Shh-producing and Hh-responsive radial glial cells in the adult hypothalamus are proliferative neural precursors. A, Schematic sagittal, transverse, and horizontal sections through the adult zebrafish brain. B, Optical sections from a whole-brain light-sheet confocal image of a Tg(GBS-ptch2:NLS-mCherry;shha:GFP) double transgenic adult showing Hh-responsive cells (Hh-resp, red) in relation to Shh-producing cells (Shh-Prod, green) in the LR and PR of the adult hypothalamic (third) ventricle. Shh-expressing cells are adjacent to Hh-responsive cells in the ventricular zone of both recesses. Yellow lines in each panel indicate the plane of section in the other panels. Video 1 shows a progression through optical sections (from posterior to anterior), and Video 2 shows a 3-dimensional rotating view of this image. C, Sagittal tissue section showing Shh-producing cells (green) and BrdU-labeled proliferative cells (red, 24-h exposure) in the adult hypothalamus. Shh-producing cells are located primarily in the dorsal portions of both the LR and PR. Shh-expressing cells in the LR send projections toward to the dorsal region of the PR. Panels at right show separated fluorescent channels from the boxed regions, with examples of co-labeled cells in both ventricular regions indicated by arrowheads. D, Sagittal tissue section through the LR and PR of a Tg(GBS-ptch2:EGFP) transgenic adult showing Hh-responsive cells and proliferative cells labeled with BrdU (red, 24-h treatment) and the anti-PCNA antibody (magenta). Panels at right show separated channels from the boxed region, with four PCNA+/BrdU+-labeled Hh-responsive cells indicated by yellow asterisks and two PCNA+/BrdU– cells indicated by white asterisks. E, Sagittal tissue section through the PR of a Tg(GBS-ptch2:EGFP) transgenic adult labeled with the anti-s100β antibody to show radial glia. Most but not all Hh-responsive cells are s100β-positive (white arrowheads). A small percentage of GFP-labeled cells that are more distant from the ventricle are s100β-negative (yellow arrowhead), suggesting these cells have differentiated but retain GFP fluorescence from previous GBS-ptch2:EGFP transgene expression. F, Transverse tissue section through the PR of the hypothalamus of a Tg(GBS-ptch2:EGFP) adult labeled with the Sox2 antibody that labels neurogenic cells. Most or all Hh-responsive cells express the Sox2 protein. Panels at right show separated channels from the boxed region with arrowheads marking co-labeled cells. G, Sagittal tissue section through the PR of a Tg(GBS-ptch2:EGFP) adult labeled with an antibody to the neuronal marker HuC/D (now called Elavl3). Double labeling of cells far from the ventricle indicates that Hh-responsive cells (green) can give rise to HuC/D-expressing neurons (red). Panels at right show separated channels from the boxed region with co-labeled cells (arrowheads). Cb, cerebellum; hy, hypothalamus; LR, lateral recess of the hypothalamic (third) ventricle; PR, posterior recess of the hypothalamic (third) ventricle; tect, tectum. Scale bars: 1 mm (A), 50 μm (B), and 20 μm (C–H).
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