 dpf, as seen in a non-UV-irradiated brain. B, Photoconverted (red) Kaede expression in hypothalamic Hh-responsive cells as seen in the brain from a fish that had been exposed to UV light for 10 min before fixation. C, Kaede expression in the hypothalamus of a fish that had been UV-irradiated 3 d before fixation and imaging. The majority of native/newly produced Kaede protein (green) is seen in the ventricular regions (e.g., inside dotted oval), consistent with continued Hh-target gene expression in proliferative cells. UV-converted Kaede protein (red) is still visible in the ventricular regions 3 d after conversion but is also present in cells more distant from the ventricle that do not contain new/native Kaede protein (arrowheads). Panels at bottom show merged and separated red and green channels as indicated. D, Dopaminergic cells and Hh-responsive cells in the ventral brain, as visualized in a Tg(slc6a3:EGFP,GBS-ptch2:NLS-mCherry) double transgenic larva. A small subset of cells expresses both the GFP and the NLS-mCherry proteins (arrowheads, circle), suggesting Hh-responsive cells can give rise to dopaminergic neurons. E, Monoaminergic neurons and Hh-responsive cells as visualized in a Tg(slc18a2:GFP,GBS-ptch2:NLS-mCherry) double transgenic larva. Again, a small subset of cells expresses both the GFP and the NLS-mCherry proteins (arrowheads), suggesting Hh-responsive cells can give rise to monoaminergic neurons. F, GABAergic neurons and Hh-responsive cells as visualized in a TgBAC(gad1b:GFP, GBS-ptch2:NLS-mCherry) double transgenic larva. A small subset of cells expresses both the GFP and the NLS-mCherry proteins (arrowheads), suggesting Hh-responsive cells can give rise to GABAergic neurons. G, Antibody labeling in a Tg(GBS-ptch2:NLS-mCherry) larval brain showing serotonin (5-HT) expression in the ventral hypothalamus. The presence of double-labeled cells is consistent with Hh-responsive cells giving rise to serotonergic neurons. H, I, Ventral views of anti-serotonin antibody labeled 7-dpf larval brains labeled cells following conditional manipulation of Hh signaling using the Tet-On transgenic system. H, Representative single transgenic [Tg(GBS-ptch2:RTTA-HA) or Tg(TETRE:shha-mCherry)] sibling larva, identified by the lack of mCherry expression, showing number of serotonergic cells in the absence of effector transgene activation. I, Representative Tg(GBS-ptch2:RTTA,TETRE:shha-mCherry) double transgenic larva, identified by mCherry expression, showing increased numbers of serotonergic cells in the PR following 2 d activation of the shha-mCherry transgene. J, Representative Tg(GBS-ptch2:RTTA,biTETRE:gli2aDR,NLS-mCherry) double transgenic larva, identified by mCherry expression, showing decreased numbers of serotonergic cells in the PR following 2 d activation of the gli2DR transgene. K, Graph showing serotonergic cell numbers, at 5–7 dpf following 1 or 2 d activation of the Tet-On system in doxycycline (see diagram at top of graph). Error bars indicate SD. Sample numbers for each experimental condition are shown on the graph, with significance determined using a one-way ANOVA; *p < 0.05, ***p < 0.001, ****p < 0.0001. Source data for graphs can be found in Extended Data Figure 8-1. A–J, Ventral views of dissected brains from 7-dpf larvae to show the hypothalamus. Dotted lines outline LR and PR on half of the brain. Small panels at right in A–D show single channel data for a single optical section in the boxed regions. hyp, hypothalamus; tel, telencephalon. Scale bars: 20 μm.
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