Fig 1.
Quantitative gene expression data in wild-type and sna::hb embryos.
(A) Diagram of the upstream half of the even-skipped locus. Enhancers are indicated in maroon, and coding sequence is indicated in grey. (B) We created transgenic lines containing lacZ reporter constructs for eve enhancers and measured gene expression in wild-type embryos and embryos misexpressing ventral hb (sna::hb embryos). Here, we show visual renderings [74] of gene expression atlas data that average measurements from multiple embryos in timepoint 4 (25–50% membrane invagination) [43]. Dorsal (D) and ventral (V) surfaces are indicated for the lateral view, as are anterior (A) and posterior (P) positions. Left: eve2+7 lacZ expression (maroon) in wild-type embryos; Right: eve2+7 lacZ in sna::hb embryos. Hb protein is shown in blue. Individual nuclei are outlined, and darker coloring indicates higher relative expression level. (C) To help visualize all relevant nuclei, we show 2-dimensional projections of expression data throughout this manuscript. Positions of individual nuclei along the dorsal-ventral (DV) axis are plotted as a function of position along the anterior-posterior (AP) axis. Darker color indicates higher expression for each nucleus. Relative expression values are normalized to the maximum value and range from 0 to 1.
Fig 2.
Hunchback directly represses eve3+7.
(A) Predicted Hb binding sites in wild-type and mutant versions of eve3+7 are depicted as vertical bars along the sequence where height is proportional to PATSER score [68]. Hb sites are indicated in blue; other regulators are in grey. (B) 2D projections of atlas data for reporter constructs expressed in wild-type or sna::hb embryos. Data is taken from timepoint 4. (C) 2D projection of a representative wild-type embryo expressing eve3+7 lacZ. We plot gene expression as a function of anterior-posterior (AP) position by averaging measurements from lateral strips in individual embryos and normalizing them to their maximum value (see E). (D) 2D projection of a representative sna::hb embryo expressing eve3+7 lacZ. To quantify the effect of ventral Hb in individual embryos, we take average measurements from lateral strips, extract the local maxima corresponding to stripes 3 and 7, and subtract those values from the corresponding peaks of the ventral strip. We perform the same analysis in wild-type embryos to account for modulation along the DV axis. A decrease in the ventral/lateral difference between wild-type and sna::hb embryos indicates Hb repression, while an increase indicates Hb activation (see F). (E) Lateral line traces from individual wild-type embryos containing eve3+7 reporter constructs (eve3+7: grey, n = 26; eve3+7mutHb: blue, n = 9). Embryos are from all six timepoints in stage 5. (F) Differences between ventral and lateral stripe peaks are plotted for individual wild-type and sna::hb embryos in all 6 timepoints in stage 5. Top: wild-type eve3+7 (wt: n = 26; sna::hb: n = 19); Bottom: eve3+7mutHb (wt: n = 9; sna::hb: n = 13). Asterisks indicate significant differences between wild-type and sna::hb embryos (p-value < 0.001, Mann-Whitney U test). Differences between wild-type and sna::hb embryos containing eve3+7mutHb were not significant (p-value > 0.4 for both stripes, Mann-Whitney U test).
Fig 3.
Hunchback indirectly activates eve2+7.
(A) Predicted Hb binding sites in eve2+7 and eve2+7mutHb. Sites are displayed as in Fig 2. (B) 2D projections of atlas data for reporter constructs expressed in wild-type or sna::hb embryos. Data is taken from timepoint 4. (C) Peak stripe expression levels for individual embryos from timepoints 2–4 (4–50% membrane invagination) containing eve2+7 (grey, n = 23) or eve2+7mutHb (blue, n = 20) were measured by normalizing lacZ expression levels using a huckebein co-stain [44] and extracting local maxima from lateral line traces. Asterisks indicate significant differences (p-value < 0.05, Mann-Whitney U test). (D) Differences between ventral and lateral stripe peaks are plotted for individual wild-type and sna::hb embryos in all 6 timepoints in stage 5. Top: wild-type eve2+7 (wt: n = 22; sna::hb: n = 26); Bottom: eve2+7mutHb (wt: n = 9; sna::hb: n = 11). Asterisks indicate significant differences between wild-type and sna::hb embryos (p-value < 0.001, Mann-Whitney U test).
Fig 4.
Hunchback is counter-repressed in eve2+7.
(A) Predicted Cad (red) and Hb (blue) binding sites in wild-type and mutant versions of eve2+7 are shown as in Fig 2. (B) 2D projections of atlas data for reporter constructs expressed in wild-type or sna::hb embryos. Data is taken from timepoint 4. (C) Lateral line traces from individual wild-type embryos containing eve2+7 reporter constructs. Embryos are from all six timepoints in stage 5. (D) Differences between ventral and lateral stripe peaks are plotted for individual wild-type and sna::hb embryos in all 6 timepoints in stage 5. Top: wild-type eve2+7 (wt: n = 22; sna: n = 26); Middle: eve2+7mutCad (wt: n = 15; sna: n = 24); Bottom: eve2+7mutCadmutHb (wt: n = 22; sna: n = 9). Asterisks indicate significant differences between wild-type and sna::hb embryos (p-value < 0.001, Mann-Whitney U test).
Fig 5.
Caudal and Hunchback binding sites co-evolve in orthologous eve stripe 2 enhancers.
(A) Enrichment scores for predicted Hb and Cad binding sites in orthologous eve stripe 2 and eve3+7 sequences from different Drosophilid and Sepsid species [51]. Scores were calculated by comparing the number of predicted sites to an expected value calculated from the genomic background [52]. Scores for Drosophila melanogaster enhancers are indicated in magenta. Spearman correlation and p-value are displayed for each set of enhancers. (B) Summary of current findings. Top: levels of Hb protein (blue), Cad protein (red) and Eve protein (grey) are plotted as a function of anterior-posterior position. Data were taken from the FlyEx database [75]. Bottom: cartoon indicating Hb function in eve2+7 at different locations in the embryo. In the anterior, Cad levels are low, so Hb represses eve2+7. In the trunk, Cad binding to eve2+7 prevents Hb repression.