Fig. 4: Two enhancers combine differently to drive P1 and P2 activation.

a Schematic of hb reporter constructs with enhancer replacements. A construct without enhancer deletion was used as a control. Two smFISH probe sets were used to label different regions of reporter mRNAs: blue, P1-intron probes; red, yellow probes. b Confocal image of a distal-enhancer-removed embryo labeled for P1-intron, yellow, and hb CDS at nc12. Scale bar, 50 μm. Inset, magnified view of a single anterior nucleus. Scale bar, 2 μm. The experiment was repeated twice, independently, with similar results. c Percentage of active nuclei as a function of the AP position for P1-intron and yellow signals in nc12 embryos of different constructs. Shadings indicate s.e.m. d Average percentage of active nuclei in the position range of 0.2–0.4 EL for P1-intron and yellow signals in different constructs during nc11–13, with two-sided t-test (ΔDist vs. Control: p = 0.048, 0.044, and 0.73 for yellow in nc11–13, respectively; p = 0.049, 0.018, and 0.13 for P1-intron in nc11–13, respectively. ΔProx vs. Control: p = 0.0035, 0.13, and 0.025 for yellow in nc11–13, respectively; p = 0.027, 0.17, and 0.18 for P1-intron in nc11–13, respectively. *p < 0.05; **p < 0.01). e Nascent P1-intron and yellow signals per nucleus as functions of the AP position in nc12 embryos of different constructs. Shadings indicate s.e.m. f The maximal signal level of the anterior expression domain for P1-intron and yellow signals in different constructs during nc11–13, with two-sided t-test (ΔDist vs. Control: p = 0.0099, 0.0091, and 0.36 for yellow in nc11–13, respectively; p = 0.19, 0.045, and 0.30 for P1-intron in nc11–13, respectively. ΔProx vs. Control: p = 0.13, 0.044, and 0.084 for yellow in nc11–13, respectively; p = 0.016, 0.033, and 0.14 for P1-intron in nc11–13, respectively. *p < 0.05; **p < 0.01). g, h The average boundary shift of the anterior expression domain for yellow (g) and P1-intron (h) signals upon removing one enhancer. Error bars were computed from the standard errors of boundary positions for enhancer-deleted and control lines using error transfer formula. Right: schematic of boundary shift. c–h Data are presented as mean ± s.e.m. ΔDist: n = 6, 4, and 4 biologically independent embryos at nc11–13, respectively; ΔDist control: n = 4 biologically independent embryos at each nuclear cycle. ΔProx: n = 5, 4, and 4 biologically independent embryos at nc11–13, respectively; ΔProx control: n = 7, 4, and 6 biologically independent embryos at nc11–13, respectively. The spatial profile of each embryo was binned from the single-nucleus data (bin size: 0.05 EL, step size: 0.025 EL). Source data are provided as a Source Data file.