The most popular conceptualization for developmental evolution is the “genetic toolkit.” Genes in the toolkit, primarily transcription factors and signaling molecules, are master regulators that govern the formation and patterning of bodies and body parts (Carroll 2005a). The finding that toolkit genes were common among diverse organisms gave rise to an apparent paradox, how did diverse body plans arise if the set of genes used to build them is the same? The solution to the paradox appears to be that developmental evolution results from changing the location and time that toolkit genes are deployed. According to the cis-regulatory view of developmental evolution, these changes in the deployment of toolkit genes results from changes in their regulatory sequences. Thus, where and when tools are used varies but the tool remains functionally the same—a hammer is a hammer. However, a growing body of data is revealing that toolkit genes have evolved novel functions, suggesting that although the developmental toolkit is common to diverse organisms, it evolves in response to each organisms functional needs—a hammer is a hammer, but it may be a mallet, a sledgehammer, a ball-pen or able to pull nails (Fig. 7). According to this view of developmental evolution, changes in gene regulation result from both changes in the regulation of toolkit genes and their functional specificities. In the remainder of this section we will highlight some examples of how novel functions emerge in toolkit genes and how the mechanisms of reducing pleiotropy discussed above have been used to facilitate functional divergence.
EVOLVING TOOLS: DOMAIN SHUFFLING
At the highest of biomolecular levels, proteins are organized into discrete structural and functional domains, which are generally defined as self-stabilizing and independently folding regions of a protein chain (Darby and Creighton 1995; Voet et al. 1999). Structural domains are generally more than 200 amino acids in length, although some such as the MADS and Hox domains are much smaller, and often are composed of smaller structural motifs. Consequently, domains are structurally and functionally independent modules. Domains have discrete activities such as catalyzing biochemical reactions and mediating molecular binding with other proteins, peptides, ligands, and nucleic acids (Voet et al. 1999). Domain structures are often well conserved across diverse organisms, often despite dramatic divergence in primary (amino acid) sequence. Because they are structurally and functionally semiautonomous units, domain swapping between proteins is common during evolution. A fascinating example of domain swapping recently been reported by Adamska and colleagues (2007). These authors have shown that the hedgehog (Hh) protein evolved in metazoans by exon shuffling between a hedge-domain containing protein ancestrally involved in intercellular communication and a hog/intein-domain containing protein, into a single gene (Fig. 8). They infer that the autocatalytic activity of the hog/intein-domain allowed the release of the hedge ligand, allowing long-range cell–cell signaling that was later coopted for complex morphogenetic patterning (Adamska et al. 2007).
Figure 8. The Metazoan Toolkit Gene Hedgehog (Hh) evolved by domain shuffling. The intein-related hog domain (black) predates the origin of the Metazoa, is auto and is associated with many proteins (white). The hedge-domain (red) arose in the ancestor of sponges and eumetazoans, and was originally part of Hedgling (red, pink, and blue). Early in metazoan evolution, domain shuffling resulted in the emergence of the conventional Hh, composed of the hedge-domain and the more ancient hog/intein-domain. The autocatalytic activity of the hog/intein-domain (black arrow) in Hh could have allowed the release of the hedge ligand and hence longer range signaling that could be used to control more complex morphogenetic patterning. Reprinted from Current Biology, 17(19), Adamska et al., The evolutionary origin of hedgehog proteins. Copyright (2007), with permission from Elsevier.
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EVOLVING TOOLS: SLiM SWITCHES
Large domains are burdened by severe structural constraints imposed by the need to fold into a stable structure and function correctly (Darby and Creighton 1995). Although many PPIs are mediated by contacts between secondary structural motifs and domains, a growing number of interactions are being identified that are mediated by short linear motifs or SLiMs (Neduva and Russell 2005). The key feature of linear motifs is their small size, usually 3–10 residues long with only two or three required to mediate the interaction, and low binding energies leading to weak interactions, thus they tend to be the primary mediator of transient interactions such as ligand docking, and the assembly of enhanceosomes and the basal transcription apparatus (Neduva and Russell 2005). In addition, SLiMs most often occur in poorly structured regions of proteins, with more than 85% of known motifs located in disordered regions, indicating they are relatively free from structural constraints (Neduva and Russell 2005, 2006; Fuxreiter et al. 2007). This feature of SLiMs is particularly advantageous because it reduces the number of potentially structurally deleterious mutations in SLiMs, thus minimizing intramolecular pleiotropic effects of amino acid substitutions.
SLiMs are particularly evolvable, their small size and lax sequence specificity means that functional linear motifs more easily appear and disappear than domains and structural motifs (Neduva and Russell 2005). Just a single mutation is often enough to convert a nonfunctional stretch of amino acids into a functional SLiM, giving them a high degree of evolutionary plasticity (Neduva and Russell 2005). Correspondingly, SLiMs are poorly conserved compared to domains even though the same kinds of motifs are used across diverse organisms. In a recent review Neduva and Russell (2005) examined the conservation of experimentally determined linear motifs across eukaryotes and found that although domain architecture was well conserved, linear motifs were poorly conserved between lineages. Because of their small size, linear motifs are also likely to evolve in unrelated proteins convergently: 1 in 20 proteins contain the SH3 binding-motif RxPxxP (Neduva and Russell 2005). Thus, SLiMs are a large source of potential interactions that can be coopted into existing regulatory or interaction networks leading to novel effects, such as acquisition of novel cofactors and target genes. These features of linear motifs, that is small size, evolutionary plasticity, and rapid turnover rates, has led them to be considered “evolutionary interaction switches” (Neduva and Russell 2005).
Perhaps the most dramatic example of a functional change in a transcription factor is the Drosophila Hox/HOM gene fushi tarazu (Ftz). Although Ftz from primitive insects functions as a homeotic gene, Drosophila Ftz has lost all homeotic functions and functions in segmentation instead (Lohr et al. 2001). To determine how Drosophila Ftz evolved from a homeotic gene into a novel segmentation gene, Löhr et al. (2001) ectopically expressed Ftz from different species in fruit flies to assess their potential to cause homeotic transformations and regulate segmentation. Although Ftz from the basal insect lineages Tribolium and Schistocerca possessed homeotic functions, for example, repressing hth and causing transformations of antenna toward leg, the Drosophila gene lost its homeotic function and only had segmentation potential.
Remarkably, although Drosophila Ftz is solely segmental, Ftz from Schistocerca and Tribolium had extremely weak and moderate segmentation potential, respectively, suggesting that the switch from homeotic to segmentation function occurred in stages (Lohr et al. 2001). This change is dependent on the ability of Ftz to interact with Ftz-factor 1 (Ftz-F1) at the nuclear receptor SLiM (LXXLL), which is present in Drosophila and Tribolium Ftz but not in Schistocerca (Fig. 9). Conversely, loss of homeotic function in Drosophila Ftz is dependent on loss of the Extradenticle (Exd) interaction SLiM YPWM upstream of the Ftz homeodomain; the YPWM motif is present in both Schistocerca and Tribolium (Fig. 9). Löhr et al. (2001) proposed a stepwise model to explain the evolution of the novel segmentation function of Drosophila Ftz: Ancestrally, all insect Ftz genes had homeotic functions dependent on the Exd interaction motif YPWM. Sometime after divergence of the Drosophila-Tribolium lineage from Schistocerca, the Ftz-F1 interaction motif evolved. Ftz in this intermediate stage had both a segmentation function, dependent on interaction with Ftz-F1, and homeotic functions dependent on interaction with Exd. Subsequent loss of the Exd interaction motif in the stem-lineage of Drosophila produced an Ftz with only segmentation functions. Thus, the evolution of a novel Ftz function was dependent upon the gain and loss of small linear motifs that mediate PPIs (Fig. 9).
Figure 9. Evolution of a novel segmentation function in Drosophila Ftz dependent on the gain and loss of Short Linear Motifs (SLiMs). The insect Ftz was ancestrally a homeotic gene, dependent on interaction with Exd at the hexapeptide motif (YPWM). In the stem-lineage of beetles and flies (Endopterygotoa) a novel SLiM (LXXLL) originated in Ftz that mediated an interaction Ftz-F1. During this intermediate stage Ftz had both homeotic function (dependent on the YPWM motif) and segmentation function (dependent on the LXXLL motif). In the Drosophila lineage, the YPWM motif was lost resulting in loss of homeotic function in Ftz.
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A particularly well-studied example of transcription factor functional divergence is the insect gene Ultrabithorax (Ubx). Ubx is a homeotic (HOM/Hox) transcription factor expressed in the third thoracic (T3) segment of insects and is necessary for proper development of T3 appendages such as hindwings in butterflies and beetles and halteres in fruit flies (Weatherbee et al. 1998, 1999; Tomoyasu et al. 2005). Averof and Akam (1995) have proposed that the insect body plan evolved from a crustacean-like plan in two phases: restriction of Ubx and AdbA expression to the proto-abdominal region followed by acquisition of repressive activities in Ubx and AdbA that suppressed thoracic-type limbs in the abdomen. Although the first stage of this transition is dependent on alterations in Ubx and AdbA expression domains (Averof and Akam 1995), the second stage could involve changes in the coding regions of Ubx and AdbA or their cofactors, the cis-regulatory regions of Ubx and AdbA or some combination of these changes.
Grenier and Carroll (2000) compared the activity of Ubx from the Onycophoran velvet worm (Acanthokara kaputensis) and fruit fly (Drosophila) using in vivo misexpression studies. The similarity of Ubx from these two species is practically nonexistent outside the highly conserved DNA-binding homeodomain, with much of the dissimilarity because of indels. In spite of these differences both proteins were able to transform antenna into legs and the forewing into a halter by repressing srf in the wing disc and activating dpp in the visceral mesoderm, respectively. The velvet worm Ubx, however, was unable to produce all the phenotypes of fly Ubx misexpression. The velvet worm gene, for example, did not transform thoracic cuticle into abdominal cuticle nor did it repress Dll expression in the leg rudiments of larval Drosophila, both typical effects of Drosophila Ubx. Homeodomain swap experiments confirmed that the results were not because of differences in their homeodomains, indicating the fly-specific activities of Ubx were not the result of differences in DNA binding. Thus, functional divergence was not dependent on cis-regulatory elements in Drosophila Ubx target genes and was likely because of differences in the ability of Drosophila and velvet worm Ubx genes to form PPIs required for normal fly Ubx function. This interpretation was supported by functional mapping that identified an insect-specific QAQAQK(A)n motif (QA-motif) C-terminal to the homeodomain in Drosophila Ubx that played a role in Dll repression and was able to confer limb repression activity when grafted onto velvet worm Ubx (Fig. 10) (Galant and Carroll 2002).
Figure 10. Evolution of a novel leg repression function in insect Ubx. The insect thorax is made of three segments, the prothorax (T1), mesothorax (T2), and the metathorax (T3). Each thoracic segment has a pair of ventral appendages (legs) and may have dorsal appendages (wings and wing derivatives). Averof and Akam (1995) have proposed that the insect body plan (Fly and Beetle) evolved from a crustacean-like plan (brine shrimp) in two phases: restriction of Ubx expression (shown in blue) to the proto-abdominal region followed by acquisition of repressive activities in Ubx that suppressed Dll expression and limbs in the abdomen (Ubx shown in red). Ancestrally, the cryptic Dll repression function of Ubx was itself repressed by amino acids downstream of the homeodomain (HD), expansion of the QA-motif in the stem-lineage of Hexapods uncovered the repression motif in the amino-terminal region of Ubx allowing Dll repression and repression of abdominal appendages likely via the recruitment of a new co-repressor (shown in green) or stabilization of other cofactor associations. Wild-type crustacean (brine shrimp) Ubx weakly represses Dll expression. Note that Ubx expression in the velvet worm is restricted to the extreme posterior region of the final body segment. Ubx is expressed in all thoracic and abdominal segments of crustaceans (brine shrimp), Ubx expression in Hexapod insets (Fly and Beetle) is restricted to the abdomen and T3 and is weakly expressed in the posterior end of T2.
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Similar results were found by Ronshaugen and colleagues (2002) by dissecting functional differences between Ubx from Drosophila and brine shrimp (Artemia). Ronshaugen and colleagues found the Dll repressor domain to be N-terminal to the homeodomain, and the QA-motif to be a facilitator of the repressor domain. Although the brine shrimp Ubx lacks the QA-motif and only has mild repression function, addition of a QA-motif in combination with experimental removal of several phosphorylation sites transforms the Artemia Ubx into strong repressor of leg development in Drosophila larva. Based on these results, Ronshaugen et al. (2002) proposed the gain of the QA-motif and the loss of serine/threonine phosphorylation sites in the common ancestor of insects uncovered a cryptic Dll-repression function in Ubx. Coupled with the restriction of Ubx expression to the posterior trunk, this novel function of Ubx contributed to the evolution of the insect body plan (Fig. 10).
Does the evolution of novel linear motifs affect global gene expression patterns, and therefore suffer the consequences of negative pleiotropy? Two recent studies of Ubx suggest they have few effects (Hittinger et al. 2005; Merabet et al. 2007). Hittinger et al. (2005) investigated the function of the QA-motif by deleting it in Drosophila using allelic replacement. Curiously, the deletion of the QA-motif (UbxΔQA) did not have an effect on Dll repression in the abdomen of UbxΔQA/UbxΔQA flies nor was it strongly pleiotropic in most other tissues. However, manipulating the dose of Ubx and AbdA (which is functionally redundant with Ubx with respect to Dll repression in the abdomen, Fig. 3) uncovered a crucial role for the QA-motif in imparting full Dll repression activity on Ubx and revealed only slightly more pronounced pleiotropic effects (Hittinger et al. 2005). Hittinger and colleagues (2005) concluded that the QA-motif was required for only a subset of Ubx-regulated developmental processes, and that the differential pleiotropy observed for the QA-motif might allow selection to alter development of characters with minimal pleiotropic fitness trade-offs.
In a similar study, Merabet and colleagues (2007) examined the ability of Drosophila Ubx to physically interact with Exd and drive expression of Ubx target genes. These authors found that mutation of the UbdA motif (UbxUbdA) dramatically reduced binding with Exd and the ability of Ubx to repress Dll, but that mutation of the hexapeptide motif (UbxHX) had no effect on the interaction with Exd or Dll repression. Although the UbxUbdA mutant lost its ability to repress Dll in the abdomen, it had no effect on the repression of the Ubx target genes spalt, Blistered/dSrf, and vestigial in the haltere. Unexpectedly, the UbxUbdA, UbxHX, and the double mutant UbxUbdA/HX all retained their ability to activate decapentaplegic, a well-characterized Ubx-Exd target gene. These results demonstrate that mutation of Ubx protein–protein interaction motifs do not have globally deleterious effects on Ubx target gene expression because different cofactor associations are used to regulate distinct sets of target genes and because Ubx-cofactor associations are tissue specific, which prevents widespread effects of mutations.
EVOLVING TOOLS: SSR KNOBS
In one of the earliest studies to demonstrate that homopolymeric amino acid repeats were functional, Gerber et al. (1994) showed that stretches of glutamine and proline could activate transcription when fused to the DNA-binding domain of the GAL4 transcription factor. In vitro, activity increased with repeat length, whereas in cell transfection assays maximal activity was achieved by 10–30 glutamines and ∼10 prolines. These authors proposed that homopolymeric amino acid stretches may be the main cause modulating transcription factor activity, but this suggestion has received little attention in developmental evolution.
Amino acid repeats are extremely rare in prokaryotes, however, in eukaryotes glutamine, asparagine, and alanine repeats are fairly common (Faux et al. 2005). Interestingly, polyglutamine repeats are common to both vertebrate and invertebrate proteins whereas polyasparagine repeats were rare in vertebrates (Faux et al. 2005). Mar Albà and Guigó (2004) analyzed repeat content in a large set (7039) of human–mouse–rat orthologs and found that a high proportion of repeats were species specific. Only 52% of mouse genes and 46.5% of rat genes had repeats conserved with human genes. Among human-specific repeats, polyalanine was most common whereas among rodent-specific repeats polyglutamine was most common (Alba and Guigo 2004).
SSRs are particularly abundant in proteins that regulate gene expression and evolve rapidly, yet few studies have examined the roles of SSRs in molecular or morphological divergence. In an elegant study of SSR variation in 17 developmental genes between 92 dog breeds, Fondon and Garner (2004) found high levels of tandem repeat variation and evidence that repeats were driven to fixation in breeds by selection. Although most of the variation between genes were small changes in repeat length, usually two or three amino acids, five genes had large expansions or contractions in SSRs, including Six-3, HoxA-7, Runx-2, HoxD-8, and Alx-4.
Although the function of most of these repeats is not known, previous developmental and biomedical studies in mice and humans suggested that mutations in Runx-2 could have phenotypic effects. The glutamine/alanine-repeat (QA-repeat) in Runx-2 is correlated with morphological divergence between closely related species, particularly the degree of dorsoventral nose bend (clinorhynchy) and midface length in dogs and other carnivores (Fondon and Garner 2004). Runx-2 regulates the rate and timing of bone development such that up regulation leads to acceleration and extension of bone development and down regulation leads to deceleration and truncation of bone development. These results suggest mutations that alter the activity of Runx-2, and not just its expression, may play a role in bone development. Opposing effects on transcriptional activity have been reported for polyglutamine and polyalanine repeats: polyglutamines have been observed to drive transcription (Gerber et al. 1994) and polyalanines to repress transcription in a length-dependent fashion (Briata et al. 1997; Brown and Brown 2004). Indeed, deletion of QA-repeat has previously been shown to dramatically reduce the transactivation function of Runx-2, with the glutamine stretch bearing the activation function and the alanine stretch possessing repression activity. Sears et al. (2007) specifically tested if variation in Runx-2 QA-repeat lengths altered its transcriptional activity assays using Runx-2 target gene reporter assays. The transcriptional activity of Runx-2 increased as the ratio of glutamines to alanines increased, thus expansion and contraction of the QA-repeat modulated Runx-2 transcriptional activity (Fig. 11). To our knowledge, this is the first experimental demonstration that SSRs actively contribute to normal developmental variation and not just disease (Stephens 2006). Interestingly, previous studies suggest that SSRs may adopt unique tertiary structures that are particularly well suited for intermolecular interactions (Hicks and Hsu 2004; Cubellis et al. 2005).
Figure 11. Simple sequence repeats in the Runx2 gene are correlated with morphological evolution in dog skull shape (A, B) and transcriptional activity (C). The ratio of glutamines to alanines is positively correlated with the degree of dorsoventral nose bend (clinorhynchy) in purebred dogs (A). Purebred bull terrier skulls from 1931 (top), 1950 (middle), and 1976 (bottom). Analysis of the Runx2 repeats in the 1931 bull terrier revealed a more intermediate allele than in the contemporary bull terrier. Data in A and B are from Fondon and Garner (2004). The ratio of glutamines to alanines in Runx2 is positively correlated with transcriptional activity as assayed in a β-reporter assay driven from the Col10 promoter (Sears et al. 2007). The size of the arrow is drawn in proportion to transcriptional activity. Pie charts indicate the ratio of glutamines (dark gray) to alanines (black), the glutamine/alanine ratio is given below each pie chart. Note that when the glutamine/alanine ratio is 1, transcriptional activity is 1% of wild-type activity (Thirunavukkarasu et al. 1998).
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Do homopolymeric amino acid repeats suffer the consequences of negative pleiotropy? Biomedical studies of repeat expansion diseases suggest that SSRs may have extremely few pleiotropic effects. For example, expansion of a polyalanine repeat in HoxD-13 by 7–14 residues causes synpolydactyly, a dominant developmental limb deformity characterized by duplication of fingers and webbing between fingers (Goodman et al. 1997; Kjaer et al. 2002; Zhao et al. 2007). No other organ or tissue systems are affected in synpolydactyly, suggesting SSR expansions have little effects on the functions of HoxD-13 outside of the autopod. Similarly, a recent study by Anan et al. (2007) found that transgenic mice missing an amniote-specific polyalanine tract in HoxD-13 had only one developmental defect: fusion of a single sesamoid bone in the wrist (Anan et al. 2007).