O P I N I O N

The molecular elements that underlie developmental evolution Claudio R. Alonso and Adam S. Wilkins

Abstract | Abundant evidence indicates that developmental evolution, the foundation of morphological evolution, is based on changes in gene function. Over the past decade a consensus has developed that transcriptional regulation, acting through enhancer sequences, is the primary level of evolutionarily significant change. Here we propose that other regulatory levels are probably as important as enhancers in developmental evolution. We also explain why these alternative regulatory levels might have been neglected, and briefly discuss ways to test our hypothesis.

In a now classic paper, Allan Wilson and Marie-Claire King postulated that much of developmental evolution involves changes in gene regulation rather than molecular evolution of protein-coding sequences1. King and Wilson implicitly identified transcrip-tion-level control as the primary site of such changes, echoing earlier and more explicit speculations2. As findings from numerous comparative studies accumulated in the following two decades, the importance of transcriptional change as an accompani-ment and driver of organismal evolution was amply confirmed3–6. Unsurprisingly, the idea that the evolution of development largely reflects the evolution of transcrip-tional regulation has been widely accepted7.

In particular, a great deal of attention has focused on enhancers, rather than on transcription-factor genes, promoter core elements or other transcriptionally relevant elements (see below) as the primary

site of transcriptional evolution7–10. In addi-tion, mutations that affect enhancers have been interpreted as the principal component of “evolutionarily relevant mutations”10 and, indeed, much evidence is consistent with that view11–13. In effect, the belief in the centrality of enhancers as the substratum of developmental evolution is built on two dis-tinct, although not always explicitly stated, assumptions. The first is that other levels of molecular regulation, what one might call ‘alternative regulatory levels’ (ARLs) have been of either negligible or secondary impor-tance in developmental evolution. The second is that the primary sites of origination of sig-nificant mutational change in developmental evolution are the enhancer sequences.

In this article we critically examine both assumptions. First we show that, on a variety of grounds, ARLs are expected to be just as effective as enhancer sites in producing muta-tional resources for developmental evolution. We then discuss two possible reasons for the relative neglect of ARL mutational events as such resources. In particular, we present argu-ments that initial genetic changes at different ARLs might either be enhanced or replaced by subsequent molecular changes at other regulatory levels. These secondary changes might include alterations at similar or other ARLs, as well as changes at the transcriptional level, mainly as a consequence of selection for either a stronger or more specific effect in the regulation of that gene. The net effect would be the conversion of initial changes at ARLs into secondary ARLs and enhancer-dependent transcriptional changes. In principle, such

derived changes might well obscure the initial selected changes that altered specific molecular elements within developmental circuits.

Possible approaches to test and explore these somewhat heterodox views are dis-cussed. Should they be validated by sub-sequent work, the results will enlarge our perspectives on developmental evolution in general and, more specifically, on its genetic and molecular underpinnings.

This is an exciting time in the field of developmental evolution: we have a good understanding of many developmental programmes in model organisms, and com-parative genomics and functional assays are progressively becoming applicable to a wider range of species. It is therefore crucial, and opportune, to establish an adequate theoreti-cal framework for studying the mechanisms that are involved in the generation of devel-opmental novelty. We believe that the com-plete spectrum of regulatory mechanisms controlling gene activity has been exploited in full, and not just partially, during animal evolution.

Systems that regulate development The phrase ‘gene expression’ most frequently brings to mind a diagram describing the dogma of molecular biology: the founda-tional view of how genes, made of DNA sequences, are copied into RNA, and how these RNA molecules are translated into proteins14. However, this view of a simplis-tic, unidirectional, linear series of discrete molecular events does not do justice to the many regulatory processes that determine the final functional output from a given gene BOX 1. Gene messages are transcribed, processed in various ways, exported from the nucleus and translated into proteins in a highly regulated manner. The specificity for all these processes is encoded in the form of sequence modules that regulate transcrip-tional kinetics, alternative-splicing profiles, degradation rates, nucleo-cytoplasmic traf-fic, protein localization and many other molecular processes. Therefore, enhancers

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PERSPECTIVES

Chromatin remodellingand memory systems Nuclear dynamics

Function

Regulation by small RNAs

RNA export

Translation regulation

Regulated polyadenylation

RNA degradation

Enhancer regulation

Basal promoterspecificity

Transcription factordegradation

RNA processing

RNA localization

Post-translational modifications(such as phosphorylation, ubiquitylation, SUMOylation and glycosylation) Protein localization

Nucleus

TranscriptionRNA

DNA

RNAsplicing

Intron Exon

Cytoplasm

mRNA

Protein

Translation

represent only a fraction of the modular, gene-specific elements that are able to affect gene activity BOX 1.

From this perspective the high regula-tory complexity of ARLs could provide a rich substrate for the evolution of devel-opment. However, is the modification of gene activity by regulation at ARLs truly relevant for development? And do the properties of particular ARLs make them more suitable for generating evolutionary and developmentally relevant changes in

gene regulation? To address these issues it is useful to first examine the attributes that make enhancers versatile control nodes for changes in developmental gene regulation.

A key feature of enhancers is their potential for modifying gene activities in a gene-specific manner. In addition, their modular structure and the combinatorial nature of their opera-tion make them especially apt for the gen-eration of functional diversity. However, these attributes are not exclusive to enhancers. The examples below highlight the modular and

combinatorial nature of many ARLs and the gene-specific, developmentally specific effects that can result from mutations at these non-transcriptional cis-regulatory modules.

Untranslated regions. Take for example the modules in the UTRs of mRNA molecules. UTR-dependent modulation of translation efficiency is essential for many developmen-tal functions. An instructive example is the mouse orthodenticle-related gene 2 (Otx2), which encodes a homeodomain-containing transcriptional regulator that controls brain morphogenesis15–17. When the structure of the 3′ UTR of Otx2 is disturbed by artifi-cially generated mutations, transgenic mice that are homozygous for these mutations show severe head abnormalities17. Mutated Otx2 mRNAs are distributed normally in the mouse embryo, but the amount of OTX2 protein is drastically diminished in particular areas of the nervous system. This is the result of the impairment of Otx2 mutated messages to form polyribosome complexes. Notably, alignment of Otx2 3′ UTR sequences from several species revealed a 140 bp cis-regulatory module that is conserved in all vertebrate Otx2 genes18. So, in the mouse, proper brain development requires regulation at a specific ARL, which ensures accurate translational control of Otx2. Interestingly, the degree of conservation of this 3′ UTR module seems to correlate with the level of complexity of vertebrate brains17,18.

Cis-regulatory UTR modules that con-trol translation efficiency are also essential for several other developmental processes, including axis formation in Drosophila melanogaster, the establishment of func-tional neuromuscular junctions, germ-line patterning in Caenorhabditis elegans, and mammalian spermatogenesis19–21.

Alternative splicing. Another powerful exam-ple of modular gene-specific regulation at ARLs is alternative splicing, a central source of protein diversity22. In vertebrates, alterna-tive splicing is actually considered to be the most important source of protein diver-sity23–25, therefore emerging as a significant mechanism for the generation of regulatory diversity.

Although the mechanistic details that underlie alternative splicing are not fully known, the general outline of the process is clear: regulatory factors interact with spe-cific sequences in pre-mRNAs to stimulate or repress exon recognition. These factors bind directly to 5′ or 3′ splice sites, or to regulatory modules that are termed exonic or intronic splicing enhancers (ESEs or ISEs)

Box 1 | A modern view of the levels of regulation that affect gene functions

Many levels of regulation affect the function of developmental genes in an enhancer-independent manner. Active genes are transcribed by complex biochemical machines, the recruitment of which is determined by the interactions between chromatin and modular elements that control transcription (that is, enhancers and silencers). These molecular interactions could be modified by other elements such as insulators. In addition, sequence elements at the core promoter, as well as particular tissue-specific auxiliary factors that participate in the transcription-initiation process, also contribute to determining the kinetics of transcription initiation. Therefore, when we consider transcription initiation, enhancer elements are only one of the many components that influence this process. But this is just the beginning. RNA transcripts are processed, largely while they are transcribed, and the nature of these processing events (for example, splicing, capping, polyadenylation, editing and trans-splicing) will have an enormous effect on the quality and quantity of the resulting mature RNA message. Processed messages are subjected to another regulatory layer by the quality control/degradation systems, and are subsequently exported from the cell nucleus — a process that could also be a further regulatory point. Some messages contain sequence modules that define their particular subcellular localization, where they will await further signals for translation release. Modules that are located in UTRs are able to affect transcript half-life and translation kinetics. Once the gene has been translated into a protein, many subequent regulatory processes will follow, including phosphorylation, glycosylation, ubiquitylation and SUMOylation (the covalent attachment of molecules of SUMO — small ubiquitin-related modifier — to substrate proteins). The rate and rhythm of all these regulatory events that affect protein function and half-life can be modulated in a gene-specific and developmentally specific manner.

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and silencers (ESSs or ISSs). These cis-regu-latory modules stimulate or repress splice-site selection22. In humans, many examples indicate that misregulation of alternative-splicing profiles by alteration of splicing regulatory modules can lead to disease. For example, ~13–20% of all mutations that cause cystic fibrosis affect the splicing of the cystic fibrosis transmembrane conductance regulator (CFTR) by direct destruction of splice-sites or by affecting ESE, ISE, ESS or ISS cis-regulatory modules25. Many of these mutations affect the levels of CFTR mRNA and therefore have a notable impact on the severity of the disease. A study on the human gene encoding the E1α subunit of the pyru-vate dehydrogenase complex (PDHA1) pro-vides another example of the developmental importance of alternative splicing. Here a mutation that creates a new ESE regulatory element leads to severe developmental delay, ataxia and mental retardation26. In another case, several mutations that affect the inter-actions between ESE, ISE, ESS and ISS mod-ules in the human microtubule-associated protein tau (MAPT) gene (which encodes the tau protein) alter the ratios between the dif-ferent MAPT splicing isoforms. These muta-tions lead to severe anomalies in neuronal function and trigger the development of a particular class of dementia (frontotemporal dementia, FTDP17) REFS 25,2729.

We therefore conclude that mutations affecting the operation of cis-regulatory elements that control alternative-splicing profiles can lead to specific developmental and physiological defects. Examples in other systems, including the regulation of the alternative-splicing profile of genes of the D. melanogaster sex-determination path-way30 and the neuron-specific alternative-splicing regulation by the fruitfly embryonic lethal abnormal vision (ELAV) protein31, demonstrate that alternative splicing is a potent ARL that can control specific devel-opmental programmes in a broad range of organisms.

Other levels of non-transcriptional regula-tion. Regulation at other ARLs also con-trols vital developmental processes. For example, the D. melanogaster bantam locus (ban) encodes a developmentally regulated microRNA that controls cell prolifera-tion and regulates the activity of apoptotic genes32. Functioning at the same level of non-transcriptional regulation, the mouse microRNA miR196a negatively regulates the expression of the homeobox gene Hoxb8 REF. 33. In addition, the regulation of the sub-cellular localization of mRNAs has been

shown to be crucial for normal development in several organisms (for a comprehensive review on this topic see REF. 20). Regulation at other less-familiar ARLs, ranging from the site-specific proteolysis of chromatin remodelling factors affecting Hox gene activity34, to the choreographed looping-out of chromatin from chromosome territories and its consequences on the collinearity of Hox gene expression in human cells35, fur-ther illustrate the extent of ARL regulation on developmental gene activity.

Evaluating the impact of ARLsIn principle, enhancer-dependent evolu-tionary change would predominate over that mediated by ARLs if sites for the former were far more abundant than for the latter

at the level of individual genes. This is an argument about relative mutational ‘target sizes’ BOX 2.

Unfortunately, the information to resolve this matter, even for one chosen organism, is still incomplete. Nonetheless, to gain approxi-mate dimensions of both enhancer associated and ARL-associated DNA target sizes, we have compiled information from different genomic databases and other sources, and have established a preliminary estimate. This information is summarized in BOX 2. It is important to consider that for this exercise, we have deliberately not considered many ARL sequences that are target sites for trans-acting ARL molecules (such as phosphor-ylation targets and ubiquitylation targets), or genes that encode trans-acting molecules

Box 2 | An estimate of the mutational sizes of enhancers and ARLs

Here we present our preliminary calculations to estimate the sequence-length of enhancers and alternative regulatory levels (ARLs).

Enhancer-related regulatory levelCalculations of enhancer-related sequences are based on the assumption that an average gene possesses seven independent enhancer modules, each one of them containing ten binding sites for transcription factors5. In turn, each transcription-factor binding site is composed of six base pairs with one base pair being nonspecific for binding6. The calculation is as follows:• Enhancer number = 7• Number of transcription-factor binding sites per enhancer = 10• Length of transcription-factor binding site = 5 bp (6 bp in total, but 1 bp is nonspecific)• Total mutational size = 7 × 10 × 5 bp = 350 bp

ARL-related regulatory levelEstimates for ARL-related dimensions are based on the average sequence length of RNA polymerase II core promoters50, the consensus signal for poly-adenylation (AATAAA), best available estimates for splice-site abundance and length — assuming a gene structure that contains four exons and three introns (see Table 1 in REF. 51). The length of splicing enhancers is considered to be six nucleotides, which is the average size for the binding sites of many RNA-binding proteins52; the model considers the presence of an average of five such enhancers per exon52,53. The length of the untranslated regulatory module has been calculated from an average of the available sequences that are compiled in REF. 20. Specific calculations are as follows:• Basal promoter (for example, TATA box, initiator, DNA-polymerase enhancer motif

(DPE)) = 35 bp• Poly-A signal = 6bp• Length of splicing acceptor (3 bp) × number of splicing acceptors (2) = 6 bp• Length of splicing donor (3 bp) × number of splicing donors (2) = 6 bp• Length of splicing enhancer (6 bp) × number of splicing enhancers (20) = 120 bp• 5′ UTR regulatory modules (for example, internal ribosome-entry site (IRES)) = 30 bp• 3′ UTR regulatory modules (for example, heat shock protein 83 (HSP83), wingless,

β-actin) = 247 bp• Total mutational size = 35 bp + (3 × 6 bp) + 120 bp + 30 bp + 247 bp = 450 bpThese preliminary sequence-length estimates support the hypothesis that enhancers and ARLs have comparable mutational sizes. Except for splicing-enhancer values — which have been calculated for vertebrate sequences52,53 — all values are derived from Drosophila melanogaster resources.

Note that phosphorylation signals, ubiquitylation signals, SUMOylation signals, nuclear retention signals, organelle retention signals, signal peptides and other regulatory signals have not been included in this calculation.

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themselves (such as kinases and ubiquitin). Consensus size estimates were not available for these components. Therefore, this calcula-tion represents a substantial underestimation of the real size of ARLs. Despite these limita-tions, our estimate indicates that ARLs and enhancers have roughly comparable dimen-sions and should, therefore, represent at least comparable sizes for mutation.

Levels of genetic variation at ARLsAs reviewed above, the large number of ARL modes of regulation has been both docu-mented and confirmed by numerous stud-ies involving mutants in D. melanogaster, C. elegans, as well as in many examples in vertebrates and human disease. These studies indicate that it is possible to identify or even induce mutations in key molecular compo-nents that are required for ARL regulation. However, the genetic dissection of ARLs only has evolutionary significance for develop-mental evolution if genetic variants for ARLs occur in natural populations at frequencies that are sufficient to allow the efficient opera-tion of natural selection. This issue can be phrased as a question: are there significant levels of standing genetic variation for ARLs to make them plausible candidates as source materials for evolution? An important point concerns what constitutes the standard for ‘significant’ levels. The appropriate com-parison, in this example, would be enhancer sequences, for which high levels of natural genetic variation have been documented13.

Contribution of cis and trans mutations to regulatory variation. The question above cannot yet be answered for ARLs because the data have not been collected. On the other hand, the idea that all or most sig-nificant genetic variation for developmental evolution resides in enhancer sequences can be tested. This hypothesis predicts that all the genetic variation that is related to expressional differences will be linked in cis to the genes that have variable expres-sion. By contrast, if significant amounts of trans-acting components were to be found in association with expressional variations (so-called expressional quantitative trait loci or eQTLs)36, it would indicate that enhancers are not the sole or even a primary source of regulatory genetic variation.

Several studies along these lines have been carried out and some of the results are summarized in TABLE 1. Although meaning-ful cross-species comparisons are not yet feasible, the clear indication from these stud-ies is that only a small fraction of eQTLs is purely cis-acting. Even in the D. melanogaster

study37, for which most of the variation in a small sample of genes has a cis-acting com-ponent, the largest fraction of this variation is not exclusively cis-acting. Furthermore, because not all the cis-acting mutations are likely to be in enhancers for the reasons given above, the true fraction of regulatory variation that is due to enhancer mutations is bound to be less than the figures given for the cis-acting variants in TABLE 1.

Contribution of transcription factors to regulatory variation. A related question is relevant to how much of the variation reflects genetic alterations in transcriptional capacity directly. Are most or all of the trans-acting variants linked to genes for specific tran-scription factors? This might be expected if enhancer-mediated transcriptional con-trol is truly the most significant level of regulatory evolution. However, the variety and number of gene products involved in regulation at ARLs makes it seem improb-able that transcription-factor genes would be the exclusive domains of mutations in trans-acting components. Although data are still sparse, the most thorough empiri-cal analysis carried out so far indicates that transcription-factor genes are, in fact, a rela-tively small fraction of the total trans-acting variants in eQTLs38.

Natural genetic variability at ARLs. So far there are no comparable studies of population levels of genetic variability for ARLs as have been carried out for enhancers11,13. However, for one specific type of ARL — RNA splicing — there is evidence that genetic variants are abundant in the population. Early estimates indicated that as much as 15% of all human genetic disease variants are due to mutations in splice sites39–41. In addition, as described above, it has become apparent that numerous sites, within both introns and exons, can func-tion as splice-site regulatory modules. When

all these elements are taken into account, the probable figure of human disease genetic variants due to alternative-splicing mutations is probably greater than 15%. In turn, because splicing variation affects all functional classes of genes, developmental defects that are due to mutations at this ARL are observed (see the previous examples).

In conclusion, the evidence summarized above indicates that regulatory genetic varia-tion for ARLs should be abundant in complex multicellular organisms. Such genetic wealth, in turn, makes it hard to see how or why it should not contribute genetic source material for developmental evolution. Furthermore, the indications that only a minor fraction of all of the trans-acting eQTLs in yeast38 are in transcription-factor genes further empha-sizes the potential of ARLs in developmental evolution.

Why have ARLs been overlooked?The conclusions discussed in the previous section seem to present a puzzle. If ARL mutations possess large evolutionary poten-tial, why has this not become apparent in the plethora of comparative studies that have been carried out? Or, to put it another way, why is there comparatively so much evidence for enhancer-dependent alterations in devel-opmental evolution, and so relatively little for significant change through mutations at ARLs? We propose that the answer involves two distinct factors.

Ascertainment bias. Transcriptional regu-lation has been considered fundamental to studies of gene expression since the founding studies that first documented gene regulation42 and, for many years, gene regulation was considered to be virtually synonymous with transcriptional regulation. Correspondingly, comparative studies began focusing on transcriptional-level changes from the early 1970s when it began to be

Table 1 | Analysis of eQTLs with effects in cis and trans

Organism Number of genes

% Cis-only % Trans-only % Cis- and trans- mixed

References

Budding yeast (Saccharomyces cerevisiae)

1,5282,294

3625 (max)

N/DN/D

6475

5438

Human cells 3,554 19 77.5 3.5 55

Drosophila (D. melanogaster vs D. simulans)

29 41 3 55 37

The studies cited here37,38,54,55 involved an analysis of a set of genes for which the sites of expressional differences could be mapped with respect to the gene for which activity has been measured. Genes in which the entire expressional differences were tightly linked to the gene in question were classified as cis-only. For two of the studies38,54, a pure trans effect could not be discriminated from a combination of cis and trans genetic alterations. eQTLs, expressional quantitative loci; N/D, not determined.

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*

*

t1

t2

t1

t2

2

a

*

b

*

*

1

2

*

Enh 1 Enh 2

Gene A

Gene A

Enh 3

Enh 1 Enh 2 Enh 3

(Weak positive effect)(Slightly negative effect)(Slightly negative effect)

Lost by recombination or segregation

ARL

Gene CGene BGene A

Gene CGene BGene A

Time

Time

Exon 1 Exon 2 Exon 3

Exon 1 Exon 2 Exon 3

ARL*

ARL*

Product of the altered gene A(Weak positive effect)

1

Low levels

High levels

High levels

possible to study the regulation of individual genes at the stages before protein production. The existence of developmental alterations in transcriptional patterns between different organisms confirmed these expectations. This route became progressively more main-stream, despite the existence of seminal work in amphibians and echinoderms indicating the importance of translational regulation in early development43–46. Accordingly, in the past decade, many of the more influential figures in the field of evolutionary develop-mental biology have stressed the importance of alterations at the transcriptional level in evolution5–7. Even between related species quantitative microarray analysis supports the finding of significant differences in tran-scriptional control of both upstream control and downstream target genes47, although the precise degree of functional significance for many of these changes has come into question48. The key point, however, is that transcriptional changes that accompany evolutionary divergence, especially in asso-ciation with enhancer sequences, have been actively sought — and found. By contrast, other types of regulatory change might have escaped notice simply because they were not looked for.

Technical limitations and biases of inter-pretation might also account for the appar-ent invisibility of ARLs. Take for example the case of RNA in situ hybridization, one of the most popular techniques in the field of evo-devo. Many comparative studies that use in situ hybridization interpret different probe patterns as an indication of transcriptional changes in enhancers, openly ignoring the possibility of post-transcriptional events that alter mRNA stability or changes in splicing profiles that affect the sequences detected by (often) a single probe. Even more modern genome-wide expression studies have seri-ous limitations in this context, as they will not necessarily detect functionally significant splicing differences or post-translational modifications.

Enhancer-dependent alterations are second-ary to ARL mutations. The second possible reason developmental evolutionary changes at ARLs have largely escaped attention is more subtle. We propose that many selected transcriptional-level changes that have occurred in evolution might be secondary events, which strengthen and fix, or replace, an initial ARL-mutational effect. In effect, we propose that many initial ARL changes that alter a developmental process and subse-quently rise to high frequency within a popu-lation can be and often are either enhanced

or superseded by later changes. As noted, ARL mutations can occur at both cis-acting and trans-acting sequences. In principle, the supplanting of initial ARL-mutant effects by secondary effects could pertain to both categories of ARL mutations. However, the mechanisms by which the secondary muta-tions would mask or replace the primary ones would necessarily differ between mutations on cis-acting and trans-acting ARLs.

Consider cis-acting ARL mutations first, the type involved, for example, in splice-site changes or 3′ UTRs. Such changes are likely to be highly context-specific, that is, they will function in a gene-specific and devel-opmentally specific manner, exerting their primary effects on the gene in which they occur. However, any gene experiencing a cis-acting ARL mutation that created a positively selectable difference would be unlikely to be expressed at optimal levels for all tissues or sites in which the gene was expressed. In effect, a weak positive effect would create selection

pressure for a stronger effect, mediated by either a gene-specific up- or downregulatory change in gene expression. For changes in either direction, ARLs might be involved. But here we also need to allow for changes at the enhancer level, because mutational alteration of enhancers can also produce gene-specific expression changes, either as increases or decreases in transcription rates7,9,10. The process visualized here of the phenotypic effect of a favoured cis-acting ARL being strengthened by a subsequent gene-specific, context-specific ARL, or by a transcriptional event (mediated by an enhancer mutation), is shown in FIG. 1a. An interesting feature of this process is that the original ARL mutation should be retained, and might well be found in the clade in which the selective effect was first manifested. Detecting it, however, would require an accurate phylogenetic reconstruc-tion of where the significant regulatory change occurred, as well as the appropriate molecular methods to detect it.

Figure 1 | Hypotheses for the evolution of regulatory mutations at ARLs. Two ways in which an initially selected alternative regulatory level (ARL) mutation (indicated by an asterisk can be hidden or superseded by a secondary mutation, which affects transcriptional or other regulatory processes. a | A cis-acting ARL, exemplified by a new alternative-splicing mutation, occurs at time t1, and affects an exon of a gene (in this example, exon 1). The new mutation creates a new gene product and the resulting phenotype is favoured by natural selection. However, the new gene product is unlikely to be expressed at the optimal level. Selection pressure would exist for a further mutation to occur at some subsequent time (t2). This secondary mutation could be in another ARL site, in a trans-acting transcription factor or in an enhancer (as shown for enh 2). In any of these cases, the new mutation would adjust the expression of the new gene product to the optimal level. Transcriptional adjustment might be more readily detectable than the original mutation. b | A new mutation in a trans-acting ARL creates a favourable effect on one gene product (for example, by increasing the stability of RNA messages transcribed from gene C) accompanied by lesser selective deficits for other gene products for which expression is affected by this gene product (that is, genes B and C). In this situation, the favourable effect is also unlikely to be simultaneously associated with optimal expression. A secondary mutation that affects transcription, either at one of the enhancers of gene C (as depicted) or in a trans-acting transcription factor affecting gene C expression, could well adjust the expression level to the optimum. However, the original mutation, if genetically unlinked, might be lost through segregation or recombination, eliminating its associated selective deficits.

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What about trans-acting ARL muta-tions? These might occur in components of ARL-processes that act on many genes. However, these can still produce seemingly gene-specific and tissue-specific effects, when combined with other modifications (consider for example, tissue-specific phos-phorylation patterns of splicing factors49). In principle, some fraction of these mutations, perhaps only a small fraction, would have the potential to create positively selectable effects, although such mutant effects might be higher in functions that are primarily inhibitory. However, virtually all such selected mutations would be accompanied by selective deficits as well. Natural selec-tion might promote the effects of the initial mutation because of its preferential effects on one gene’s activity but, at the same time, there would be selection pressure to eliminate the associated negative effects, potentially involving many unrelated genes. If the initial effect were weak but positive, a gene-specific strengthening of expression of the affected gene could be achieved again by a gene-specific cis-ARL, or through an enhancer-mediated effect. Subsequently, the initial trans-acting mutant could be lost through recombination or chromosome segregation without (necessary) loss of the positively selected gene-specific effects. Therefore, for trans-acting ARL mutations, secondary ARL and enhancer mutations are likely to arise at multiple loci, and could, in principle, replace the initial ARL mutation. This situation con-trasts with that of cis-acting selected ARL mutations, which would tend to be retained, as described above. The proposed sequence of events for a favoured trans-acting ARL mutation is shown in FIG. 1b.

Therefore, triggering molecular events at ARLs might lead to secondary regulatory events that involve both ARLs and enhanc-ers. This ‘indirect feeding’ into secondary enhancer alterations by initial changes at ARLs, in combination with the already dis-cussed enhancer-centric views in the field, might simply account for the preponderate description of developmental changes related to enhancer activity in modern comparative studies.

How might these ideas be tested? One possibility is to use the selective power that is inherent in yeast genetics. ARL mutations, either acting in cis or trans, could be created and conditions devised under which their phenotype could be selected in the labora-tory. If the effects were weak, then over time, new, second-site variants should arise that are more strongly selected, showing up as larger colonies. Some fraction of these

should, if our hypothesis is correct, be found to be in enhancer units, which affect gene transcription. Initial cis-acting ARLs would be maintained, whereas initial trans-acting ARLs would be expected, over several sexual generations, to be lost.

But ideally, because these ideas have been advanced with respect to develop-mental evolution, we would also want to test them in multicellular organisms. As most developmental mutants in C. elegans or D. melanogaster are unfit compared with the wild type, it would be difficult to devise selective conditions that would favour devel-opmental novelties over the wild type. For these two experimental animal systems, how-ever, we could start with mutants that show non-lethal developmental defects, select for partial suppressors and identify those that are in ARL functions. If stronger selection for full reversion is applied, second-generation suppressors could be isolated and genetically mapped. If the hypothesis advanced above is correct, a fraction of these mutations should be in enhancer units.

ConclusionsA fundamental belief in modern evolution-ary developmental biology is that enhancer units are the primary sites for developmental evolution. Here we have argued that this view might be misleading and incomplete. In particular, we have examined the evidence that other molecular regulatory processes affecting gene expression would be expected to contribute genetic source material for developmental evolution. We have termed the collective set of other processes alterna-tive regulatory levels or ARLs.

If ARLs have been important in this way, however, why has there not been more evidence for their contributions to developmental evolution? We suggest that one important reason is that because such contributions have not been looked for, they have largely escaped notice. A second possibility is also explored here. Because many ARL mutations would be sub-optimal or even involve selective deficits, an ARL mutant with some favourable evolutionary potential would, in many cases, be subjected to further selective pressure; this, in turn, would select for second-site mutations that accentuate the positive effect(s). In a fraction of cases, these second-site mutations would be expected to be in enhancer units. The nature of the original ARL mutation would, however, largely predetermine whether it was retained or eliminated by these subsequent selection events. In either case, however, a secondary ARL or enhancer-driven change

might supersede an initial ARL-mutational event. We suggest that part of the ‘invisibility’ of ARL-mutation contributions to develop-mental evolution reflects this process and we discuss some general approaches that might be useful in testing these ideas.

We now know that various regulatory mechanisms can strongly affect the nature of the information expressed from developmen-tal genes. We believe that this knowledge must be incorporated in full to the analysis of the genetic basis for developmental evolution.

Claudio R. Alonso is at the Laboratory for Development and Evolution, Department of Zoology, University of Cambridge, Downing

Street, Cambridge CB2 3EJ, UK. Adam S. Wilkins is at the BioEssays Editorial

Office, 10–11 Tredgold Lane, Napier Street, Cambridge CB1 1HN, UK.Correspondence to C.R.A.

e-mail: cra21@hermes.cam.ac.ukdoi:10.1038/nrg1676

Published online 10 August 2005

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AcknowledgementsC.R.A. wishes to thank The Wellcome Trust for support, and members of the Laboratory for Development and Evolution

for helpful comments and discussions on the ideas in this manuscript. The authors wish to thank the comments of two anonymous referees, which have contributed to improving the quality of this manuscript.

Competing interests statementThe authors declare no competing financial interests.

Online links

DATABASESThe following terms in this article are linked online to:Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=geneban | CFTR | Hoxb8 | MAPT | Otx2 | PDHA1OMIM: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIMcystic fibrosis

FURTHER INFORMATIONClaudio Alonso’s web page: http://www.zoo.cam.ac.uk/zoostaff/members/alonso.htmlAccess to this interactive links box is free online.

E S S AY

Early American genetics journalsJames F. Crow

Abstract | Before the Second World War, there were only two North-American journals exclusively devoted to genetics — the Journal of Heredity and Genetics. In the late 1940s, Genetics spawned two progeny — the American Journal of Human Genetics and Evolution. This article recounts the early days of these journals, their influential and often colourful founding editors, and their contents. It emphasizes the contrast between those years, when a reader had a realistic chance of keeping up with the whole field, and the current plethora of journals that makes it impossible to keep up with even the tables of contents.

Sewall Wright once said that when he entered graduate school in 1912, and for many years after, he read every article in the field of genetics. What a contrast to the pres-ent, when one despairs of even scanning the tables of contents. In the early days, there was only a handful of journals, the pages were smaller and fewer than those today, and the issues appeared less frequently.

Of course, Wright was unusual. He had been introduced to genetics by read-ing Reginald Punnett’s famous article on Mendelism in the 1907 edition of the Encylopedia Britannica1. In addition to an extensive acquaintance with biology, Wright had a working knowledge of mathematics and

enzymology and was therefore in a much better position than most biologists to keep up with the field of genetics. Another person who kept up with all of the current literature was the even broader British geneticist, J.B.S. Haldane. Like Wright, he was inter-ested in enzymes and wrote a book on the subject2.

The first volume of the leading North-American journal Genetics, published in 1916, had 22 articles. At the time, the total number of journals on both sides of the Atlantic could be counted on the fingers of one hand. Not every geneticist was like Wright or Haldane, but it is likely that a reasonably conscientious reader could keep abreast of the field by scanning three or four articles a week.

Until after the Second World War, there were only two North-American journals devoted exclusively to genetics — the Journal of Heredity and Genetics. In the late 1940s, two journals split off from Genetics — Evolution and the American Journal of Human Genetics. The deluge of new jour-nals came later. Although I focus on North-American journals in this article, similar trends in genetics publishing occurred in Britain and the rest of Europe BOX 1.

In this article, I look at the early years of genetics publishing, focusing on North America. The emphasis here is on the content of the journals and the role of their

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