Evolving spots

Here's what seems to be a relatively simple problem in evolution. Within the Drosophila genus (and in diverse insects in general), species have evolved patterned spots on their wings, which seem to be important in species-specific courtship. Gompel et al. have been exploring in depth one particular problem, illustrated below: how did a spot-free ancestral fly species acquire that distinctive dark patch near the front tip of the wing in Drosophila biarmipes? Their answer involves dissecting the molecular regulators of pattern in the fly wing, doing comparative sequence analyses and identifying the specific stretches of DNA involved in turning on the pigment pattern, and testing their models experimentally by expressing novel gene constructs in different species of flies.

Expression of the Yellow protein prefigures adult wing pigmentation. The conspicuous spot of dark pigmentation present at the tip of the male wing of Drosophila biarmipes (left) is a new trait evolved among species of the Drosophila melanogaster group (about 15 Myr of divergence; divergence time is 60-80 Myr for the family Drosophilidae), superimposed on the ancestral pattern of uniform grey shading and darker veins found both in D. melanogaster and in D. pseudoobscura, a species from the sister D. obscura group (25 Myr of divergence). In all three species the male pupal distribution of Yellow in the wing, revealed by a specific antibody (right), foreshadows the adult pigmentation.

The particular gene of interests is calledl yellow (y), which is required for the production of black pigments (why is a gene for black pigments called yellow? Because genes are often named for their effect when mutated. Break the yellow gene with a mutation, and the resulting mutant animal can't make dark pigments, and looks yellowish.) Yellow is normally turned on at a low level everywhere in the fruit fly wing, pigmenting the wing an overall light gray. In D. biarmipes, there is an additional patch of elevated yellow expression in one corner of the wing. What activates this gene in just that one place?

Cis or trans?

The jargon starts already. Cis and trans are terms that refer to the mechanisms of gene regulation. There are two broad classes of genetic elements that control transcription. One class is the trans elements. These are transcription factors, genes that are transcribed and translated into proteins that can bind to DNA. They are called trans (from the Latin, "across") because the proteins can bind different strands of DNA than the one that contains their own gene, so that mutations to a trans acting transcription factor may be seen as changes in the levels of activity of multiple other genes.

Cis is also Latin, meaning "on this side". The cis elements are regulatory regions associated with and in relatively close proximity to a specific gene. They are the spots of DNA to which those transcription factors, and other proteins, may bind. For instance, every gene has a region called the promoter which is located close to the start of the gene, and is the site where the RNA polymerase that copies the gene into a strand of messenger RNA binds. In addition, there are other sites that may be significantly farther away called enhancers and suppressors. When a transcription factor that recognizes the sequence in an enhancer is available in the cell, it binds to it and facilitates and upregulates the activity of the gene. Suppressors, obviously have the opposite effect: when a transcription factor binds a suppressor, it inhibits transcription of the gene.

Each gene has a promoter and multiple enhancers and suppressors. Whether a gene is active or not will depend on the precise mix of transcription factors that are active in the cell. If there are no transcription factors present that bind to the enhancers, or if there are transcription factors that bind to the suppressors, the gene will be quiet. If there are factors floating around in the environment that bind to the enhancers, then the gene will be active.

In the case of the spot in the wing of D. biarmipes, there are a couple of ways that could have evolved. One is that there could have been a change in the expression of trans-acting transcription factors: a gene that turns yellow on could have acquired a new zone of activity. Alternatively, and more economically, the yellow gene could have acquired a new or modified cis regulatory element that makes it more sensitive to an existing transcription factor that is already expressed in the desired pattern. That's more economical, because transcription factors tend to act on many different genes, so changing the pattern of one can have pleiotropic effects. Tweaking the yellow gene alone with a new cis regulatory region is more discrete.

There's a relatively simple way to test whether cis or trans factors have been modified in D. biarmipes. Extract a large region of DNA 5' to the yellow gene, containing all or most of the D. biarmipes cis regulatory elements. Connect that to a reporter gene, in this case Green Fluorescent Protein, or GFP. Then inject that into a D. melanogaster embryo. What will happen?

If the spot is generated by differences in D. biarmipes trans factors, the reporter gene should be expressed in a pattern similar to the D. melanogaster pattern, that is, uniform and at low level, because only the D. melanogaster trans factors are present. If the spot is generated by unique cis elements, the D. biarmipes reporter construct in the D. melanogaster environment should go ahead and produce a spot on the wing. And here is the result:

The entire 5' region of D. biarmipes y, comprising sequences between the coding sequences of y and the closest predicted gene (CG3777), is sufficient to drive reporter GFP expression in D. melanogaster at a time and in a pattern similar to those of y expression in native D. biarmipes.

A 5' D. biarmipes yellow regulatory region produces a D. biarmipes-type wingspot in D. melanogaster. That suggests that D. biarmipes has acquired some new cis element somewhere within the large chunk of DNA the researchers extracted.

Where is the regulator?

The next step was to narrow down the area of interest from the whole 5' chunk of cis elements to the smaller, specific region responsible for invoking yellow expression in just the spot. The diagram below illustrates the subset of the cis region that they examined.

In the top 3 figures, for D. melanogaster, D. pseudoobscura, and D. biarmipes, the solid line represents the DNA strand, while the thick black bars on the right are the exons of yellow—the actual coding regions for the yellow protein. On the left side of each, there is a set of gray boxes. These represent regions of the DNA that were extracted and coupled to a reporter gene for the next experiments. This is fairly straightforward stuff; just break the regulatory region down into smaller and smaller chunks, stick it back into a fly, and look to see which chunk drives gene expression within the region of the spot. One especially cool bit about this experiment, though, is that they also used orthologous chunks from each of the three species, so they are looking for a piece of D. biarmipes DNA that regulates expression in a specific spot, while the comparable pieces from D. melanogaster and D. pseudoobscura do not.

In particular, they identified a region called wing that is important in generating yellow expression in the wing. They pulled out a copy of this region from D. melanogaster (wing^mel), D. pseudoobscura (wing^pse), and D. biarmipes (wing^bia). They also broke each wing region into a left and right piece, and coupled each fragment to a marker gene.

Cis-regulatory changes at the yellow locus are responsible for species-specific differences in Yellow distribution. a, The organization of the y locus is similar in Drosophila melanogaster, Drosophila biarmipes and D. pseudoobscura. Black boxes, coding sequence; grey boxes, fragments analysed in transgenic constructs.

Next step: take each of those fragments+marker, inject them into the developing D. melanogaster, and ask where the existing network of trans-acting regulatory factors turn them on. And presto, here are the results.

The cis-regulatory sequences governing spot formation evolved in the context of an ancestral wing enhancer. a, Conservation of the wing element sequence between D. biarmipes (bia) and D. melanogaster (mel) or D. pseudoobscura (pse) determined by Vista 47 with a 10-base-pair window length; only conservation above 75% is shown as solid boxes. Arrows show the boundaries of the left and right fragments. b, Reporter expression driven by the orthologous wing elements and its subfragments left and right (columns) of D. melanogaster (top), D. biarmipes (middle; the wing bia large element is shown) and D. pseudoobscura (bottom), all expressed in D. melanogaster. The ubiquitous expression driven by the outgroup species wing pse element (expression is present in vein cells at a lower levels comparable to those in left pse) shows that the sequences responsible for the spot pattern in D. biarmipes have evolved in the context of an ancestral wing regulatory element. The sequences controlling the spot pattern are separable from those controlling general expression in D. biarmipes (left and right). Note that the posterior boundary of activity of the left bia construct lies near or at the anterior-posterior compartment boundary.

In the left column, the wing element from all of the species is turned on all over the wing; the wing^bia element alone also turns on most strongly in the anterior tip.

The middle column shows what just the left element does. Left^bia is turned on in only the anterior spot.

The right column shows the pattern of expression of just the right element, and in all species (except D. melanogaster) it triggers diffuse, weak expression all over the wing.

The interpretation is that in D. biarmipes, the wing cis regulatory element has become more specialized, where one part (right) mediates general wing expression, while another part (left) responds to signals in the wing to elevate expression in just the pigment spot.

What it all means is illustrated in the diagram below. The authors propose that there is a "conserved wing regulatory landscape"…that is, there is an overlapping array of transcription factors (the red and the green areas) that impose a constant frame of reference for the drosophiliid lineage. Individual genes can acquire new cis regulatory elements that respond to that environment in a logical way. For instance, the yellow gene of Drosophila biarmipes has an enhancer that is bound by the red transcription factor, and a suppressor which is bound by the green transcription factor. It has in effect added a little logical module that says, "turn on yellow if the red factor is present, and the green factor is not," which then produces the localized patch of wing pigment.

Cryptic prepatterns and the evolution of novel gene expression patterns through the evolution of cis-regulatory sequences. a, The upper panel shows a model of the conserved landscape of transcriptional regulators that pattern and shape the Drosophila wing (green and pink represent repressor and activator, respectively). The evolution of binding sites for a subset of these regulators in the yellow wing cis-regulatory element (coloured stars) co-opts them to modify yellow expression (lower panel). Combined with other regulatory changes at other loci, the changes at the y locus result in a novel pigmentation spot. b, Wing pigmentation patterns similar to D. biarmipes (left) or D. guttifera (right) evolved independently in other fly families (here Otitidae and Lauxaniidae).

These results explain a couple of things. One is the way some patterns emerge again and again in flies, as in the Euxesta example above, which also develops a wingtip spot like D. biarmipes—they are all building on a common framework of conserved transcription factors and are lighting up homologous domains. Another is how these patterns can so readily emerge: the underlying rules are relatively simple, and require only the swapping in of short DNA sequences into the regulatory regions of a gene to give it a novel and specific pattern of expression.

Gompel N, Prud'Homme B, Wittkopp PJ, Kassner VA, Carroll SB (2005) Chance caught on the wing: cis-regulatory evolution and the origin of pigment patterns in Drosophila. Nature 433:481-487.