Fig. 5: BMAL1 regulates dopaminergic neurogenesis by modulating progenitor proliferation and differentiation in human neuroepithelial stem cells.

a, Schematic of the differentiation protocol used to assess the role of BMAL1 in mesDA neurogenesis. BMAL1-KD and BMAL1-OE were performed in hLT-NES cells. BMAL1-OE was induced with doxycycline (Dox) at 2 μg ml⁻¹ on day 4 and 1 μg ml⁻¹ on day 5. Asterisks indicate additional BMAL1 induction days used in follow-up experiments. b–g, BMAL1-OE increases proliferation, neurogenesis and the yield of TH⁺ neurons. Representative immunofluorescence images showing Th⁺EdU⁺ neurons (arrowheads) in CTRL and BMAL1-OE hLT-NES. Cells were pulsed with 10 µM EdU for 4 h on day 2 (d), day 4 (b) or day 6 (f), followed by analysis on day 8. Arrowheads indicate cells undergoing neurogenesis (Th⁺EdU⁺) neurons. Box plots (c,e,g) showing the percentage of TH⁺ neurons⁺ (TH ⁺/DAPI⁺), neurogenic cells (TH⁺EdU⁺/DAPI⁺) and proliferating progenitors (EdU⁺TH⁻/DAPI⁺) in response to BMAL1-OE on day 4 (c), day 2 (e) or day 6 (g) in CTRL and BMAL1-OE hLT-NES, n = 3 independent differentiations, 2 wells per condition. TH⁺/DAPI⁺ = 17.72 ± 0.33% (BMAL1-OE) versus 13.67 ± 1.39% (CTRL), P = 0.047; TH⁺EdU⁺/DAPI⁺ = 4.30 ± 0.26% (BMAL1-OE) versus 2.52 ± 0.30% (CTRL), P = 0.020; EdU⁺/DAPI⁺ = 58.60 ± 1.66% (BMAL1-OE) versus 35.45 ± 6.79% (CTRL), P = 0.037; mean ± s.e.m. (c). TH⁺/DAPI⁺ = 15.19 ± 0.84% (BMAL1-OE) versus 9.11 ± 1.84% (CTRL), P = 0.040; TH⁺EdU⁺/DAPI⁺ = 2.78 ± 0.12% (BMAL1-OE) versus 0.47 ± 0.15% (CTRL), P = 0.0003; EdU⁺TH⁻/DAPI⁺ = 33.61 ± 3.20% (BMAL1-OE) versus 23.26 ± 1.54% (CTRL), P = 0.043; mean ± s.e.m (e). TH⁺/DAPI⁺ = 12.40 ± 0.36% (BMAL1-OE) versus 12.08 ± 2.12% (CTRL), P = 0.891; TH⁺EdU⁺/DAPI⁺ = 0.88 ± 0.14% (BMAL1-OE) versus 1.04 ± 0.44% (CTRL), P = 0.746; EdU⁺TH⁻/DAPI⁺ = 38.51 ± 1.44% (BMAL1-OE) versus 21.89 ± 0.36% (CTRL), P = 0.0004; mean ± s.e.m (g). NS, not significant. h–k, BMAL1-KD impairs DA neurogenesis and reduces progenitor proliferation. Representative immunofluorescence images of TH⁺EdU⁺ neurons in shCTRL and shBMAL1 hLT-NES following a 4-h EdU pulse on day 4, analyzed on day 8 (h). Arrowheads indicate cells undergoing neurogenesis (TH⁺EdU⁺) neurons. Box plots showing the percentage of TH⁺ neurons, neurogenic (TH⁺EdU⁺/DAPI⁺) and proliferating cells (EdU⁺TH⁻/DAPI⁺) on day 8 (i). EdU⁺TH⁻/DAPI⁺ = 33.55 ± 1.19% (shBMAL1) versus 51.46 ± 4.28% (shCTRL), P = 0.016; TH⁺/DAPI⁺ = 3.73 ± 0.46% (shBMAL1) versus 4.12 ± 0.62% (shCTRL), P = 0.638; TH⁺EdU⁺/DAPI⁺ = 0.05 ± 0.03% (shBMAL1) versus 0.27 ± 0.05% (shCTRL), P = 0.020; mean ± s.e.m. Representative immunofluorescence images of TH⁺ and Ki67⁺ cells in shCTRL and shBMAL1 hLT-NES on day 4. Box plots showing the percentage of TH⁺ neurons and proliferating cells (Ki67⁺/DAPI⁺) on day 4 (k). Ki67⁺/DAPI⁺ = 28.91 ± 2.31% (shBMAL1) versus 42.52 ± 2.43% (shCTRL), P = 0.015; TH⁺/DAPI⁺ = 2.28 ± 0.39% (shBMAL1) versus 0.54 ± 0.11% (shCTRL), P = 0.013; mean ± s.e.m. Box plots display IQR, median (line), whiskers (1.5× IQR) and individual data points (scatter) (h,k). Asterisks indicate statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001), determined using two-tailed Student’s t test. n = 3 independent differentiations, two wells per condition. l–o, qPCR analysis following BMAL1-OE or BMAL1-KD. Gene expression was measured after BMAL1-OE on day 2 (m), day 4 (n), day 6 (o) or stable BMAL1-KD (l). Expression levels are shown as relative FC (ΔΔCt), normalized to day 0 ± s.e.m. from n = 3 independent differentiations. Arrows in m–o indicate Dox treatment timing. Asterisks denote statistical significance (*P < 0.05; **P < 0.01; ***P < 0.001), determined using two-tailed Student’s t test, with exact P values provided in Supplementary Table 2. FC, fold change.

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