Fig. 3: Bcd activates P1 and P2 through different regulatory relations.

a Nascent P1–5′UTR and P2–3′UTR signals per nucleus as functions of the AP position in a 1× bcd embryo at nc12 (solid line, bin size: 0.05 EL, step size: 0.025 EL) were compared with that in the WT (dashed line, from Fig. 1e). Shadings indicate s.e.m. b The boundary position of the anterior expression domain for P1–5′UTR and P2–3′UTR signals in WT and 1× bcd embryos during nc11–13. 1× bcd data are presented as mean ± s.e.m. WT: n = 8, 5, and 7 biologically independent embryos at nc11–13, respectively; 1× bcd: n = 5, 3, and 4 biologically independent embryos at nc11–13, respectively. Two-sided t-test was applied between strains (P1–5′UTR: p = 6.7 × 10⁻⁴, 2.6 × 10⁻³, and 3.8 × 10⁻⁴ for nc11–13, respectively; P2–3′UTR: p = 4.3 × 10⁻³, 8.2 × 10⁻⁴, and 2.6 × 10⁻⁵ for nc11–13, respectively. **p < 0.01; ***p < 0.001; ****p < 0.0001). c Nascent P1–5′UTR (upper) and P2–3′UTR (lower) signals in individual nuclei of a single embryo were plotted against nuclear Bcd concentration (single WT embryo, >1200 nuclei, 0.25–0.75 EL). The single-nucleus data were binned along the Bcd axis (mean ± s.e.m., bin size: 16 nM, step size: 2 nM) and fitted to Hill functions. d The Hill coefficient of the gene regulation function for different probe signals during nc11–13, with two-sided t-test (P1-Intron vs. CDS: p = 0.011, 0.010, and 0.046 for nc11–13, respectively; P1–5′UTR vs. P2–3′UTR: p = 2.8 × 10⁻⁴, 1.1 × 10⁻³, and 0.041 for nc11–13, respectively. *p < 0.05; **p < 0.01; ***p < 0.001). e The ratios of the concentration threshold between P1-intron and CDS signals, and between P1–5′UTR and P2–3′UTR signals, during nc11–13. f The average number of Bcd molecules bound at P1- and P2-active hb loci in the anterior expression domain (0.2–0.35 EL) during nc11–13, with two-sided t-test (p = 0.039, 2.9 × 10⁻⁷, and 0.067 for nc11–13, respectively. *p < 0.05; ****p < 0.0001). Inset: an anterior nucleus labeled for P1–5′UTR and P2–3′UTR of hb mRNAs and Bcd protein. The enriched Bcd signal in the vicinity of the promoter (yellow and purple circles) was measured. Scale bar, 2 μm. g Bcd binding at P1- and P2-active hb loci as a function of nuclear Bcd concentration at nc12. Data were pooled from n = 5 biologically independent embryos and were binned by nuclear AP position (bin size: 0.035 EL, step size: 0.01 EL). The binned data were fitted to multi-Hill functions. Dashed lines highlight discrete binding plateaus for each promoter. d–f Data are presented as mean ± s.e.m. P1–5′UTR and P2–3′UTR: n = 8, 5, and 7 biologically independent embryos at nc11–13, respectively; P1-Intron and CDS: n = 5 biologically independent embryos at each nuclear cycle. Source data are provided as a Source Data file.