Dense and pleiotropic regulatory information in a developmental enhancer

Abstract

Changes in gene regulation underlie much of phenotypic evolution¹. However, our understanding of the potential for regulatory evolution is biased, because most evidence comes from either natural variation or limited experimental perturbations². Using an automated robotics pipeline, we surveyed an unbiased mutation library for a developmental enhancer in Drosophila melanogaster. We found that almost all mutations altered gene expression and that parameters of gene expression-levels, location, and state-were convolved. The widespread pleiotropic effects of most mutations may constrain the evolvability of developmental enhancers. Consistent with these observations, comparisons of diverse Drosophila larvae revealed apparent biases in the phenotypes influenced by the enhancer. Developmental enhancers may encode a higher density of regulatory information than has been appreciated previously, imposing constraints on regulatory evolution.

Extended Data Fig. 1 ∣. Distribution of mutations in the E3N enhancer library.

a, Mutant enhancer variants of E3N were created via degenerate PCR and integrated into the placZattB plasmid, which contains a minimalized core hsp70 promoter and the lacZ reporter gene. Plasmids were integrated into the Drosophila genome at the attP2 site. b, Pie chart depicting base-pair composition of the WT E3N enhancer. c, (Left) Histogram for all 749 mutants (dark red) is approximately normal with an average of 7 mutations per mutant. Magenta bars denote lines antibody stained (117 total), and blue lines indicate lines that were also Beta-Galactosidase stained (274 total). (Right) pie chart shows probability of mutation normalized to ATCG composition (see b). d, Manhattan plot shows the summation of all mutations within the E3N library. e, Unsmoothened “footprinting scores” from Fig. 1h. Scores plotted linearly over transcription factor binding motifs (colored and shaded regions) across the E3N genomic sequence.

Extended Data Fig. 2 ∣. An automated platform for fixing, staining, and imaging Drosophila embryos.

a–d, Collecting Drosophila embryos. (a) Custom fly chambers were made, holding up to 24 different strains. (b) An explosion-view of the fly chambers. Embryo meshes (red) can attach and detach from the fly chambers and are suspended above an apple juice-agar plate. (c) Embryos are collected onto the embryo meshes and washed with saline solution and bleached. (d) Embryos are loaded into a fixation plate. e–h, Components of the robot. (e) The fluid-dispensing manifold. Seven pneumo-hydraulic syringe pumps are coupled to the fluid-dispensing manifold; one pump for priming chemicals into the fluid-dispensing manifold, and six pumps for dispensing chemicals into the fixation plate. (f) The fluid-separating manifold uses 24 small syringes to aspirate fluid from the isotonic shocking attachments. (g and g’) Different components of the robot. (h) Cross-section of the fixation plate and aspiration tips and syringes. 24 small aspiration tips draw fluid from the top of each well within the isotonic shocking attachment and six main dual-purpose tips dispense and aspirate fluid into and out of the bottom of the wells. i–k, The adaptive feedback imaging pipeline. (i) Samples are mounted on multi-well slides. (j) An overview tile-scan of each well is taken and x,y coordinates for embryos (green) are identified either manually or computationally. (k) For each coordinate, a fast, low-resolution confocal stack is automatically acquired. An algorithm determines the embryo’s z position and rotation, yielding a bounding box within which a high-resolution, 3D stack of the entire embryo is acquired. See also Methods. l, Control E3N WT embryos were fixed and stained on the robot. A single embryo in the same orientation and age from each well was selected and the individual nuclear fluorescence intensities were measured in AU, arbitrary units of fluorescence intensity. In plots, centre line is mean, upper and lower limits are standard deviation.

Extended Data Fig. 3 ∣. Methods of image and data analysis.

a, Images acquired from automated imaging are compiled into a large montage image. b–d, Registering multiple images using fiducials. An embryo acquired during automated imaging (b) can be automatically rotated in 3D space using ELAV (teal) as a fiducial. Once properly rotated, maximum projections of the ventral half can be computed (c). Finally, the 2-D projections can be elastically registered – or deformed – to align multiple samples (d). e–g, Methods of measuring expression patterns. (e) Sliding window analysis. A box is drawn between A2 and A3 and centred within A2. Multiple measurements are taken, sliding the box across the stripe. Each point on the boxplot represents one measurement within the box. In box plots, centre line is mean, upper and lower limits are standard deviation and whiskers show 95% CIs. (f) State method analysis. A row of cell-sized regions of interest are dragged down across the A2 stripe. Each point on the boxplot represents a single cell. (g) Plot profile analysis. A box is drawn from the A1 to A5 and the mean intensity is taken for each column of pixels and plotted (N = 10 embryos). Shaded areas indicate ± 1 SD, solid line is the mean expression. Scale bars, 100 μm. Embryos are matched to scale respectively in (a) and in (b-e), and (g).

Extended Data Fig. 4 ∣. Single base pair mutations and E3N conservation.

a–s, Example embryos carrying individual E3N::lacZ variants with single mutations. Constructs are ordered from smallest to largest effect sizes. t, u, PhyloP scores across the E3N enhancer sequence. Locations of the single mutations and their PhyloP scores are highlighted as magenta bars. v, E3N sequence alignment between 10 Drosophila species. Scale bars, 100 μm (a). Embryos are matched to scale respectively (a – s).

Extended Data Fig. 5 –. Testing additional Hth-Exd motifs in E3N.

a, b, Embryos carrying E3N::lacZ reporter constructs in a WT w¹¹¹⁸ background (a) and hth homeodomain-less (HthHM) hth^100.1 background stained with anti-β-Galactosidase (b). c–p, Embryos carrying E3N::lacZ reporter constructs stained with anti-β-Galactosidase adjacent to their respective expression plot profiles. Constructs contain mutations in Hth1 (CTGGCA → CCCCCC), Hth2 (TGACAA → CCCCCC), Hth3 (TTGTCG → CCCCCC), and Hth4 (TGAGAG → CCCCCC). (c and d) E3N WT. (e and f) E3N with Hth1 site changed. (g and h) E3N with Hth2 site changed. (i and j) E3N with Hth1 and Hth2 sites changed. (k and l) E3N with Ubx3 site changed (CATAATTTGT → CAGGGTTTGT). (m and n) E3N with Hth3 and Hth4 sites changed. (o and p) E3N with Hth1, Hth2, Hth3, and Hth4 sites changed. In all plots, the black and magenta lines denote the average expression driven by the wild-type and modified enhancers, respectively (n = 10 for each genotype). Shaded areas indicate ± 1 s.d. AU, arbitrary units of fluorescence intensity. q, top, Schematic for the E3N enhancer, denoting binding sites and possible protein-to-protein interactions. q, bottom, Schematic for different E3N fragments tested. r, Multiple-species alignment of Hth1, 2 and the UBX-Exd site. s–y, Electromobility shift assays (EMSA) for different fragments of E3N denoted in (q). All EMSAs were run on native (non-denatured) gels. HthHM/Exd is the homeodomain-less (HthHM) isoform of Hth incubated with Exd. HthFL/Exd is the Hth isoform with a homeodomain, incubated with Exd. Fragments tested with the WT Hth binding site and a mutated form. (s) EMSA for fragment-f with Hth2 mutated (t) additionally with increasing Ubx concentrations. (u) EMSA for fragment-a with Hth1 and 2 mutated. (v) EMSA for fragment-a and fragment-b with Hth3 and Hth4 mutated. (w) EMSA for fragment-a, fragment-c, and fragment-d. (x) EMSA for fragment-e with Hth1 mutated. (y) EMSA for fragment-g with Hth2 mutated. Scale bars, 100 μm. Embryos are matched to scale.

Extended Data Fig. 6 ∣. The effects of Ubx affinity on morphology.

a, b, Schematic output from NRLB shows predicted binding affinity for Exd::Ubx heterodimers across the E3N sequence, where black peaks are on the 5′ strand and red peaks respectively on the 3′ strand. Affinity plots are shown for Drosophila melanogaster (a) and Drosophila virilis (b). c, d, Drosophila cuticle preps for flies with WT E3N driving svb cDNA (c), or E3N with increased Ubx binding affinity driving svb cDNA (d). Trichomes were counted within a region of interest (teal box) defined by anatomic epithelial sensory cells (*). Arrows and brackets demarcate ectopic trichomes. e, Boxplots comparing trichome numbers in the A1 segment in the region of interest from panels (c) and (d) (n = 13, P < 0.02), see also Tsai et al., 2018. In box plots, centre line is mean, upper and lower limits are standard deviation and whiskers show 95% CIs. Scale bars, 25 μm each.

Extended Data Fig. 7 ∣. Extensive pleiotropic effects across the E3N enhancer.

a, b, Plot comparing the percent of lines with pleiotropic or ectopic expression versus the number of mutations based on antibody staining (a) and Beta-Galactosidase staining (b). c–j, A subset of mutants with pleiotropic effects. (c) Line 145-2 drives ectopic expression in the developing wing and haltere discs (7/7 embryos). (d) Line 139-6 drives wider stripes and increased expression, as well as ectopic expression between the stripes and (e) on the dorsal side, (5/5 embryos). (f) Line 40-8 drives a split stripe pattern, where the middle row of nuclei within the stripes is not active (6/6 embryos). (g) Line 93-4 expression varies along the anterior-posterior axis (5/5 embryos). (h) Line 77-9 drives ectopic expression in the salivary glands (5/5 embryos). (i) Line 81-7 drives expression in the developing mouth hooks (5/5 embryos.) (j) Line 15-2v activates expression at stage 10 and drives ectopic expression throughout the embryo in multiple developmental stages (14/14 embryos). k, Plot of footprinting scores versus E3N sequence. Magenta is the footprinting score (σ_i, see methods). The higher the peak, the higher probability that a mutation will change expression. Gray plots are the mutation coverage for the number of lines screened per base (M_i, see methods). l, EWAC scores represent p-values from a log of odds ratio test for the association of a mutation changing expression. Dashed lines denote p- and q-values, respectively. See Materials and Methods. m, Plot comparing the percent of lines with changed expression for mutations in the overlapping Pan/Hth site. n, Quantification of the staining intensities in the stripe and naked domains with the indicated reporter construct using the “sliding window” technique (Extended Data Fig. 3e). N = the number of embryos per line measured, from left to right: 10, 10, 7, 9, 10, 7, 10, 6, 10, 8, 4, 8, 10, 10, 10. In box plots, centre line is mean, upper and lower limits are standard deviation and whiskers show 95% CIs. Scale bars, 100 μm. Embryos are matched to scale (c – j).

Extended Data Fig. 8 ∣. Cuticle preps from 60 Drosophila species across approximately 100 million years of evolution.

a, Phylogenetic tree of Drosophila species studied here, spanning approximately 150 million years of evolution. Red indicates a loss of trichomes. b, Representative cuticle preps for Drosophila species. See also Fig. 4. Scale bars, 25 μm each.

Fig. 1 ∣. Most nucleotide mutations in E3N alter gene expression.

a, Subset of E3N mutagenesis library and schematic of the reporter construct used for integration of enhancers into the Drosophila genome (Extended Data Fig. 1). b, The liquid-handling robot (Extended Data Fig. 2). c, Percentage of lines with no expression plotted against number of mutations per line. d, Lines were scored as 1 (positive) or 0 (no visible expression defects; see Methods). e, Footprinting scores plotted along the E3N sequence. Magenta line, footprinting score (σ_i, see Methods). Higher peaks show a higher probability that a mutation will change expression. Grey histogram, number of mutations per base for the screened lines (M_i, see Methods). f, EWAC scores represent P values (t-test, two-tailed) from a log of odds ratio test for the association of a mutation with changing expression (see Methods). g, Examples of fly embryos with single-mutant E3N::lacZ reporter constructs. WT, wild-type; numbers are line ID numbers. h, Schematic of possible changes to expression outputs. i, Top, nuclear intensity changes associated with single mutations compared to wild-type E3N (n = 212 nuclei; 8 embryos). Mean ± s.d.; ***P < 0.01, two-tailed t-tests. See Methods for sample sizes. AU, arbitrary units of fluorescence intensity. Bottom, Changes in expression output for the single mutations (Extended Data Fig. 3). j, k, Pearson correlations between mutation effect sizes and PhyloP scores for 27 (j) and 124 species (k) with least squares linear regression, and R² values. g, Scale bar, 100 μm; embryo in h matched for scale.

Fig. 2 ∣. Mutational scanning identifies a Hth binding site associated with a changed evolutionary phenotype.

a, Ventral abdominal segments from lines with mutations in an Hth2 binding site (left) and individual cell-staining intensities (right). b, Sequences of Hth2 binding site in lines tested. c, Conservation of E3N in Drosophila, highlighting conserved binding sites (coloured areas). d–g, Embryos bearing E3N::lacZ constructs in a D. melanogaster background, stained with anti-β-galactosidase. d, D. melanogaster (D. mel.) wild-type E3N::lacZ reporter construct. e, D. melanogaster E3N::lacZ reporter construct with a mutated Hth2 site. f, Drosophila virilis E3N::lacZ reporter construct. g, D. virilis E3N::lacZ reporter construct with rescued Hth2 site (5′-TGACAA). h, Single-cell quantification of staining intensities in embryos bearing indicated constructs. D. vir., D. virilis. Magenta crosses, mean; green squares, median. Two-tailed t-test; ***P < 0.01, NS not significant (n = 50, 10 embryos). Left to right: P < 0.01, P = 0.34, P < 0.01. i, j, Cuticle preparations for D. melanogaster (k) and D. virilis (l) showing ventral regions. Scale bars, 50 μm. Teal rectangle highlights trichomes that are absent in D. virilis. f, Scale bar, 100 μm; embryos in d–g matched for scale.

Fig. 3 ∣. Ubx mutations often simultaneously change levels, timing, and locations of E3N expression.

a–d, Embryos carrying E3N::lacZ reporter constructs stained with anti-β-galactosidase. a, b, Wild-type E3N::lacZ at stages 15 and 14. c, d, E3N::lacZ construct with increased Ubx affinity at stages 15 and 14. e, Measurements of stripe (intra), naked (inter-stripe), and anterior (ectopic) nuclear expression by wild-type E3N::lacZ and Ubx high-affinity E3N::lacZ. Centre line, mean; upper and lower limits, s.d.; whiskers, 95% confidence intervals (CIs). Two-tailed t-test, ***P < 0.01 (n = 50, 10 embryos). f, Change in expression between mutant lines and wild-type, comparing total Ubx affinity (shades of green and circle size), anterior intensity, stage 14 intensity, and stage 15 intensity. Inset, model for Ubx affinity and linked traits. a, Scale bar, 100 μm; embryos in a–d are matched for scale.

Fig. 4 ∣. E3N enhancer architecture may constrain evolution.

a, Ventral views of wg-deficient (wg^CX4) cuticle preparations. b, Top, model of wg expression in naked cuticle regions that represses svb expression through Pan (Tcf) activity. Bottom, schematic of the E3N enhancer denoting binding motifs for Ubx, Hth, and Pan. c, d, Embryos carrying E3N::lacZ reporter constructs stained with anti-β-galactosidase; c, wild-type; d, 97-3::lacZ line (Extended Data Fig. 4). e, Nuclear expression in naked and stripe regions for a subset of Pan mutation lines. Centre line, mean; upper and lower limits, s.d.; whiskers, 95% CIs. Two-tailed t-test; ***P < 0.01, NS not significant (n = 50, 10 embryos) (Extended Data Fig. 7). f, Changes in intensities compared to wild-type in inter- and intra-stripe regions with anterior expression. Inset, model of the relationship between Pan affinities and effects on multiple traits. g, Phylogenetic tree of Drosophila species (including Hirtodrosophila pictiventris) examined here noting losses (red) of trichomes in the naked region (there were no examples of gains). h–j, Cuticle preparations from D. navojoa (h), D. fraburu (i), and D. munda (j) (Extended Data Fig. 8). Scale bars, 100 μm (c, d); 25 μm (a, h-j).