J. Neurogenetics, 27(3): 8696 Copyright 2013 Informa Healthcare USA, Inc. ISSN: 0167-7063 print/1563-5260 online DOI: 10.3109/01677063.2013.

789512

Review

The Role of Human-Specic Gene Duplications During Brain Development and Evolution
Takayuki Sassa
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

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Abstract: One of the most fascinating questions in evolutionary biology is how traits unique to humans, such as their high cognitive abilities, erect bipedalism, and hairless skin, are encoded in the genome. Recent advances in genomics have begun to reveal differences between the genomes of the great apes. It has become evident that one of the many mutation types, segmental duplication, has drastically increased in the primate genomes, and most remarkably in the human genome. Genes contained in these segmental duplications have a tremendous potential to cause genetic innovation, probably accounting for the acquisition of human-specific traits. In this review, I begin with an overview of the genes, which have increased their copy number specifically in the human lineage, following its separation from the common ancestor with our closest living relative, the chimpanzee. Then, I introduce the recent experimental approaches, focusing on SRGAP2, which has been partially duplicated, to elucidate the role of SRGAP2 protein and its human-specific paralogs in human brain development and evolution. Keywords: brain development, gene duplication, human genome, SRGAP2

INTRODUCTION How the human brain has acquired its cognitive ability through evolution is a fundamental issue in evolutionary biology. The brain is formed through highly coordinated processes, including neurogenesis, synapse formation, and wiring of the neural circuitry. In addition to an increase in size, human brain exhibits a prolonged maturation or neoteny. Neoteny is a key feature that allows our brain to be modied through interaction with the environment and probably underlies the emergence of higher cognitive skills. How can we address the issue of human brain evolution? The genetic program for developing the human brain to acquire the traits that are unique to or most prominent in humans must be embedded in our genome. By taking advantage of the power of comparative genomics, it has become possible to identify the humanspecic sequences or regions in the human genome. The effects of such sequences or regions on brain development can then be tested experimentally utilizing various in vitro and in vivo model systems. The purpose of this review is to emphasize the importance of such an evodevo approach. In the second part, the genes duplicated

specically in the human lineage, identied by comparative genomics, are introduced. In the following part, the analysis of the role of one of such genes, SRGAP2, in brain development, and particularly in dendritic spine maturation, is outlined.

THE ARCHITECTURE OF HUMAN SEGMENTAL DUPLICATION A comparison of human and chimpanzee draft genome sequences estimated approximately 1% sequence divergence between the two genomes (Chimpanzee Sequencing and Analysis Consortium, 2005). Moreover, orthologous proteins are highly similar, typically differing by only a few amino acids, and approximately 29% are identical (Chimpanzee Sequencing and Analysis Consortium, 2005; King & Wilson, 1975). These initial analyses suggest that the differences in gene expression level or pattern, which are coordinated by regulatory regions such as promoters, enhancers, or noncoding RNA, may be important for the phenotypic differences. However, the identication of segmental duplications (SDs) increased the estimate of

Received 26 February 2013; accepted 21 March 2013. Address correspondence to Takayuki Sassa, PhD, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. E-mail: tasasa@pharm.hokudai.ac.jp 86

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total differences between the two genomes to approximately 4% (Britten, 2002; Cheng et al., 2005). Importantly, SDs often contain duplicated genes. Gene duplication underlies the evolution of novel biological functions; this raises the attractive hypothesis that these recently duplicated human lineage genes have signicantly contributed to the evolution of human-specic phenotypic traits. SDs are dened as duplicated DNA fragments that are more than 90% identical and larger than 1 kb (Jiang et al., 2007; Marques-Bonet et al., 2009). Their high degree of conservation has made it challenging to detect, map, and resolve the structure of the human-specic SDs. For example, in the genomes assembled using whole-genome shotgun (WGS) sequencing, SDs are underrepresented in comparison with the genomes assembled using BAC clone-order-based approach. SDs that are larger than 15 kb and have more than 97% homology are almost absent in the genomes assembled using the WGS sequencing (She et al., 2004). WGS sequences are not long enough to nd and assign an overlapping sequence from highly similar sequences during genome assembly. Thus, the recent SDs that are most likely to be relevant to human-specic gene duplications are the most difcult to resolve. To overcome these problems, a computational method that identies SDs in an assembly-independent manner has been developed (Bailey et al., 2002; Marques-Bonet et al., 2009). In this method, WGS sequence reads of nonhuman primates are aligned against a human genome, which is most reliable and used as a reference assembly. SDs are detected as an excess or shortage of WGS sequence read-depth. The method is particularly sensitive for detecting high-identity duplications, and therefore suitable not only for comparisons between humans and nonhuman primates but also for comparisons between human populations to identify disease-associated SDs (Lupski, 1998; Sharp et al., 2006). These studies have revealed several features of human and nonhuman primate SDs. First, large human SDs ( 20 kb) are mostly (66%) separated by 1 Mb of unique sequences (Bailey et al., 2001; Cheng et al., 2005; She et al., 2004). This is in sharp contrast with the mouse, in which the large SDs are predominantly (88%) organized in tandems (She et al., 2008). Second, human SDs distribute nonrandomly in the genome. Most human SDs cluster to form approximately 400 blocks (She et al., 2004). Each block consists of a complex mosaic of different SDs, suggesting that it has been formed by multiple rounds of duplication (Johnson et al., 2006). These blocks map to the euchromatic portions as well as subcentromeric and subtelomeric regions (Linardopoulou et al., 2005; She et al., 2004). Third, intrachromosomal SDs tend to show higher identity than interchromosomal SDs (She et al., 2006). Using this difference in the degree of substitution, it has been estimated that interchromosomal duplication reached its peak around 25 million years ago (mya), when the ancestral human lineage separated from the Old World

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monkeys, whereas intrachromosomal duplication activity peaked around 10 mya and began to decline around 2 mya (She et al., 2006). Global genome-wide comparison of SDs of humans and chimpanzees revealed that, of the human SDs larger than 20 kb, 33% (26.5 Mb) are unique to the human and 66% (53.4 Mb) are also duplicated in the chimpanzee (Cheng et al., 2005; Chimpanzee Sequencing and Analysis Consortium, 2005). The chimpanzee-only SDs and copy number differences between the shared SDs account for the genetic difference between the two species of approximately 2.7%. This difference is larger than the difference (approximately 1.2%) caused by single-base-pair substitutions, supporting the hypothesis that gene duplication underlies the evolution of phenotypic traits unique to humans (OBleness et al., 2012; Varki et al., 2008).

GENES SPECIFICALLY DUPLICATED OR EXPANDED IN THE HUMAN LINEAGE The average length of large human-only SDs ( 20 kb) is 54.6 kb (Cheng et al., 2005). Thus, genes in SD can be duplicated as a whole or partially. In the study mentioned above, the genome-wide comparison of SDs between human and chimpanzee has identied 177 genes duplicated in the human but not in chimpanzee (Cheng et al., 2005). About half of the gene duplications found in the study are partial, although the precise structure and copy number of the duplicated genes remain unresolved. Using array comparative genomic hybridization against human cDNA array, other studies compared the human genome not only with that of chimpanzee but also with the genomes of other nonhuman primates, including bonobo, gorilla, and orangutan (Dumas et al., 2007; Fortna et al., 2004). This approach has identied 134 genes as candidates for human lineage-specic gene duplications (Fortna et al., 2004). The study has found that about half of the genes that have an increased copy number in humans in comparison with chimpanzees have also increased their copy number in other nonhuman primates. This nding highlights the importance of comparing multiple species to identify human-specic gene duplication events. More recently, a sequence read-depth obtained using the next-generation sequencing has been applied to predict the copy number of duplicated genes (Sudmant et al., 2010). The study has compared 159 human genomes with those of a gorilla, chimpanzee, and orangutan, and identied 23 genes with human-specic duplications (diploid in the three nonhuman primates) as well as 30 genes with duplications in the primate lineage and further expansions in the human lineage. A current list of duplicated genes identied by these genome-wide studies is presented in Table 1 (Cheng et al., 2005; Dumas et al., 2007; Fortna et al., 2004; Sudmant

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Table 1. List of genes duplicated or expanded specically in the human lineage.

Gene FCGR1A RBM8A HIST2HBF SRGAP2 NCRNA00152 PTPN20A FRMPD2 GPRIN2 C10orf57 CHRFAM7A ARHGAP11A ARHGEF5 HYDIN GTF2H2 SERF1B SMN2 NAIP DUSP22 GTF2IRD2 FAM115C LOC154761 ZNF322 WASH2P CROCCL1 MSTP2 AMY1A FLJ39739 PDE4DIP NBPF14 NCF1 LOC645166 GOLGA6L10 GIYD1 LRRC37A4 C2orf78 RPL23AP53 LOC728323 POM121L4P ZNF595 DRD5 LOC100272216 GUSBL1 LOC100170939 C6orf41 STAG3L1 GTF2IP1 SPDYE5 AQP7 KGFLP1 FAM95B1 LOC642929 ARHGAP15 NBEA ARHGAP42 NEK2 PMP2 SLC6A13 FGF7 OCLN Chromosome 1 1 1 1 2 10 10 10 10 15 15 7 16 5 5 5 5 6 7 7 7 6 2 1 1 1 1 1 1 7 1 15 16 17 2 8 2 22 4 4 5 6 5 6 7 7 7 9 9 9 9 2 13 11 1 8 12 15 5 Copy number 5.64 3.52 5.49 5.36 3.88 3.68 3.94 4.40 4.06 4.27 3.55 5.94 3.85 4.49 3.60 3.58 5.04 3.98 5.50 3.93 3.94 3.85 20.81 10.23 13.85 9.91 11.83 7.20 244.66 6.43 12.50 29.81 6.90 16.22 10.88 27.08 19.18 50.71 10.63 11.34 63.64 13.20 9.73 11.48 8.61 6.81 37.77 11.70 11.29 16.19 14.62 ND ND ND ND ND ND ND ND Status D D D D D D D D D D D D D D D D D D D D D D E E E E E E E E E E E E E E E E E E E E E E E E E E E E E ND ND ND ND ND ND ND ND Full or partial Full ND ND Partial ND ND Partial ND ND Full Partial Full Partial Full Full Full ND Full Full ND ND ND ND ND ND Full Full Partial ND Full ND ND ND ND ND ND ND ND Partial Full ND ND ND ND ND ND ND Full ND ND ND ND ND ND ND ND ND ND Partial Proposed function IgG binding mRNA binding DNA binding GTPase activation noncoding RNA Tyrosine phosphatase phosphatidylinositol binding Neurite outgrowth Unknown CHRNA7-FAM7A fusion protein Rho GTPase activation Guanine-nucleotide exchange Cilia motility Transcriptional regulation Unknown Biogenesis of snRNPs Inhibition of apoptosis Dual specicity protein phosphatase Transcriptional regulation Unknown Unknown Transcriptional regulation WAS protein family homolog Ciliary protein Hepatocyte growth factor-like 1,4 alpha-glucosidase Unknown Golgi/Centrosome localization Neuroblastoma breakpoint family NADPH oxidase F-actin binding Golgin subfamily Structure-specic endonuclease Unknown Unknown Ribosomal protein Unknown Nucleoporin Transcriptional regulation Dopamine signaling Unknown Degrades glycosaminoglycans Degrades glycosaminoglycans Non-protein coding RNA Meiotic chromosome pairing Transcriptional regulation Unknown Water, glycerol and urea transport Keratinocyte growth factor Unknown Transcriptional regulation Rho GTPase activation A-kinase anchoring Rho GTPase activation Serine/threonine protein kinase Myelination GABA reuptake Growth factor Cell adhesion (Continued)

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Table 1. (Continued).
Gene USP10 ANAPC1 PAK2 CDH12 FXYD2 MST1 E2F6 DDX11 RAB6C ABCC6 GPR116 EIF3A MPPE1 J Neurogenet Downloaded from informahealthcare.com by UNICAMP on 04/14/14 For personal use only. Chromosome 16 2 3 5 11 3 2 12 2 16 6 10 18 Copy number ND ND ND ND ND ND ND ND ND ND ND ND ND Status ND ND ND ND ND ND ND ND ND ND ND ND ND Full or partial ND Partial ND ND ND Full ND Full ND ND Partial ND ND Proposed function Ubiquitin pathway Cell cycle Protein kinase Cell adhesion Transport Immune response Transcription RNA helicase Trafcking Transport G protein signaling Translation GPI anchor biosynthesis

Note. Genes listed are chosen from Fortna et al. (2004) and Sudmant et al. (2010). From Fortna et al. (2004), only genes conrmed by Dumas et al. (2007), which employed essentially the same experimental approach, were adopted. Copy number refers to total copies per diploid genome, including both ancestral and duplicated copies. Status refers to whether the gene is duplicated specically in the human lineage (D), i.e., after the divergence of human and chimpanzee lineages, or the gene had duplicated in a common primate ancestral lineage followed by further expansion specically in the human lineage (E). Full or Partial refers to whether the gene duplication includes entire exons (Full) or lacks more than one exon (Partial). Note that not all duplicated copies of a particular gene are entirely Full or Partial, and it remains to be determined whether both full duplication and partial duplication had occurred from one ancestral gene. ND not determined.

et al., 2010). These genes are distributed on several chromosomes: chr1, chr2, chr5, chr7, chr9, and chr10. The chromosomal distribution of duplicated genes is very similar to that of high-identity ( 95%) segmental duplications. Each duplicated gene may undergo neofunctionalization, subfunctionalization, or degradation, or exert a dosage effect by the virtue of its high identity with the ancestral gene (Hurles, 2004; Lynch & Katju, 2004; Ohno, 1970). Many genes with human-specic duplication or expansion could be involved in brain development, function, and diseases. HYDIN is a agellar central pair protein that is required for ciliary motility. HYDIN mutation in mice impairs ciliary motility and results in hydrocephaly (Lechtreck et al., 2008). Ancestral HYDIN is located on 16q22.2 and its duplicated paralog is inserted in the 1q21.1 region, which is susceptible to microdeletion and microduplication (Doggett et al., 2006). Notably, microdeletion and microduplication were correlated with microcephaly and macrocephaly, respectively, suggesting that the copy number of HYDIN may regulate the brain size (Lechtreck et al., 2008). CHRNA7 is located on 15q13 and encodes the 7 subunit of the nicotinic acetylcholine receptor, which is highly expressed in the nervous system. A recurrent 680-kb deletion within this region that encompasses CHRNA7 has been associated with neurodevelopmental abnormalities, including mental retardation and seizures (Shinawi et al., 2009). Humanspecic partial duplication of CHRNA7 in this region has generated a novel chimeric gene, CHRFAM7A, which is a hybrid between CHRNA7 and FAM7A (Riley et al., 2002). Using homozygous haplotype mapping, CHRFAM7A has

been identied as one of the candidate genes associated with autism-spectrum disorders (Sinkus et al., 2009). It remains to be determined whether CHRFAM7A has acquired a novel function or exerts its effect through interaction with CHRNA7. DRD5 and SLC6A13, encoding dopamine D5 receptor and -aminobutyric acid (GABA) transporter, respectively, are possibly involved in the development of the brain and the formation of cognitive abilities through regulation of synaptic transmission and plasticity (Centonze et al., 2003; Hglund et al., 2005; Lemon & Manahan-Vaughan, 2006). GPRIN2 interacts with guanosine triphosphate (GTP)-bound form of Go or Gz in vitro, and induces neurite formation in Neuro2A cells (Chen et al., 1999). GTF2IRD2, GTF2I, and GTF2IRD1 constitute the GTF2I family of putative transcription factors with multiple helix-loop-helix domains known as I repeats (Gunbin & Ruvinsky, 2013; Palmer et al., 2012). Interestingly, these three genes are all located in a narrow region of 7q11.23. Microdeletions in this region, including GTF2I and GTF2IRD1, are associated with the pathogenesis of the cognitive and behavioral phenotypes of Williams-Beuren syndrome (Makeyev et al., 2004; Porter et al., 2012). Patients with slightly larger deletions encompassing GTF2IRD2 are more cognitively impaired in executive function (Porter et al., 2012). NAIP genes encode the nucleotide-binding domain and leucinerich repeat (NLR) protein family, which suppress apoptosis through the inhibition of procaspase-9 activation (Davoodi et al., 2010; Mercer et al., 2000; Perrelet et al., 2002). Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease in which the motor

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neurons in the anterior horn of the spinal cord degenerate (Hamilton & Gillingwater, 2013). SMA is associated with mutations in SMN1. SMN2, a human-specic gene, is very similar to SMN1 and encodes a protein identical to the product of SMN1 (Lorson et al., 2010). However, a synonymous point mutation in the coding sequence of SMN2 results in the skipping of exon 7, which makes most of the SMN2 transcripts inactive (Lorson et al., 1999). SMN2 has been characterized as a modier of SMA; an increase in SMN2 copy number reduces the severity of the disease (Zheleznyakova et al., 2011). OCLN encodes a transmembrane protein involved in the formation of tight junctions (Steed et al., 2010). Mutations in OCLN cause band-like calcication with simplied gyration and polymicrogyria (BLC-PMG), an autosomal recessive neurological disorder characterized by microcephaly, polymicrogyria, and gray matter calcication (ODriscoll et al., 2010). The list of genes duplicated or expanded in the human highlights the genes related to signal transduction mediated by Rho family of GTPases (Ridley, 2012), including SRGAP2 (ARHGAP34), ARHGAP11A, ARHGEF5, ARHGAP15, ARHGAP42, and PAK2. SRGAP2 is the rst human-specic duplication gene for which the precise nucleotide sequence of the duplicated copies has been determined and the function experimentally demonstrated (see next section) (Charrier et al., 2012; Dennis et al., 2012). PAK2 is a member of the p21 (Cdc42/Rac)-activated serine/ threonine kinase family (Boda et al., 2006). Among the six human PAK genes, PAK3 is associated with X-linked nonsyndromic forms of mental retardation, which are characterized by the cognitive decit (Allen et al., 1998). PAK3 preferentially binds to and is activated by Cdc42 and regulates the dendritic spine morphogenesis, synapse formation, and plasticity (Boda et al., 2004; Kreis et al., 2007). PMP2 (FABP8), a member of the fatty acidbinding protein (FABP) family (Smathers & Petersen, 2011), is expressed mainly in peripheral myelin. PMP2 is one of the major proteins in peripheral myelin, and immunization of rats with PMP2 induces experimental autoimmune neuritis, an animal model of acute inammatory demyelinating polyradiculoneuropathy/Guillain-Barr syndrome (Rostami et al., 1984). DUSP22 is a member of dualspecicity phosphatases that can dephosphorylate both tyrosine and serine/threonine residues (Patterson et al., 2009). DUSP22 protein is associated with actin cytoskeleton, dephosphorylates focal adhesion kinase (FAK) and regulates cell motility (Li et al., 2010). Which of these duplicated genes may be involved in human brain evolution? One point to be considered is the copy number stability of the duplicated gene among human populations. Some of these recently duplicated genes are highly variable in terms of copy numbers. The existence of normal individuals having the same gene copy number as nonhuman primates makes it less likely that such genes played a role in the human brain evolution. Of the genes

mentioned above, HYDIN, GTF2IRD2, SRGAP2, and ARHGAP11A are almost copy numberxed in human populations and therefore may be attractive candidates for further experiments. Because HYDIN dosage positively regulates the brain size, the duplication may have contributed to the expansion of the human cortex. Rho family GTPases are implicated in neuronal morphogenesis, including axon guidance and synapse formation. Thus, SRGAP2 and ARHGAP11A may regulate the formation of neural circuits and their subsequent maturation unique to the human brain.

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ROLE OF SRGAP2 DUPLICATION IN BRAIN DEVELOPMENT As mentioned above, SRGAP2 is emerging as one of the candidate genes whose duplication may have contributed to the human brain evolution. This has stimulated research on the structure and function of duplicated as well as ancestral SRGAP2 (Figure 1). The results show that the human SRGAP2 has been partially duplicated thrice. One of the duplicated copies is xed among human populations and has acquired a novel function, i.e., antagonizing ancestral SRGAP2 protein function. Heterogeneous introduction of duplicated SRGAP2 in mouse brain induces neoteny in dendritic spine maturation, a trait observed in human prefrontal cortex (Figure 2). Thus, the duplication of SRGAP2 is the rst example that supports the hypothesis that the human-specic gene duplication may play a key role in human brain evolution. These results are discussed in more detail in the following paragraphs. Human-specic duplication of SRGAP2 was rst demonstrated in 2004 by Fortna et al. (Fortna et al., 2004). In that study, array-based comparative genomic hybridization (aCGH) was used to perform the whole-genome comparison of copy number variation between humans and nonhuman primates. Another work, using short-read

Figure 1. Schematic representation of SRGAP family proteins. SRGAP2A, SRGAP1, and SRGAP3 possess F-BAR, Rho-GAP, and SH3 domains. SRGAP2B and SRGAP2C are present only in humans and consist of truncated F-BAR domain followed by a unique VRECYGF sequence. In addition, SRGAP2B and SRGAP2C have several amino acid substitutions compared with SRGAP2A.

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Figure 2. Schematic representation of changes in dendritic spine morphology induced by expression of SRGAP2C in mouse neocortical pyramidal neurons in vivo. SRGAP2A localizes in dendritic spines and promotes spine maturation (its activity is shown in red). SRGAP2C forms dimers with SRGAP2A and antagonizes its spine maturation activity, which results in higher density of spines with longer necks and smaller heads.
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mapping depth to human reference genome, has independently identied SRGAP2 duplication in humans but not in nonhuman primates (Sudmant et al., 2010). However, the position, structure, and extent of the duplication remain to be determined. Fluorescent in situ hybridization (FISH) has identied three SRGAP2 loci on human chromosome 1: 1q32.1, 1q21.1, and 1p12 (Dennis et al., 2012). In nonhuman primates, only one signal on 1q32.1 has been identied, which has established 1q32.1 as the ancestral locus. Notably, ancestral copy of SRGAP2 (named SRGAP2A) as well as duplicated copies (SRGAP2B on 1q21.1 and SRGAP2C on 1p12) have been misassembled and contained sequence gaps in the human reference genome (GRCh37/hg19), suggesting that the identities between allelic and paralogous copies were too high to distinguish them in the process of genome assembly. Dennis et al. have resolved this problem by constructing a bacterial articial chromosome (BAC) library covering a haploid genome (Dennis et al., 2012). From this BAC library, sequence contigs corresponding to ancestral and duplicated copies have been unambiguously generated. Moreover, a fourth copy (SRGAP2D), not found in the reference genome, has been identied in the BAC library and mapped by FISH on 1p21.1, proximal to SRGAP2B (Dennis et al., 2012). Charrier et al. also succeeded in generating BAC contigs corresponding to SRGAP2A, SRGAP2B, and SRGAP2C by identifying BAC clones that covered the regions missing in the reference genome (Charrier et al., 2012). These studies, together with an additional method to obtain haplotype-resolved genome information (Kitzman et al., 2011), present a strategy applicable to other genes that have undergone human-specic duplication or expansion. Combining multiple-sequence alignment, phylogenetic analysis, and FISH, Dennis et al. revealed essentially the whole picture of SRGAP2 duplication events during human evolution (Dennis et al., 2012). The rst event duplicated the promoter and the rst nine exons of SRGAP2A to 1q21.1 for generating SRGAP2B around 3.4 mya. Two larger duplications copied the entire SRGAP2B and adjacent

regions to 1p12 (SRGAP2C) and 1q21.1 (SRGAP2D) approximately 2.4 and 1 mya, respectively. SRGAP2D was later made nonfunctional by a 115-kb deletion including exons 2 and 3. The copy number of SRGAP2C is xed at a diploid copy of 2, whereas that of SRGAP2B is variable. Moreover, the identication of normal individuals with homozygous deletions of SRGAP2B has left SRGAP2C as the only candidate. What is the function of SRGAP2C? Before addressing this question, it is necessary to overview the function of ancestral SRGAP2. Mammalian SRGAP family consists of three members, SRGAP13 (Figure 1). SRGAP1 has been originally identied as Rho GTPase-activating protein (GAP) that may participate in the Slit-Robo signaling pathway involved in axon guidance and cell migration (Wong et al., 2001). SRGAPs are expressed dynamically during the development of various regions of nervous system, including the neocortex (Bacon et al., 2009; Guerrier et al., 2009). SRGAP protein is composed of three domains from the N-terminus to C-terminus: F-BAR (Bin, Amphiphysin, Rvs) domain (Frost et al., 2009), GAP domain, and Src homology 3 (SH3) domain (Figure 1). F-BAR domains of SRGAPs form homodimers or heterodimers and can regulate membrane deformation and lopodia formation in vitro (Coutinho-Budd et al., 2012). The GAP domain of SRGAP2 and SRGAP3 is specic for Rac1 (Guerrier et al., 2009; Soderling et al., 2002). Accumulating evidence indicates that ancestral SRGAP2 plays a pivotal role in brain development. SRGAP2 is highly enriched in the neuritis. Overexpression of mouse SRGAP2 in radially migrating neocortical neurons induces the formation of highly dynamic neurites and branches, which destabilize the leading process and impair directional cell body translocation (Guerrier et al., 2009). F-BAR domain of SRGAP2 is necessary and sufcient for this effect, suggesting that the ability of this domain to induce membrane protrusions is required for appropriate neuronal migration and morphogenesis. Consistently with the overexpression analysis results, knockdown of SRGAP2 reduces branching in the leading process and accelerates neuronal migration (Guerrier et al., 2009). High-level expression of SRGAP2 continues at the later stages of brain development such as the synapse formation (Bacon et al., 2009; Charrier et al., 2012; Guerrier et al., 2009). Endogenous SRGAP2 is a postsynaptic protein accumulated in the heads of dendritic spines (Charrier et al., 2012). Analysis of SRGAP2-knockout (KO) mice has demonstrated that SRGAP2 promotes spine maturation and limits spine density (Charrier et al., 2012) (Figure 2). Notably, these changes are dosage dependent; the phenotypes of heterozygous individuals is intermediate between wild-type and KO phenotype. Thus, at low SRGAP2 levels, spine maturation is neotenic, i.e., its duration is extended; the adult neurons form immature spines with a longer neck. These changes possibly have

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a profound impact on neuronal connectivity and synaptic input integration (Bourne & Harris, 2007; Matsuzaki et al., 2004; Yuste, 2011). SRGAP2C is predicted to encode a protein of 459 amino acids (a.a.) (Figure 1). The rst 452 a.a. of SRGAP2C correspond to the rst 452 a.a. of the F-BAR domain of SRGAP2A (a.a. 1501) and the remaining 7 a.a. are derived from intron 9. Thus, SRGAP2C consists of a truncated F-BAR domain. SRGAP2C can either heterodimerize with SRGAP2A or homodimerize with SRGAP2C. Whereas expression of SRGAP2A in COS7 cells induces lopodia, SRGAP2C expression does not (Charrier et al., 2012; Guerrier et al., 2009). Interestingly, coexpression of SRGAP2C and SRGAP2A efciently inhibits the ability of SRGAP2A to induce lopodia (Charrier et al., 2012). There can be two explanations for the observed effects based on the two differences between SRGAP2C and SRGAP2A (Figure 1). First, SRGAP2C is C-terminally truncated by 49 a.a. residues compared with the F-BAR domain of SRGAP2A. Second, SRGAP2C has several amino acid substitutions in its truncated F-BAR domain. Whereas some of these substitutions are the same in different human individuals, others are variable (Dennis et al., 2012). Thus, SRGAP2C differs from SRGAP2A, in up to ve residues in the corresponding region. To test the relative contribution of these two types of mutations to the inhibitory effect on SRGAP2A-mediated lopodia induction, two individual mutants of the ancestral F-BAR domain were generated: F-BAR-D49, which lacks the C-terminal 49 a.a. residues, and F-BAR-D5R, which has ve amino acid substitutions (all to arginine) corresponding to the most varied form of SRGAP2C. Both mutants lose the ability to induce lopodia. However, only F-BAR-D49 exhibited the ability to antagonize SRGAP2A-mediated lopodia induction (Charrier et al., 2012). Thus, SRGAP2C is able to interact with and inhibit the ancestral SRGAP2A, primarily due to its truncated form of F-BAR domain. These results are interesting from the evolutionary point of view: if the duplication had been slightly larger and included a few more exons, then the F-BAR domains of the duplicated paralog would not have acquired such an inhibitory property. The expression of SRGAP2C in the developing mouse cortex essentially phenocopied the SRGAP2 deciency. The introduction of SRGAP2C into the mouse neocortex by in utero electroporation reduces the leading process branching and accelerates neuronal migration. This phenomenon is very similar to the changes observed in shRNA-mediated knockdown of endogenous SRGAP2 (Charrier et al., 2012). Such an increase in the rate of neuronal migration may be benecial to the development of human neocortex. In the human brain, the postmitotic neurons have to travel a longer distance before reaching the nal destination, as the neocortex is substantially thicker than in nonhuman primates or

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rodents (Dehay & Kennedy, 2007; Rakic, 2009; Sidman & Rakic, 1973). Charrier et al. introduced SRGAP2C by in utero electroporation in the mouse cortex and examined the transfected neurons during the juvenile period, after the completion of neuronal migration. During this period, in SRGAP2C-expressing neurons, the spine maturation slows down; the spine head is smaller and the neck longer, and the density increases (Figure 2) (Charrier et al., 2012). In adult animals, SRGAP2C-expressing neurons attain a spine head width similar to that in controls, but the spine neck length and the density remain at the juvenile level. Thus, the human-specic paralog of SRGAP2 functionally antagonizes the ancestral SRGAP2 and induces neoteny during spine maturation. These morphological and temporal changes induce in the mouse dendritic spines the characteristics observed in human dendritic spines (Benavides-Piccione et al., 2002; Elston et al., 2001; Petanjek et al., 2011). Since dendritic spines undergo morphological changes to increase the connectivity, isolate the inputs from each other, and enable input-specic plasticity (Bloodgood & Sabatini, 2005; Bourne & Harris, 2008; Matsuzaki et al., 2004; Yuste, 2011; Yuste & Bonhoeffer, 2001), it will be particularly interesting to investigate whether the properties or functions of neural circuits are changed in the presence of duplicated SRGAP2.

IMPLICATIONS OF HUMAN-SPECIFIC GENE DUPLICATION AND THE FUTURE PROSPECTS The development of new methods for genome analysis, such as hybridization-based microarray and sequencingbased computational approaches, is largely responsible for the current advances in the evolutionary biology eld (Alkan et al., 2011). The list of genes that have undergone human-specic duplication or expansion is continuously modied to accommodate new biological annotations as well as the discovery of new genes. SRGAP2 is the rst gene for which the signicance of its duplication in human brain evolution has been experimentally examined (Charrier et al., 2012; Dennis et al., 2012). SRGAP2C protein modulates spine maturation through inhibition of SRGAP2A (Figure 2). Dendritic spines receive most of the excitatory presynaptic input in the cortex. Spines are heterogeneous in their morphology, stability, and density; those with large heads are mature and stable, and contribute to strong synaptic connections, whereas the spines with small heads and long necks are immature, motile, and unstable, and contribute less to synaptic connections (Kasai et al., 2003; Yuste, 2011). The spines are initially overproduced and subsequently selectively stabilized or eliminated in an activitydependent manner. In the human prefrontal cortex, this

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process continues beyond adolescence and throughout the third decade of life (Petanjek et al., 2011). The production of SRGAP2C and induction of neoteny during spine maturation may prevent the human brain from becoming hard-wired, and allow the embedded neural circuits to be diverged and modied by experience. This process undoubtedly contributes to our higher cognitive abilities such as language acquisition and creativity. Many of the genes that have undergone humanspecic duplication or expansion, including SRGAP2, are associated with neurological diseases or other disorders. A de novo balanced translocation t(1;9)(q32;q13), which disrupts SRGAP2 on 1q32, has been reported in a patient with early infantile epileptic encephalopathy and severe psychomotor disability, suggesting that SRGAP2 has a conserved role in the human brain development (Saitsu et al., 2011). Another balanced de novo translocation t(X;3)(p11.2;p25), which disrupts SRGAP3 on 3p25, has been reported in a patient with hypotonia and severe mental retardation (Endris et al., 2002). Although the involvement of genes other than SRGAPs in the pathogenesis of these two cases remains possible, SRGAP2 and SRGAP3 may play essential and nonoverlapping roles in human brain development in a dosage-sensitive manner. This is consistent with the observation that intellectual disability is most consistently associated with spine dysgenesis (van Bokhoven, 2011). We can conclude that even subtle changes in the dosage of genes encoding the key molecules in spine morphogenesis and function can be either benecial or detrimental. The copy number of most duplicated genes varies considerably, indicating the continuing genomic gain and loss among human populations (Sudmant et al., 2010). It is possible that a particular copy number may change as a result of positive, neutral, or negative selection depending on whether its effect is advantageous, neutral, or harmful. A gain or loss of duplicates beyond a certain threshold may manifest itself as a disease or creation. A model of evolution by gene duplication proposed by Ohno assumes whole-gene duplication, i.e., the duplicated gene is at rst identical to the original, then acquires a novel function over time by accumulating mutations (Lynch & Katju, 2004; Ohno, 1970). Alternatively, as exemplied by SRGAP2C, partial gene duplication can immediately confer a novel function. Testing the role of duplicated genes in human brain development and evolution requires analyses at multiple levels, including biochemical, cell biology, and behavioral approaches. The functionality and expression of the duplicated gene must be carefully examined because partial gene duplication may miss some elements such as promoters, enhancers, or exons encoding indispensable domains. Although the mouse is evolutionarily more distant from the human than nonhuman primates, it will remain an important experimental model animal.

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Examining the phenotype of transgenic mice that harbor duplicated genes is one of the most straightforward approaches to obtain insights into the in vivo role of duplicated genes in the human brain development and function. Another promising strategy is the use of embryonic stem (ES) cells and induced pluripotent stem (iPS) cells derived from human and nonhuman primates as well as rodents (Liu et al., 2008; Takahashi et al., 2007; Thomson et al., 1998; Yamanaka & Blau, 2010). Induced differentiation of ES or iPS cells into cells or tissues forming the brain will provide an invaluable source of new information (Eiraku et al., 2011; Gaspard et al., 2008; Han et al., 2011). Gene targeting of the duplicated genes in ES or iPS cells will enable to test the function of the duplicates directly (Zou et al., 2009). Future studies of the effect of human-specic gene duplications on normal brain development and pathological conditions will provide a wealth of insights into the evolutionary genetic basis of what makes us human.

ACKNOWLEDGMENTS I would like to thank Dr. Kaumudi Joshi for the comments on the initial version of the manuscript. I would like to thank Enago for the English language review. Declaration of interest: The author reports no conicts of interest. The author alone is responsible for the content and writing of the paper. REFERENCES
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