Human Molecular Genetics Advance Access originally published online on May 10, 2006
Human Molecular Genetics 2006 15(13):2031-2037; doi:10.1093/hmg/ddl123
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Sonic Hedgehog, a key development gene, experienced intensified molecular evolution in primates
1 Department of Human Genetics, Howard Hughes Medical Institute, 2 Committee on Genetics, University of Chicago, Chicago, IL 60637, USA and 3 Center for Reproduction of Endangered Species, Zoological Society of San Diego, CA 92112, USA
* To whom correspondence should be addressed. Tel: +1 7738344393; fax: +1 7738348470; Email: blahn{at}bsd.uchicago.edu
Received May 2, 2006; Accepted May 3, 2006
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ABSTRACT |
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Sonic Hedgehog (SHH) is one of the most intensively studied genes in developmental biology. It is a highly conserved gene, found in species as diverse as arthropods and mammals. The mammalian SHH encodes a signaling molecule that plays a central role in developmental patterning, especially of the nervous system and the skeletal system. Here, we show that the molecular evolution of SHH is markedly accelerated in primates relative to other mammals. We further show that within primates, the acceleration is most prominent along the lineage leading to humans. Finally, we show that the acceleration in the lineage leading to humans is coupled with signatures of adaptive evolution. In particular, the lineage leading to humans is characterized by a rampant and statistically highly non-random gain of serines and threonines, residues that are potential substrates of post-translational modifications. This suggests that SHH might have evolved more complex post-translational regulation in the lineage leading to humans. Collectively, these findings implicate SHH as a potential contributor to the evolution of primate- or human-specific morphological traits in the nervous and/or skeletal systems and provide the impetus for additional studies aimed at identifying the primate- or human-specific functions of this key development gene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Primates and especially humans show a number of morphological features that set them apart from other species. Many of these, such as enlarged brain, upright posture, opposable thumb and reduced face, pertain to evolutionary changes in the nervous system and the skeletal system. It is therefore reasonable to postulate that development genes involved in the patterning of the nervous and skeletal systems might have played important roles in the evolution of primate- or human-specific phenotypes (1,2).
The mammalian SHH gene encodes a signaling molecule that plays a central role in the developmental patterning of the nervous and skeletal systems (3). In particular, SHH has been shown to play critical roles in the patterning of the brain, the spinal cord, craniofacial elements, the axial skeleton, limbs and digits (3), all are structures that have undergone salient morphological changes during the evolution of primates, especially humans.
During embryonic development, SHH is expressed from a highly restricted set of cells in the notochord, the neural tube and limb buds. The protein product of SHH undergoes autocatalytic cleavage to generate an N-terminal peptide known as SHH-N. The cleavage reaction is coupled with fatty acylation on both ends of the peptide. The acylated SHH-N is then secreted from source cells and acts as a diffusible signaling molecule that drives the morphogenesis of downstream cell (4,5). The autocatalytic activity of SHH resides entirely within the carboxyl portion of the protein known as SHH-C (5). This domain's critical function is evidenced by the fact that humans heterozygous for loss-of-function mutations in SHH-C suffer from holoprosencephaly, a condition characterized by severely reduced brain size, fusion of the two forebrain hemispheres and defects of craniofacial elements (6). The protein sequence of the SHH-N signaling molecule is extraordinarily conserved among mammals, and even between mammals and arthropods. In contrast, the SHH-C autoprocessing domain is much more divergent among taxa. In this study, we examine the molecular evolution of SHH, focussing on the region encoding SHH-C. We show that the rate of non-synonymous changes is significantly accelerated in primates, especially along the lineage leading to humans. We further show that the pattern of sequence changes is consistent with adaptive evolution. Our data thus suggest that SHH may have contributed to the evolution of primate- or human-specific morphological features in the nervous and/or skeletal systems.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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SHH evolution in diverse mammalian orders
The rate of protein sequence evolution as scaled to neutral mutation rate is commonly estimated by the ratio of non-synonymous (Ka) to synonymous (Ks) substitution rates. We first calculated the Ka/Ks ratio of SHH-C in catarrhine primates by comparing orthologous sequences between human and the Old World monkey (OWM) macaque. We then examined the Ka/Ks ratio in four non-primate mammalian orders: rodents (by comparing mouse and rat orthologs), artiodactyls (sheep and cow), perissodactyls (zebra and rhinoceros) and carnivores (cat and dog). We also examined New World monkeys (NWM) (also known as platyrrhines, which is a primate clade more basal to catarrhines) by comparing squirrel monkey and owl monkey orthologs. We found that among these six pairs of species, Ka/Ks is by far the highest in catarrhine primates (Fig. 1A). The rate difference between catarrhines and other mammals is statistically highly significant (P<0.002 by two-tailed Fisher's exact test), whereas the differences among other mammals are not significant. These results strongly suggest that the rate of protein sequence evolution of SHH-C is significantly accelerated in catarrhines relative to other mammals.
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We next performed a sliding-window analysis to investigate whether the acceleration in catarrhines is concentrated within specific subregions of the gene or is more uniformly distributed. This analysis identified a 5' subregion where Ka/Ks is exceptionally high only in catarrhines but is near background level in all other mammals (Fig. 1B). Furthermore, in this subregion, the catarrhine Ka/Ks exceeds 1, which is suggestive of positive selection (7), though this in itself is not conclusive evidence of positive selection. Despite its high rate of evolution, this subregion is clearly functionally important for human development because missense mutations within the subregion result in holoprosencephaly (6). There is also a 3' subregion that shows high Ka/Ks in most species, indicating its high variability across taxa (Fig. 1B).
SHH evolution within primates
To address whether the rate acceleration in catarrhines occurred predominantly on the human branch or the OWM branch, we used the two NWM species as outgroups to assign human-OWM nucleotide substitutions onto either branch. Notably, the acceleration in catarrhines is almost entirely attributable to the human branch (Fig. 1A). The Ka/Ks ratio of the OWM branch is only moderately higher than that of non-catarrhine mammals. In contrast, Ka/Ks of the human branch is four to 10 times greater than any of the other species examined, a disparity that is statistically highly significant (P<0.001).
Next, we obtained SHH-C sequences in additional primates representing key positions of the primate phylogeny, including four apes (chimpanzee, gorilla, orangutan and gibbon) and a prosimian (lemur) (for alignment, see Supplementary Material, Fig. S1). Using all available primate sequences, we constructed two phylogenetic trees: one depicting the rate of synonymous substitutions (Fig. 2A), the other depicting the rate of non-synonymous changes (Fig. 2B). We found that synonymous substitution rates are roughly comparable across various branches with modest levels of fluctuation, although the OWM branch appears somewhat higher than the other branches, which may be due to higher mutation rates in OWM as previously reported (8). In contrast, non-synonymous rates vary among branches in a systematic way, with the lineage from the last simian ancestors to humans showing significantly more substitutions than other branches. This pattern reflects an ongoing evolutionary acceleration along the lineage leading to humans. Two analyses were performed to further investigate this acceleration. First, Ka/Ks was calculated for each branch of the primate phylogenetic tree (Fig. 2B). It showed that branches with the highest Ka/Ks values all reside along the lineage leading to humans (highlighted in Fig. 2B). In particular, the internal branch between the last catarrhine ancestors and the last ape ancestors has a Ka/Ks ratio much greater than 1, which suggests (though does not prove) positive selection. Secondly, we performed a series of pairwise sequence comparisons, with each pair consisting of human and another primate. Using the next closest primate as the outgroup, we assigned base substitutions between human and the other primate onto either evolutionary branch. This revealed that, regardless of which primate was compared with human, Ka/Ks is consistently higher in the branch leading to humans than the branch leading to the other primate (data not shown). Collectively, these observations indicate that within the primate phylogenetic tree, the amino acid replacement rate of SHH-C is specifically and dramatically accelerated in the lineage from the last simian ancestors to humans.
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Rampant and statistically highly non-random gain of serines/threonines in the lineage leading to humans
The classical and most commonly used method for detecting positive selection is to examine whether the rate of amino acid changes in a gene exceeds the neutral expectation. Another approach is to ask whether the types of amino acid changes are highly non-random relative to the neutral expectation (9). To this end, we inspected all the amino acid replacements in SHH-C that have occurred within primates. Surprisingly, among 13 replacements between the last simian ancestors and modern humans, eight resulted in the addition of either serines or threonines, residues that are potential substrates of covalent post-translational modifications (discussed subsequently), yet none led to a loss of serine or threonine (Fig. 2C).
This observation appears highly non-random. To assess it more quantitatively, we performed two statistical analyses. First, we assumed a model where non-synonymous changes in SHH-C are assumed to be selectively neutral; we then calculated the probability that 13 non-synonymous substitutions in the gene would result in a gain of eight or more serines/threonines (see Materials and Methods). This analysis showed that the observed gain of serines/threonines is a significant departure from the neutral expectation (P<0.0001). In the second analysis, we compared SHH-C with empirical data. In the lineage from the last catarrhine ancestors to humans, there are 10 amino acid replacements resulting in a gain of six serines/threonines. Given the availability of whole-genome sequences of human, macaque and mouse, we were able to examine the gain or loss of serines/threonines for a large number of genes in the lineage from the last catarrhine ancestors to humans. Using 2939 genes that contained at least six amino acid replacements in said lineage, we obtained the empirical distribution of rates by which genes gained or lost serines/threonines (Fig. 3). SHH-C is a clear outlier in this distribution (P<0.001), demonstrating that its rampant gain of serines/threonines is highly non-random even when compared with empirical data. We further note that the only other primate branches in Fig. 2C for which SHH-C has gained or lost any serines/threonines are the OWM branch (gaining one serine and one threonine, but losing two serines at other positions) and the NWM branch (gaining one serine). Additionally, none of the amino acid replacements in SHH-C between the two NWM species used in this study (i.e. squirrel monkey and owl monkey) involves serine or threonine (data not shown). Thus, the rampant gain of serines/threonines is a distinguishing feature of SHH-C evolution in the primate lineage leading to humans. The significant departure of this finding from the neutral expectation and from empirical observations of other genes provides a compelling argument that positive selection is responsible for driving the gain of serines/threonines.
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Glycosylation and phosphorylation of serines or threonines are well-attested means of post-translational regulation (10,11). Computational analysis of the eight serines and threonines added in the lineage from the last simian ancestors to humans suggests that four are potential glycosylation sites and three are potential phosphorylation sites (Supplementary Material, Fig. S1). Given that SHH is a secreted protein, glycosylation may be the more likely of the two possibilities (10). We note that SHH-C in mouse and chicken has indeed been shown to undergo glycosylation (12). Additionally, an in silico three-dimensional structure constructed for partial human SHH-C polypeptide revealed that the sites of serine/threonine additions appear in regions sterically accessible to potential modifications (data not shown). Finally, we note that a human point mutation known to impair SHH function and cause holoprosencephaly reside immediately upstream of an added serine (6) and that the mutation is predicted to impair glycosylation of this serine (Supplementary Material, Fig. S1). These observations corroborate the possibility that SHH has evolved more complex post-translational modifications in the lineage leading to humans.
Human polymorphism at the SHH locus
One signature of recent positive selection is depressed polymorphism with an excess of rare alleles at the affected locus (13). To assess whether SHH shows such a signature, we sequenced a 25 kb region encompassing the gene in a panel of 42 humans (84 chromosomes) from diverse populations. The heterozygosity (
) of the 10 kb transcribed region of SHH is 0.0004 (0.0005 if coding sequence is excluded), much lower than the genome average of 0.0008–0.001 (14–17). Tajima's D of the same region is –0.9 (–0.7 if coding sequence is excluded). Negative Tajima's D indicates an excess of rare alleles relative to the neutral expectation, and similar to low heterozygosity, is also a signature of recent positive selection (18). Thus, the values of both heterozygosity and Tajima's D in the transcribed region of SHH are suggestive of positive selection of this gene during recent human evolution. To investigate this possibility further, we performed a sliding-window analysis of heterozygosity and Tajima's D along the 25 kb region surrounding the SHH locus (Fig. 4). We found two regions of particularly pronounced depression for both of these parameters: one corresponding to exon 3 which encodes the entire SHH-C and the other to intron 1 which contains important enhancer elements (19). These depressions are outliers relative to a set of neutrally evolving genomic regions recently surveyed in a population panel comparable to ours (16) (Fig. 4). Although these depressions do not provide definitive proof of positive selection in light of the complex demographic history of humans (20), they are nevertheless suggestive of recent selective sweeps that resulted from positive selection operating at the SHH locus (13).
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Molecular evolution of other members of the Hedgehog family
SHH is one of three genes in the mammalian Hedgehog family. The other two are Indian Hedgehog (IHH) and Desert Hedgehog (DHH). We obtained IHH and DHH sequences in human, macaque, mouse, rat and several other primate and non-primate species. We found that these two genes are highly conserved across these various species (data not shown). Thus, the accelerated evolution of SHH in the primate lineage leading to humans appears to be a distinct feature of this member of the Hedgehog family.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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In this study, we showed that the molecular evolution of the key development gene SHH (more specifically, its autoprocessing domain) is dramatically accelerated in primates relative to other mammals. Within primates, the acceleration is most pronounced along the lineage leading to humans. Particularly striking is the observation that the lineage leading to humans is characterized by a rampant and statistically highly non-random gain of serines and threonines. Finally, certain polymorphism features at the SHH locus in humans, such as depressed heterozygosity and Tajima's D, are consistent with signatures of recent positive selective sweeps.
We argue that, collectively, these observations are indicative of positive selection rather than relaxed functional constraint based on the following reasons. First, the loss of one functional copy of SHH in humans results in profound developmental abnormalities of the brain and craniofacial elements (6). In comparison, losing one copy of SHH in mice has no detectable phenotype (21). These observations are consistent with the critical importance of SHH in human development and do not support functional relaxation. Secondly, as shown in Figure 1C, the higher rate of SHH evolution in primates is largely due to a localized rate increase within a small subregion of the gene, where Ka/Ks exceeds 1 in primates but is very low in all other mammals examined. This argues against a gene-wide relaxation of constraint, and instead suggests adaptive evolution within this subregion. The functional importance of this subregion in human development is further supported by the fact that missense mutations in this subregion result in holoprosencephaly (6). Thirdly, the majority of amino acid replacements along the primate lineage leading to humans results in the addition of serines or threonines, residues that can potentially be modified by glycosylation or phosphorylation. Statistical test demonstrated that such a rampant gain of serines and threonines is a significant departure from the neutral expectation; it is also an outlier when compared with empirical data collected from a large number of genes in the genome. The highly non-random nature of this amino acid replacement pattern is clearly inconsistent with relaxed constraint (which should result in a much more random replacement pattern), and instead supports the functional significance of these replacements. The dramatic increase in the number of serines/threonines in SHH-C in the evolutionary lineage leading to humans suggests that human SHH-C may be subject to more complex post-translational modifications, which in turn may lead to more complex regulation of SHH-N production. Interestingly, serine/threonine replacements in the homeotic gene Ubx were recently shown to be critical for the evolutionary modification of the insect body plan (22). Finally, the polymorphism patterns of the human SHH locus are suggestive of recent positive selective sweeps at this locus. In their totality, these multiple lines of evidence argue compellingly that SHH has experienced adaptive evolution in the primate lineage leading to humans.
SHH is centrally involved in the patterning of multiple tissues, most notably the nervous system (including the brain and the spinal cord) and the skeletal system (including craniofacial elements, the axial skeleton, limbs and digits) (3). Its accelerated and highly unusual patterns of molecular evolution in the lineage leading to humans may therefore bear relevance to the emergence of certain primate- or human-specific features in the nervous and/or skeletal systems, a possibility that is potentially testable in future studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Sequence acquisition and analysis
Genomic DNA was obtained from the following species: common chimpanzee (Pan troglodytes), lowland gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), white-handed gibbon (Hylobates lar), crab-eating macaque (Macaca fascicularis)*, Bolivian squirrel monkey (Saimiri boliviensis)*, owl monkey (Aotus spp.)*, black and white ruffed lemur (Varecia variegata variegata)*, sheep (Ovis aries)*, cow (Bos taurus)*, domestic cat (Felis cattus)*, domestic dog (Canis familiaris)*, Grant's zebra (Equus burchelli boehmi)* and northern white rhinoceros (Ceratotherium simum cottoni)*. Total RNA was also extracted from brain specimens where available (indicated by asterisks) and primed by random hexamers or poly(T) oligomers to synthesize first-strand cDNA. Standard PCR conditions were used to amplify SHH from genomic DNA (100–500 ng per 30 µg reaction) and cDNA (0.5–5 ng per reaction). PCR primers were initially based on human SHH sequence until species-specific sequence was obtained. All sequencing was performed using standard dye-terminator chemistry on PCR products. SHH sequences of human (Homo sapiens; GenBank accession no. NM_000193), mouse (Mus musculus; GenBank accession no. NM_009170) and rat (Rattus norvegicus; GenBank accession no. NM_017221) were obtained from NCBI databases. Nucleotide sequences were aligned in-frame using the Megalign program in the DNASTAR software package (DNASTAR, Madison, WI, USA). In multiple-species alignments, the ancestral sequence was deduced using parsimony. The Diverge function from The Wisconsin Package v10.2 (Accelrys Inc., San Diego, CA, USA) was used to analyze evolutionary divergence (i.e. number of synonymous and non-synonymous substitutions and Ka and Ks rates) according to Li's method (23). Similar results were obtained using the PAML method (24). For sliding-window analysis of Ka/Ks ratio, Ka was calculated for window size of 90 and sliding increment of three base pairs, and Ks of the entire region was used as denominator to avoid problems associated with stochastic variation that sometimes leads to division by zero.
Statistical analysis
To assess the statistical significance that the Ka/Ks ratio of one lineage is distinct from that of another lineage, the numbers of synonymous and non-synonymous substitutions corrected for multiple hits and transition/transversion bias were calculated for both lineages using Li's method (23) as implemented by The Wisconsin Package v10.2. The resulting four values were placed in a 2x2 contingency table and assessed for statistical significance by the two-tailed Fisher's exact test.
To assess the statistical significance associated with the gain of serines and threonines along the primate lineage leading to humans, a putative SHH-C sequence for the last simian ancestors was constructed by parsimony. A codon usage table was then generated from this sequence. For each codon, the probability of a non-synonymous substitution resulting in a serine, threonine or some other residue was calculated. The probability that none of the 13 non-synonymous substitutions occurred on an ancestral serine or threonine was approximated as:
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S/T) is the average probability that a non-serine/threonine residue would mutate to serine or threonine and P(XX) is the average probability that a non-serine/threonine residue would mutate to another non-serine/threonine. The probability that of the 13 non-synonymous changes, none would fall on a ancestral serine or threonine but eight or more would result in a change to serine or threonine was calculated as the product of the two equations.
Prediction of functional sites
Likely glycosylation and phosphorylation sites were identified by online prediction engines at http://www.cbs.dtu.dk/services/NetOGlyc and http://www.cbs.dtu.dk/services/NetPhos, respectively, based on neural network algorithms as previously described (25,26).
Analysis of human polymorphism
Contiguous double-stranded sequence was obtained from 42 human individuals (84 chromosomal copies) for 25 kb centered around the SHH locus on human chromosome 7q36.3. These individuals were chosen from the Coriell Human Variation Panels to represent a diverse selection of world-wide populations (six North Africans, six South Africans, six Chinese, six Russians, three Basques, three Iberians, three Pacific Islanders, three Andeans, three Southeast Asians and three Southwest Asians). Sequence chromatograms were generated by the ABI 3700 sequencer (Applied Biosystems, Foster City, CA, USA) and aligned by the Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA). Polymorphism was detected by direct inspection of sequence chromatograms as displayed by Sequencher. A sliding-window analysis of nucleotide diversity () and Tajima's D was conducted using the DNAsp software (27), with window size of 2500 bp and sliding increment of 50 bp. This window size was chosen to be compatible with a previous human polymorphism survey (16), which was used in this study as a reference. Similar results were obtained with window size of 1000 or 2000 bp. A sliding-window analysis of sequence conservation between human and mouse was done with the VISTA software (28), with window size of 200 bp and sliding increment of 100 bp.
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SUPPLEMENTARY MATERIAL |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Supplementary Material is available at HMG Online.
Conflict of Interest statement. None declared.
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