Extended Data Fig. 9: Manipulation of Eminens gene regulatory network.
From: Comprehensive single-cell transcriptome lineages of a proto-vertebrate
a, Pseudotemporal expression profiles of regulatory genes and signalling components in Eminens neurons. b, Diagram of the Prop regulatory sequences with their length indicated on the left. A representative embryo is shown for the different fusion genes (GFPCAAX, green). The minimal Prop enhancer has weak expression in Eminens neurons (arrow). When the binding site for FoxH-a was mutated (260 bp FoxH-a mut), these regulatory sequences show even less activity. c, Bar plot of the percentage of the embryos that express GFP shown in b. Numbers on the right of the column correspond to the percentage of GFP+ embryos. χ2 test with four degrees of freedom was performed (P < 2.2 × 10−16), followed by two-sided Fisher’s exact test with Bonferroni adjustment for multiple comparisons. P values: 900 bp versus 700 bp, P = 1.05 × 10−7; 900 bp versus 300 bp, P = 3.47 × 10−13; 900 bp versus 260 bp, P = 2.36 × 10−4; 900 bp versus 260-bp FoxH-a mut, P = 1.81 × 10−19; 700 bp versus 300 bp, P = 0.011; 700 bp versus 260 bp, P = 0.36; 700 bp versus 260-bp FoxH-a mut, P = 0.088; 300 bp versus 260 bp, P = 5.59 × 10−6; 300 bp versus 260-bp FoxH-a mut, P = 0.69; 260 bp versus 260-bp FoxH-a mut, P = 1.27 × 10−7. Numbers of embryos: 900 bp, n = 207; 700 bp, n = 300; 300 bp, n = 160, all pooled over 2 electroporation experiments; 260 bp, n = 440, 260-bp FoxH-a mut, n = 750, all pooled over 3 electroporation experiments. d, Overexpression of Prop using Dmrt1 regulatory sequences causes supernumerary Prop+ cells (bottom panel) compared to control embryos expressing LacZ (top). The 2-kb Prop reporter gene shows specific expression in Eminens neurons (H2B–YFP, green). The images show representative embryos for both conditions. e, Quantification of Prop+ cells from the experiments in d. Dmrt1>LacZ, n = 269 embryos; Dmrt1>Prop, n = 210 embryos, pooled over 3 electroporation experiments. The orange dots indicate the mean and the bars indicate the s.d. Dmrt1>LacZ, 1.5 ± 1.4 cells; Dmrt1>Prop, 4.2 ± 4.8 cells. Mann–Whitney U-test, P = 3.65 × 10−9. Scale bars, 20 μm.
