Hh-responsive cells are more highly proliferative than other radial glia in the hypothalamus. A, Sagittal tissue section through the PR of a TgBAC(nes:EGFP) transgenic adult brain showing PCNA antibody-labeled proliferative cells. Nestin-expressing cells are predominantly PCNA negative. B, Sagittal tissue section through the PR of a Tg(ptch2:EGFP) transgenic adult brain showing PCNA antibody labeled proliferative cells. Arrowheads indicate examples of PCNA-labeled (proliferative) Hh-responsive cells. Panels at right show separated channels from the boxed region. C, Hh-responsive cells (red) are largely distinct from Nestin-expressing cells (green) in the PR of the adult hypothalamus, as visualized in Tg(GBS-ptch2:NLS-mCherry;nes:EGFP) double transgenic fish. However, 17% of cells (74 cells of 442 total) contained both GFP and mCherry (arrowheads, n = 9 tissue sections from 3 double transgenic fish) with GFP fluorescence substantially lower in double-labeled cells. Panels at right show separated channels from the boxed region. D, E, BrdU pulse-chase experiment. Schematic above panels shows timing of pulse and chase, with 47-dpf adult TgBAC(nes:EGFP) or Tg(GBS-ptch2:EGFP) fish being exposed to 10 μm BrdU in fish water for 2 d; 32 d later fish were killed, and tissue sections were labeled using anti-BrdU (red) and anti-PCNA (magenta) antibodies. D, Representative sagittal section through the PR of a TgBAC(nes:EGFP) adult, insets show single channel data for the boxed region. A small number of Nestin-expressing cells in the PR retained the BrdU label after one month. These cells did not express PCNA (arrowheads), indicating they were not in G1/S/G2 of the cell cycle at the time of fixation. E, Representative sagittal section through the PR of a Tg(GBS-ptch2:EGFP) adult, insets show single channel data for the boxed region. Most Hh-responsive cells failed to retain the BrdU label after one month, and many of these cells expressed PCNA (arrowheads), indicating active cell cycling at the time of fixation. F, Graph showing the percentage of Nestin-expressing or Hh-responsive cells that co-labeled with the BrdU or PCNA antibodies. Tg(nesGFP): n = 3 fish, 13–16 tissue sections per fish. Tg(GBS-ptch2:EGFP): n = 2 fish, 13–16 sections per fish. G, Quantification of BrdU label intensity in Nestin-expressing and Hh-responsive cells showing BrdU labeling intensity was significantly lower in Hh-responsive cells compared with nestin-expressing cells. H–K, Representative images of PCNA labeling in single confocal optical sections from sagittal sections through the hypothalamus in 7-dpf larvae expressing four different transgenes. Top panels show merged images (transgene reporter + PCNA + DAPI) and bottom panels show single channels. H, In Tg(GBS-ptch2:NLS-mCh) larvae over 60% of NLS-mCherry-expressing cells in the hypothalamic ventricular regions are labeled with the PCNA antibody (arrowheads). In the LR, 214 of 288 NLS-mCherry+ cells labeled with PCNA (74%), while in the PR 110 of 117 (62%) cells were double labeled (n = 12 sections from 3 larvae). I, Shh producing cells were predominantly found in the PR (dotted oval); 19 of 186 GFP+ cells expressed PCNA (10%, arrowheads; n = 15 sections from 3 larvae). J, Notch responsive cells, as revealed in the Tg(Tp1bglob:GFP) line, were also localized to the PR (oval); 27 of 113 GFP+ cells were PCNA+ (24%, arrowheads; n = 11 sections from 3 larvae). K, Wnt-responsive cells are confined to the PR (oval). None of the 266 cells examined expressed PCNA (n = 18 sections from 3 larvae). L, Graph showing the percentage of each transgene-expressing cell type found to also express PCNA; ***p < 0.001, ****p < 0.0001. Source data for graphs can be found in Extended Data Figure 7-1. All panels show 0.5-μm single optical sections of 20-μm tissue sections. Scale bars: 20 μm.
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