Evolution acts on enhancer organization to fine-tune gradient threshold readouts

Abstract

The elucidation of principles governing evolution of gene regulatory sequence is critical to the study of metazoan diversification. We are therefore exploring the structure and organizational constraints of regulatory sequences by studying functionally equivalent cis-regulatory modules (CRMs) that have been evolving in parallel across several loci. Such an independent dataset allows a multi-locus study that is not hampered by nonfunctional or constrained homology. The neurogenic ectoderm enhancers (NEEs) of Drosophila melanogaster are one such class of coordinately regulated CRMs. The NEEs share a common organization of binding sites and as a set would be useful to study the relationship between CRM organization and CRM activity across evolving lineages. We used the D. melanogaster transgenic system to screen for functional adaptations in the NEEs from divergent drosophilid species. We show that the individual NEE modules across a genome in any one lineage have independently evolved adaptations to compensate for lineage-specific developmental and/or genomic changes. Specifically, we show that both the site composition and the site organization of NEEs have been finely tuned by distinct, lineage-specific selection pressures in each of the three divergent species that we have examined: D. melanogaster, D. pseudoobscura, and D. virilis. Furthermore, by precisely altering the organization of NEEs with different morphogen gradient threshold readouts, we show that CRM organizational evolution is sufficient for explaining changes in enhancer activity. Thus, evolution can act on CRM organization to fine-tune morphogen gradient threshold readouts over a wide dynamic range. Our study demonstrates that equivalence classes of CRMs are powerful tools for detecting lineage-specific adaptations by gene regulatory sequences.

Figure 1. Identification and Characterization of NEE-Driven Loci in Diverse Drosophilid Genomes

(A) NEE sequences in many Drosophila genomes share a bipartite Su(H)/Dorsal motif and one or two pairs of linked Dorsal and CA-core E-box [E(CA)] motifs. (B) NEE sequences are found at diverse and unrelated loci in dipteran genomes. Other species shown are Anopheles gambiae and Tribolium castaneum. Gray boxes indicate loci; X indicates that a locus is not found; NEE indicates that the locus contains an NEE. My, million years. (C–E) Endogenous expression of the NEE-bearing rho loci is depicted for D. melanogaster (C), D. pseudoobscura (D), and D. virilis (E) stage 5 embryos. All images are lateral views of embryos with anterior pole to the left and dorsal side up. (F) Measurements of the width of lateral stripe of expression in number of nuclei at 50% egg-length at relative stage 5 cellularizing embryos is shown for four NEE-bearing loci in all three species.

Figure 2. Configurations of D. melanogaster, D. pseudoobscura, and D. virilis NEE Sequences

Sequences of NEE cis-elements described in this study were aligned from the vnd, rho, vn, brk, and sog loci from D. pseudoobscura (top aligned sequence), D. melanogaster (middle aligned sequence), and D. virilis sequences (bottom aligned sequence). Particular details (circled numbers) are discussed in the text. Dorsal motifs are shown in blue, Twist CA-core E-boxes are depicted in green, and Su(H) motifs are depicted in red. Overlap between Dorsal and Su(H) motifs are depicted in purple. Only the regions containing these motifs are shown. Ellipses (“…”) indicate intervening sequences that are not shown. See Table S1 for the full-length sequence.

Figure 3. Analysis of NEE Activities in Transgenic D. melanogaster Embryos

(A–D) NEE-driven lacZ transgenes assayed in D. melanogaster embryos from the brk (A), vn (B), rho (C), and vnd (D) loci demonstrate that D. virilis enhancers tend to drive broader stripes than D. melanogaster enhancers. Similarly, D. melanogaster enhancers tend to drive broader stripes than D. pseudoobscura enhancers. (E) Quantification of the widths of lateral stripes of expression at 50% egg length across multiple embryos from several lines supports this general trend. The in situ staining experiments in this figure were conducted in parallel and with the same anti-sense lacZ probe to facilitate comparisons.

Figure 4. Determination of Dorsal/Ventral Borders of Expression of NEE-Driven Transgenes

Fluorescent double-labeling in situ hybridization experiments were carried out using probes for snail, which marks the mesoderm (purple), and lacZ, which is driven by the indicated enhancers in the lateral regions of the embryo (green). All enhancers span D/V expression domains that abut the sharp snail border in the mesoderm and continue to more dorsal nuclei. (A) D. melanogaster sim mesectodermal enhancer (MEE) driving lacZ shows that NEE driven lacZ expression (B–F) enters the mesectoderm because both NEE and MEE activities equally abut the mesodermal border. (B) D. melanogaster rho NEE driving lacZ. (C) D. melanogaster vein NEE driving lacZ. (D) D. melanogaster vnd NEE driving lacZ. (E) D. melanogaster sog NEE driving lacZ. (F) D. melanogaster brk NEE driving lacZ.

Figure 5. Fluorescent Double-Labeling of rho NEE-Driven Transgenes

(A–C) Double-staining (lacZ and snail expression in green and purple, respectively) for rho NEE driven transgenes from D. pseudoobscura (A), D. melanogaster (B), and D. virilis (C) and snail (sna) expression in stage 5 embryos. The sharp snail border of expression provides a landmark to align expression patterns across embryos. (D) All three enhancers drive expression patterns of similar intensity although the D/V axis although the width of the stripe is narrower for D. pseudoobscura, and wider for D. virilis than the D. melanogaster NEE driven transgene.

Figure 6. Precise Changes in NEE Organization Determine Lineage-Specific Threshold Readouts of Morphogen Gradient

(A–E) Minimal modification of the D. melanogaster brk NEE configuration so that it resembles the D. virilis spacing (A) is sufficient to expand expression to levels seen for the D. virilis brk NEE-driven transgene (B–E). Asterisk (*) indicates that the spacing has been mutated in an otherwise wild-type D. melanogaster brk NEE. (F–J) A series of minimal modifications to the D. melanogaster vein (vn) NEE configuration (F) so that it differs by −1 bp, 0 bp (wild-type), +1 bp, and +2 bp, which is similar to the broadly expressed D. melanogaster sog NEE configuration, yields a series of monotonically increasing widths for lateral stripes of expression (G–J). The in situ staining experiments in this figure were conducted in parallel and with the same anti-sense lacZ probe to facilitate comparisons.

Figure 7. Fluorescent Double-Labeling of brk NEE Driven Transgenes

(A–L) Double-staining (lacZ and snail expression in green and purple, respectively) for brk wild-type and mutated enhancers from D. melanogaster and D. virilis. (A) D. melanogaster brk NEE driving lacZ. (B) D. virilis brk NEE driving lacZ. (C) D. melanogaster brk NEE deletion mutant (*) driving lacZ (as in Figure 4) is depicted. The spacing between both pairs of Dorsal and Twist sites has been adjusted to resemble D. virilis brk NEE spacing (see Figure 6A). (D) The D. melanogaster brk enhancer with adjusted Dorsal–Twist spacing drives lacZ expression over a similar width and at similar intensities to the D. virilis brk enhancer. For a wide-range of signal intensity thresholds, the width of the stripe is greater in these enhancers with optimal spacers is much greater than the wild-type D. melanogaster brk NEE. (E) The same data as in D normalized to peak intensity shows that there is still a measurable difference in stripe width in embryos carrying enhancers with optimal brk Dorsal–Twist configuration.