... CECILIA CONACO,*† DANIELLE S. BASSETT,‡§ HONGJUN ZHOU,*† MARY LUZ ARCILA,*† SANDIE M. DEGNAN, BERNARD M. DEGNAN, AND KENNETH S. KOSIK*†#...

... Assembly of a functioning neuronal synapse requires the precisely coordinated synthesis of many proteins. To understand the evolution of this complex cellular machine, we tracked the developmental expression patterns of a core set of conserved synaptic genes across a ... continue to operate seamlessly within the synapses of modern animals. This work shows that the application of network techniques to emerging genomic and expression data can provide insights into the evolution of complex cellular machines such as the synapse....

... Biology, ‡Department of Physics, and §Sage Center for the Study of the Mind, University of California, Santa Barbara, CA 93106; and School of Biological Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. #To whom correspondence should be addressed. E-mail: kosik@...

... (Porifera), generally recognized as the oldest surviving metazoan phyletic lineage (Fig. 1.1B), occupy a highly informative position for understanding the evolution of features that uniquely characterize animals (Srivastava et al., 2010). The synapse, a cellular machine formed through the dynamic ... that signal back to an electrical impulse in the postsynaptic neuronal cell. Surprisingly, the genome of the Poriferan demosponge, Amphimedon queenslandica, contains an almost complete set of genes homologous to those found in mammalian synapses (Fig. 1.1A), although the organism does not assemble any ... morphologically resembling a synapse (Sakarya et al., 2007; Srivastava et al., 2010). Although limited gene innovation and the invention of new protein interaction sites can partially explain how preexisting genes came together to form the synaptic complex (Sakarya et al.,...

... Changes in conserved transcriptional programs arising from modification of instructions encoded in the genome have contributed to our understanding of animal evolution (Barabási and Oltvai, 2004; Oldham et al., 2006, 2008; Brawand et al., 2011). Specific patterns of expression can ... discrete tissues, cell types, and even functional protein complexes. Genes with similar expression patterns often have similar function (Eisen et al., 1998). Furthermore, when ... constraints (Carlson et al., 2006). Thus, changes in coexpression relationships for any group of genes may contain information on the assembly and evolution of cellular machines. To understand the evolutionary transition leading to the emergence of a functional synapse, we used network analysis ... in representative species from diverse phylogenetic positions. We show that “protosynaptic” genes have an inherent modular structure and that the coregulatory links between these modules characterize species with functional synapses. In contrast, ancient eukaryotic cellular machines, ... as the proteasome and nuclear pore, already operate in early metazoans, and their associated genes display highly correlated expression patterns over development. These findings...

... ancestor of origin for each gene, as indicated in B. (B) Evolutionary relationships among animal phyla. The names of representative species are shown. [Note: Figure can be viewed in color in the PDF version of this volume on the National Academies Press website, www.nap.edu.]...

... [Fig. 1.2A; Conaco et al. (2012, Fig. S1A)], we obtained the expression profiles of sponge synaptic gene homologues by sequencing the A. queenslandica transcriptome at four developmental stages from larva to adult. For comparison, expression data were also obtained for the same set of synaptic ... .1B). Animal species included in this study were the cnidarian coral, Acropora millepora; invertebrate bilaterians, Caenorhabditis elegans (nematode) and Drosophila melanogaster (arthropod); and vertebrates,...

... FIGURE 1.2 Structure of protein interactions within the (A) synaptic, (B) epithelial, (C) NPC, and (D) 26S proteasome networks. Each node represents a gene. Node size represents the number of interactions formed by a protein, and edge length is ... of evidence for a functional link between two proteins. Network structures are based on the human interactome annotated in STRING (Snel et al., 2000) and visualized by using Cytoscape (Shannon et al., 2003). (E) Degree distribution patterns of gene networks based on the human interactome. The frequency ...

... Danio rerio (zebrafish) and Xenopus tropicalis (frog) (Hillier et al., 2009; Domazet-Lošo and Tautz, 2010; Graveley et al., 2011; Meyer et al., 2011; Yanai et al., 2011). The correlation matrix for synaptic gene homologues from each species ... all pairs of gene expression profiles across development (Fig. 1.3A). The correlation matrix represents a network in which the genes are nodes and the correlations between gene expression patterns are edges. We averaged all elements of the correlation matrix to obtain a measure of connectivity ... coregulation, R (Fig. 1.3B, D, F, and H). By using a community detection algorithm (Girvan and Newman, 2002; Blondel et al., 2008; Porter et al., 2009), the modularity, Q, of each network was computed by determining the optimal partition of the ... into communities whose nodes were more connected to other nodes inside of their own community than expected in a random null model (Fig. 1.3C, E, G, and I). The modularity, Q, can be interpreted as a measure of the cohesiveness of coregulation: higher Q values indicate more segregation between ... groups. To determine the statistical significance of our results, we computed the same properties (R and Q) for various random control models....

... showed even greater coregulation compared with sponge or coral. Synaptic genes showed significantly increased correlation compared with permuted and random controls in all species [Fig. 1.3B; Conaco et al. (2012, Table S1)]. To verify the observed differences in expression coregulation, we performed ... comparisons of subsets of synaptic genes common between species. Comparison of genes found in sponge and the other five species showed that the increased correlation in eumetazoans was significant [P < 1 × 10-5, two-tailed t test; Conaco et al. ( ... , Table S2)]. Pairwise comparison of average coregulation for genes common between coral and each of the other species further revealed significantly greater correlation in bilaterian organisms (P < 1 × 10-10, two-tailed t test). ... pairwise correlation values were significantly greater than coregulation within three separate random control models (P < 0.05, two-tailed t test; Materials and Methods). However, Q values for most of the synaptic gene networks did not show the ... is composed of subsets of genes with distinguishable differences in their developmental expression patterns, similar to what we would expect from a random collection of genes taken from the transcriptome. These distinct modules may be performing...

... same tendency was also true for the synaptic vesicle genes in most bilaterians. In contrast, sponge synaptic genes in these functional complexes showed a more heterogeneous expression pattern that appeared to follow a different regulatory logic than that of functional synaptic networks, as ... is tightly coregulated even in sponge, suggesting a gain of functionality long before animal divergence (Finnigan et al., 2012). It should be noted, however, that, although we did not see similar module enrichment patterns for these functional complexes in the frog, we did observe a strong correlation ...

... FIGURE 1.3 Correlation and modularity analysis for gene networks in six organisms. (A) The strength of genetic coregulation for any two genes in a network was estimated by ... represent N×N correlation matrices for genes in each network in each species (red, positive correlation; blue, negative correlation). (B, D, F, and H) Average correlation, R, was computed from the matrices in A. (C, E, G, and I) The presence of distinct coregulated modules was estimated by the Q ... (Blondel et al., 2008). The computations for each true network (red circles) were also performed on control datasets: time-permuted (1,000 randomly scrambled versions of the correlation matrix, orange diamonds), random gene set (100 gene sets of size N randomly sampled from the entire ... , blue triangles), and random number matrix (100 matrices generated with the same gene number and developmental stages as the true network, green squares). The number of genes included in the analysis for each network in each species is shown in ... . Error bars represent SD of R and Q; some SDs are smaller than the marker size. Asterisks indicate a significant difference from the random gene set control (P < 0.05, two-tailed t test). [Note: Figure can be viewed in color in the PDF version of this volume on the National Academies ...

... the organism or have no available expression data. Dashed circles represent the approximate boundaries of the postsynaptic density, synaptic vesicle, and vATPases. (B) Percent of genes in each functional complex that belong to coregulation modules detected by modularity optimization. Colors correspond ...

... network also lacks a morphological correlate in the sponge. In epithelial cells, the adherens junction links to apical-basal polarity genes and Wnt/planar polarity genes [Fig. 1.2B; Conaco et al. (2012, Fig. S1B)]. Although A. queenslandica expresses many orthologues of epithelial genes, the ... exhibits only rudimentary features of a functional epithelium (Adams et al., 2010; Fahey and Degnan, 2010). As in the synaptic gene set analysis, we extracted the expression patterns of epithelial genes from six species and calculated the ... correlation, R, and modularity, Q, of the coregulation network (Fig. 1.3D and E). The epithelial network in all species that were tested showed significantly greater R when compared pairwise vs sponge [P < 1 × 10-8, ... t test; Conaco et al. (2012, Table S2)]. As in the synaptic network, the modularity of epithelial networks was not consistently lower compared with random controls for most of the species tested....

... Neurons and epithelial cells and their defining cellular machines appear in eumetazoans after sponges diverged from other animals. We asked whether genes drawn from more ancient ... the origin of animals. We performed a similar modularity optimization on transcriptome data for homologues of genes in the nuclear pore complex (NPC) and the 26S proteasome [Fig. 1.2C and D; Conaco et al. (2012, Fig. S1C and D)]. These networks are highly interconnected and exhibit a negatively skewed ... distribution, which differs from the relatively large hubs and positively skewed degree distribution observed in mammalian synaptic and epithelial networks (Fig. 1.2E)....

... The nuclear envelope is a defining feature of eukaryotic cells (Wente and Rout, 2010). Transport of molecules between the nucleus and cytoplasm is mediated by the NPC, which is made up of approximately 30 nucleoporin genes. Coregulation analysis of nucleoporin homologues represented ... the transcriptome set revealed higher average correlation and generally lower modularity compared with the synaptic or epithelial networks in the same species (Fig. 1.3F and G). Most of the NPC networks showed ... greater R and lower Q compared with permuted or random size-matched data, suggesting that the components of the NPC act as a single functional unit (Conaco et al., 2012, Table S1). In contrast, greater ... of the synaptic and epithelial polarity networks suggests a requirement for some modularity in the operation of these machines, perhaps as a result of the presence of ...

... et al., 1999). Coregulation analysis of homologues of proteasomal genes revealed that, like the NPC, the proteasome has higher average correlation and lower modularity...

... compared with the synaptic or epithelial networks within each species (Fig. 1.3H and I). All eumetazoans showed significantly higher correlation when compared pairwise vs. sponge (P < 1 × 10-52, two-tailed t test; Conaco et ... ., 2012, Table S2). Coregulation and modularity of proteasomal genes differed significantly from permuted or random data, except in the sponge (Conaco et al., 2012, Table S1). Nevertheless, in all species tested, including the sponge, the proteasome gene set ... as a distinct community when analyzed together with NPC genes (Fig. 1.5) and is therefore likely to represent a functionally significant module....

... In a unicellular eukaryote, like the yeast, Saccharomyces cerevisiae, the NPC and proteasome gene networks exhibit high correlation and low modularity that are quite similar to the average values observed for the metazoans (Conaco et al., 2012, Table S1). These findings further ... the hypothesis that gene networks that establish their modern function long before the origin of metazoa exhibit significantly higher correlation and lower modularity, consistent with a greater and more homogeneous connectivity between genes....

... function from the unknown functions of these same gene sets in the common animal ancestor. This reorganization appears as increased connectivity and a change in the network structure with functional complexes clustering into coregulated modules. In contrast, more ancient machines, such as the ... and the NPC, show a cohesiveness of expression as far back as the eukaryotic ancestor....

... to the affinities among their various interaction domains. Among the core features of synapses are scaffolding proteins that position receptors and ion channels in register with synaptic vesicles across the synaptic cleft and link the pre- and postsynaptic elements to intracellular signaling ...

... detects biologically relevant gene communities. (A) Heat maps represent the N×N Pearson correlation matrices for union networks of NPC and proteasome genes (red, positive correlation; blue, negative correlation). Average partition similarities (ave. part. sim.) computed from permutation ... with 1,000 iterations showed that, compared with the randomly scrambled gene set, genes in the union network clustered into communities that more closely recapitulated the true partition between networks (P & ... ; 0.05). Color bars to the right of the heat maps indicate the boundaries of detected coregulation modules (Modules) and the relative location of NPC (orange) and proteasome (blue) genes within the detected communities (Genes). (B) Box plots show the developmental ... patterns of genes within the NPC (Left) and prote-asome network (Right) for each of the six representative species. [Note: Figure can be viewed in color in the PDF version of this volume on the ...

... of these highly connected proteins (Sakarya et al., 2010), an observation consistent with network growth by link dynamics, that is, link detachment and attachment (Berg et al., 2004). Just as mutations in coding sequences can change link dynamics and enable new protein–protein interactions, ... in cis regulatory sequences can lead to the evolution of new transcriptional linkages and coexpression of gene batteries that were not previously associated. In fact, the sponge already possesses homologues of genes that function in ... to be determined if these factors were responsible for a biological unit originating in the sponge ancestor that was selected for an unknown function and later exapted to assemble the synapse (Richards et al., 2008). These conserved bilaterian developmental and neurogenic genes are associated with ... regulatory mechanisms in vertebrates placed many synaptic genes under the control of the transcriptional repressor, REST, thus ensuring exclusive and coordinated expression in neurons (Schoenherr and Anderson, 1995; Bruce et al., 2004; Otto et al., 2007)....

... signaling networks that control the body plan are thought to evolve by changes in cis regulatory regions resulting in changes in timing, level, and location of gene expression (Peter and Davidson, 2011). In contrast, the network edges of cellular machines represent physical interactions rather ... a cascade of signaling events (Spirin and Mirny, 2003). Nevertheless, the resolution of a signaling or interaction network depends on the extent of coregulatory data available to inform the ... edges. Our analysis required that we compare the coregulation and modularity of the same set of genes; however, inclusion of genes linked to the synaptic network that are not shared between the comparison groups ... likely improve the coregulation signal as gene innovation and duplication can affect network structure through dynamic interactome rewiring (Arabidopsis Interactome Mapping Consortium, 2011). Although these ... increase the likelihood of detecting biologically spurious correlations and may contribute to the apparent modularity observed in some random gene sets, the ability of the community detection algorithm to partition genes into their respective cellular machines indicates a functional ... of the structural communities derived simply from transcriptional coregulation (Fig. 1.5). The generation of more transcriptomes at finer temporal and spatial resolution and the sequencing of genomes from other basal metazoans, as well as improved homologue detection, may strengthen or weaken an ... explanation that the gene expression patterns in A. queenslandica represent a loss of more ancient gene regulatory patterns....

... networks is a key facet of organismal complexity. Several publications have claimed that gene expression networks are scale-free (Barabási and Oltvai, 2004), and although no rigorous proof of the claim exists, many gene expression networks do display a tail in their degree distributions, ... the presence of large hubs. Interestingly, one particular model of scale-free network growth suggests that (i) networks expand continuously by the addition of new nodes, and (ii) new nodes attach preferentially to sites that are already well connected (Udny Yule, 1925). Gene ...

... of global coregulation compared with eumetazoans with functional nervous systems. Protosynaptic gene expression modules from the sponge, A. queenslandica, which lacks synapses and a nervous system, but possesses a nearly complete complement of synaptic genes, are organized into independent ... . These findings suggest that functional synapses evolved through the exaptation of preexisting genes and smaller cellular machines, presumably by modification of regulatory circuitries resulting in coordinated neuronal expression. This work demonstrates ... the modularity approach based on network theory provides a very simple and data-driven method for the identification of gene communities, linking this study to a larger array of network diagnostics that could be used in ...

... MATERIALS AND METHODS...

... Genes in the synaptic, epithelial, NPC, and 26S proteasome networks were compiled from the literature (Voges et al., 1999; Fahey and Degnan, 2010; Srivastava et al., 2010; Wente and Rout, 2010). Protein interaction...

... networks were based on the human interactome annotated in STRING (Snel et al., 2000) and visualized by using Cytoscape (Shannon et al., 2003). Homologues for genes in these networks were determined by reciprocal best-hit BLAST alignments ... . Expression data for gene homologues were extracted from transcriptomes obtained by RNA sequencing of four developmental stages in sponge, A. queenslandica (Conaco et al., 2012, SI Materials and Methods); six experimental treatments of coral larvae, A. millepora (Meyer et al., 2011); four ... stages in worm, C. elegans (Hillier et al., 2009); and 15 developmental stages in fly, D. melanogaster (Graveley et al., 2011). Microarray data for 70 developmental stages in zebrafish, D. rerio (Domazet- ... šo and Tautz, 2010), and 14 developmental stages in frog, X. tropicalis (Yanai et al., 2011), were also included. Microarray expression data for the yeast, S. cerevisiae, ...

... Coregulation and Modularity Analysis...

... strength for all possible pairs of genes, we constructed organism-specific N×N coregulation networks in which genes were represented by nodes and connections between genes were weighted by the correlation between their expression levels over development. These coregulation networks were ... by two diagnostic variables: the average correlation, R, and the modularity, Q, as defined in the following paragraphs....

... be viewed as a complex network in which gene–gene edges are signed (i.e., positive or negative correlations) and weighted (correlations range from -1 to 1). In each organism’s coregulation network, we tested for the presence of uniquely coregulated groups ... genes by using the community detection approach (Porter et al., 2009) of optimizing modularity (Girvan and Newman, 2002) by using the Louvain method (Blondel et al., 2008) [note that a second heuristic, spectral optimization (Newman, 2006), gave nearly ... results: r = 0.9960, P < 0.01; Conaco et al. (2012, Table S3)]. We define the correlation matrix A and then define wij+ to be an N×N matrix containing the positive elements of Aij and wij- to be an N×N matrix containing only the negative ...

... where gi is the community to which node i is assigned, gj is the community to which node j is assigned, γ+ and γ- are resolution parameters, and the following equation applies (Traag and Bruggeman, 2009):...

... in the optimization of modularity for such a signed, weighted network exist (Gómez et al., 2009): the resolution parameters γ+ and γ- (Fortunato and Barthélemy, 2007). For simplicity in the present analysis, we chose the traditional value of γ+ of 1.0 and set & ... positive correlations in the coregulation matrix for two reasons. First, we noted that most gene sets had significantly more positive correlations, and in fact some gene sets had no negative correlations at all (e.g., worm NPC). To ensure that our analysis was consistent across both organisms and ... organization than the negative correlations (Conaco et al., 2012, Fig. S2). Further details are provided in Conaco et al. (2012, SI Materials and Methods)....

... Robustness and Statistical Validity...

... To examine the robustness and statistical validity of our findings, we assessed the reliability of the group partitions and tested our results against three separate postmodularity-optimization null models as described in the following paragraphs....

... heuristics that we used produce robust results, that is, that the partitions found by iterative optimizations are highly similar. For each organism and each machine, we calculated the partition similarity (Danon et al., 2005) (which is bounded in [0,1]) between 100 separate optimizations. We found ... the average partition similarity was >0.8 for most organisms and machines, with the mean over organisms and networks being even higher (Conaco et al., 2012, Table S1)....

... to quantifying the reliability of our findings, we examined the statistical validity of our results by comparing the diagnostic variables (Rʹ and Qʹ) derived from the true network to those derived from networks constructed from three separate random null models: true random (random number ... ), time-permuted, and random gene set. The true random null-model network is constructed by generating uniformly distributed random numbers for the same number of genes and developmental stages found in the true dataset (100 instantiations). A coregulation matrix is then ... and Rʹ and Qʹ are calculated. The time-permuted null-model network is constructed by randomly scrambling the order of expression for each gene within the network (1,000 instantiations), recomputing the coregulation matrix, and calculating ... ʹ and Qʹ. The random gene set null-model network was constructed by extracting the expression data for an identically sized randomly chosen set of genes from the whole transcriptome (100 instantiations). Further details are provided in Conaco et al. (2012, SI Materials and ... ). The statistical significance of the true R and Q values was examined by using a one-sample t test in comparison with the R’ and Q’ values, respectively, for each random null model (Conaco et al., 2012, Table S1). We noted that the level of background correlation and ... observed within sets of N genes randomly selected from each of the transcriptomes is variable (Conaco et al., 2012, Fig. S4). One possible explanation for these differences is that the ...

... matrix R2 between the combined gene set of the proteasome and NPC for each species. We optimized the modularity quality function to partition this combined matrix into groups in a data-driven manner. We next ... whether this data-driven partition was statistically similar to the true partition of the genes into the two groups of proteasome genes and NPC genes. To answer this question, we computed the partition similarity between the data-driven partition and the true partition and used ... testing to determine whether this similarity was statistically significant. The permutation test was implemented by randomly reassigning genes to the two groups of “proteasome” and “NPC,” recomputing the correlation matrix R2ʹ, partitioning ... genes in the correlation matrix into modules, and computing the similarity between this partition and the true partition. This process was repeated 1,000 times to construct a distribution of similarity values expected under the null hypothesis that ... coregulation patterns between proteasome and NPC genes do not differ. For each species, the P value to reject this null hypothesis was computed as follows: the number of similarity values ...

... We thank Boris Shraiman, Mason Porter, Adel Dayarian, and Marija Vucelja for invaluable suggestions and comments; Scott Grafton and Jean Carlson for facilitating the collaboration; and Marc Kirschner and Leonid Peshkin for sharing Xenopus tropicalis microarray data. This work was ... by gifts from Harvey Karp and Gus Gurley (K.S.K.); David and Lucile Packard Foundation (D.S.B.); Public Health Service Grant NS44393 (to D.S.B.); Institute for Collaborative Biotechnologies Contract W911NF-09-D- ... from the U.S. Army Research Office (to D.S.B.); and the Australian Research Council (S.M.D. and B.M.D.)....