Fig. 4: Resolving the dynamic regulation of Kr by multiple TFs from fixed embryos.

a Confocal image of a fixed WT embryo labeled for Bcd protein, Hb protein, Kr mRNA, and DNA at nc13, with magnified views of a nucleus. Scale bars, 50 μm (embryo), 5 μm (nucleus). Bottom: Bcd and Hb concentrations and number of nascent Kr mRNAs per nucleus as a function of the AP position. Data from 1312 nuclei were binned along the AP axis (bin size: 0.1 EL, step size: 0.05 EL) to show mean ± s.d. The experiment was repeated six times, independently, with similar results. b Spatiotemporal profiles of nuclear Bcd concentration, Hb concentration, and Kr transcription in nc11–13. Data were binned along the AP axis (bin size: 0.1 EL, step size: 0.05 EL) and time (bin size: 1 min, step size: 0.25, 0.25, and 0.5 min for nc11–13, respectively). c Maximum Bcd and Hb concentrations along the AP axis and average Kr transcription in the expression band (0.4–0.6 EL) as functions of time. Data from individual embryos were binned over time (bin size: 2 min, step size: 0.5 min) to show mean (line) ± s.e.m. (shading). d Schematic of endogenous Kr regulation through cooperative Bcd and Hb binding at enhancers. e, Regulatory relationship between Kr, Bcd, and Hb over time in nc13. AP-binned data from (b) (red dots) were fitted to a time-dependent thermodynamic model (surfaces). b–c, e Data from n = 25, 36, and 76 fixed WT embryos for nc11–13, respectively. f Kr mRNA production rate over time. Colors mark distinct expression periods. g, h Comparison of Hill coefficients (g) and concentration thresholds (h) for Bcd and Hb. Values from different expression periods (dots) were summarized as mean ± s.d. Bcd and Hb concentrations at average Kr boundaries for WT embryos (n = 68) during the expression periods in nc11–13 (mean ± s.d.) are shown as reference. i Schematic showing the determination of anterior and posterior Kr expression boundaries by the spatial profiles of Hb and Bcd, respectively. Source data are provided as a Source Data file.