Metazoan Complexity Research Articles

What distinguishes the elephant from E. coli: Causal spreading and the biological principles of metazoan complexity

What distinguishes the elephant from E. coli: Causal spreading and the biological principles of metazoan complexity

Low oxygen seawater and major shifts in the paleoenvironment towards the terminal Ediacaran: Insights from the Portfjeld Formation, North Greenland

Earth’s environment during the diversification of metazoans in the Late Ediacaran was characterised, amongst other factors, by changing oxygen levels and an extreme negative carbon isotope (δ13C) excursion, the Shuram Excursion. The Portfjeld Formation, North Greenland, deposited in the Franklinian Basin of eastern Laurentia, presents a unique opportunity to investigate a previously geochemically unstudied succession of Ediacaran marine carbonates to better understand the environment at this time. The succession encompasses a δ13C excursion (from +4 to −8‰) comparable to the Shuram Excursion, as well as the Portfjeld Biota, fossils akin to the earliest putative metazoan life forms, the Weng’an Biota. We present a multiproxy geochemical study of 44 samples including rare earth element (and yttrium; REE + Y), chromium isotope (δ53Cr) and strontium isotope (87Sr/86Sr) compositions.The Portfjeld Formation reveals that low oxygen waters existed beneath oxic surface waters in a stratified early Franklinian Basin. Samples from an interval including bituminous cherty dolomites are enriched in cerium relative to its neighbouring REE+Y’s, expressed as positive cerium anomalies (Ce/Ce* = 1.15–1.38), as well the redox sensitive element molybdenum (EF(Mo) = 23–179). This indicates deposition in waters with active metal oxyhydroxide dissolution, which necessitates dissolved oxygen levels as low as <10 μM. This has potential implications for the habitable environment of early putative metazoan forms, as the Portfjeld Biota fossils are found in this interval.The δ53Cr composition of carbonates can be used to track redox related paleoenvironmental changes. A stratigraphic increase in the estimated authigenic δ53Cr composition (from +0.75 to +1.28‰) occurs throughout the nadir and recovery of the succession’s δ13C carbon excursion. This δ53Cr shift coincides with an increase in detrital element concentration and 87Sr/86Sr values. Therefore, in this instance the δ53Cr composition of the carbonates of the Portfjeld Formation appears to have responded to elevated on-land weathering, and thus nutrient input, which may have induced further fractionation associated with surface water Cr (VI) back reduction by primary producers, in line with modern ocean observations. These geochemical changes indicate that Ediacaran carbon isotope excursions may be tied to environmental shifts that eventually led to the dramatic increase in metazoan complexity that ensued.

Phylogenetic analysis of the Tc1/mariner superfamily reveals the unexplored diversity of pogo-like elements

BackgroundTc1/mariner transposons are widespread DNA transposable elements (TEs) that have made important contributions to the evolution of host genomic complexity in metazoans. However, the evolution and diversity of the Tc1/mariner superfamily remains poorly understood. Following recent developments in genome sequencing and the availability of a wealth of new genomes, Tc1/mariner TEs have been identified in many new taxa across the eukaryotic tree of life. To date, the majority of studies focussing on Tc1/mariner elements have considered only a single host lineage or just a small number of host lineages. Thus, much remains to be learnt about the evolution of Tc1/mariner TEs by performing analyses that consider elements that originate from across host diversity.ResultsWe mined the non-redundant database of NCBI using BLASTp searches, with transposase sequences from a diverse set of reference Tc1/mariner elements as queries. A total of 5158 Tc1/mariner elements were retrieved and used to reconstruct evolutionary relationships within the superfamily. The resulting phylogeny is well resolved and includes several new groups of Tc1/mariner elements. In particular, we identify a new family of plant-genome restricted Tc1/mariner elements, which we call PlantMar. We also show that the pogo family is much larger and more diverse than previously appreciated, and we review evidence for a potential revision of its status to become a separate superfamily.ConclusionsOur study provides an overview of Tc1-mariner phylogeny and summarises the impressive diversity of Tc1-mariner TEs among sequenced eukaryotes. Tc1/mariner TEs are successful in a wide range of eukaryotes, especially unikonts (the taxonomic supergroup containing Amoebozoa, Opisthokonta, Breviatea, and Apusomonadida). In particular, ecdysozoa, and especially arthropods, emerge as important hosts for Tc1/mariner elements (except the PlantMar family). Meanwhile, the pogo family, which is by far the largest Tc1/mariner family, also includes many elements from fungal and chordate genomes. Moreover, there is evidence of the repeated exaptation of pogo elements in vertebrates, including humans, in addition to the well-known example of CENP-B. Collectively, our findings provide a considerable advancement in understanding of Tc1/mariner elements, and more generally they suggest that much work remains to improve understanding of the diversity and evolution of DNA TEs.

Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan mitochondrial tRNAs

Metazoan complexity and life-style depend on the bioenergetic potential of mitochondria. However, higher aerobic activity and genetic drift impose strong mutation pressure and risk of irreversible fitness decline in mitochondrial (mt)DNA-encoded genes. Bilaterian mitochondria-encoded tRNA genes, key players in mitochondrial activity, have accumulated mutations at significantly higher rates than their cytoplasmic counterparts, resulting in foreshortened and fragile structures. Here we show that fragility of mt tRNAs coincided with the evolution of bilaterian animals. We demonstrate that bilaterians compensated for this reduced structural complexity in mt tRNAs by sequence-independent induced-fit adaption to the cognate mitochondrial aminoacyl-tRNA synthetase (aaRS). Structural readout by nuclear-encoded aaRS partners relaxed the sequence constraints on mt tRNAs and facilitated accommodation of functionally disruptive mutational insults by cis-acting epistatic compensations. Our results thus suggest that mutational freedom in mt tRNA genes is an adaptation to increased mutation pressure that was associated with the evolution of animal complexity.

Nonprotein-Coding RNAs as Regulators of Development in Tunicates.

Tunicates, or urochordates, are a group of small marine organisms that are found widely throughout the seas of the world. As most plausible sister group of the vertebrates, they are of utmost importance for a comprehensive understanding of chordate evolution; hence, they have served as model organisms for many aspects of the developmental biology. Current genomic analysis of tunicates indicates that their genomes evolved with a fast rate not only at the level of nucleotide substitutions but also in terms of genomic organization. The latter involves genome reduction, rearrangements, as well as the loss of some important coding and noncoding RNA (ncRNAs) elements and even entire genomic regions that are otherwise well conserved. These observations are largely based on evidence from comparative genomics resulting from the analysis of well-studied gene families such as the Hox genes and their noncoding elements. In this chapter, the focus lies on the ncRNA complement of tunicates, with a particular emphasis on microRNAs, which have already been studied extensively for other animal clades. MicroRNAs are known as important regulators of key genes in animal development, and they are intimately related to the increase morphological complexity in higher metazoans. Here we review the discovery, evolution, and genome organization of the miRNA repertoire, which has been drastically reduced and restructured in tunicates compared to the chordate ancestor. Known functions of microRNAs as regulators of development in tunicates are a central topic. For instance, we consider the role of miRNAs as regulators of the muscle development and their importance in the regulation of the differential expression during the oral siphon regeneration. Beyond microRNAs, we touch upon the functions of some other ncRNAs such as yellow crescent RNA, moRNAs, RMST lncRNAs, or spliced-leader (SL) RNAs, which have diverse functions associated with the embryonic development, neurogenesis, and mediation of mRNA stability in general.

An atlas of alternative splicing profiles and functional associations reveals new regulatory programs and genes that simultaneously express multiple major isoforms.

Alternative splicing (AS) generates remarkable regulatory and proteomic complexity in metazoans. However, the functions of most AS events are not known, and programs of regulated splicing remain to be identified. To address these challenges, we describe the Vertebrate Alternative Splicing and Transcription Database (VastDB), the largest resource of genome-wide, quantitative profiles of AS events assembled to date. VastDB provides readily accessible quantitative information on the inclusion levels and functional associations of AS events detected in RNA-seq data from diverse vertebrate cell and tissue types, as well as developmental stages. The VastDB profiles reveal extensive new intergenic and intragenic regulatory relationships among different classes of AS and previously unknown and conserved landscapes of tissue-regulated exons. Contrary to recent reports concluding that nearly all human genes express a single major isoform, VastDB provides evidence that at least 48% of multiexonic protein-coding genes express multiple splice variants that are highly regulated in a cell/tissue-specific manner, and that >18% of genes simultaneously express multiple major isoforms across diverse cell and tissue types. Isoforms encoded by the latter set of genes are generally coexpressed in the same cells and are often engaged by translating ribosomes. Moreover, they are encoded by genes that are significantly enriched in functions associated with transcriptional control, implying they may have an important and wide-ranging role in controlling cellular activities. VastDB thus provides an unprecedented resource for investigations of AS function and regulation.

Recent advances on the functional and evolutionary morphology of the amniote respiratory apparatus.

Increased organismic complexity in metazoans was achieved via the specialization of certain parts of the body involved in different faculties (structure-function complexes). One of the most basic metabolic demands of animals in general is a sufficient supply of all tissues with oxygen. Specialized structures for gas exchange (and transport) consequently evolved many times and in great variety among bilaterians. This review focuses on some of the latest advancements that morphological research has added to our understanding of how the respiratory apparatus of the primarily terrestrial vertebrates (amniotes) works and how it evolved. Two main components of the respiratory apparatus, the lungs as the "exchanger" and the ventilatory apparatus as the "active pump," are the focus of this paper. Specific questions related to the exchanger concern the structure of the lungs of the first amniotes and the efficiency of structurally simple snake lungs in health and disease, as well as secondary functions of the lungs in heat exchange during the evolution of sauropod dinosaurs. With regard to the active pump, I discuss how the unique ventilatory mechanism of turtles evolved and how understanding the avian ventilatory strategy affects animal welfare issues in the poultry industry.

Pre-metazoan origins and evolution of the cadherin adhesome.

ABSTRACTVertebrate adherens junctions mediate cell–cell adhesion via a “classical” cadherin–catenin “core” complex, which is associated with and regulated by a functional network of proteins, collectively named the cadherin adhesome (“cadhesome”). The most basal metazoans have been shown to conserve the cadherin–catenin “core”, but little is known about the evolution of the cadhesome. Using a bioinformatics approach based on both sequence and structural analysis, we have traced the evolution of this larger network in 26 organisms, from the uni-cellular ancestors of metazoans, through basal metazoans, to vertebrates. Surprisingly, we show that approximately 70% of the cadhesome, including proteins with similarity to the catenins, predate metazoans. We found that the transition to multicellularity was accompanied by the appearance of a small number of adaptor proteins, and we show how these proteins may have helped to integrate pre-metazoan sub-networks via PDZ domain–peptide interactions. Finally, we found the increase in network complexity in higher metazoans to have been driven primarily by expansion of paralogs. In summary, our analysis helps to explain how the complex protein network associated with cadherin at adherens junctions first came together in the first metazoan and how it evolved into the even more complex mammalian cadhesome.

Predicting Spatial and Temporal Gene Expression Using an Integrative Model of Transcription Factor Occupancy and Chromatin State

Precise patterns of spatial and temporal gene expression are central to metazoan complexity and act as a driving force for embryonic development. While there has been substantial progress in dissecting and predicting cis-regulatory activity, our understanding of how information from multiple enhancer elements converge to regulate a gene's expression remains elusive. This is in large part due to the number of different biological processes involved in mediating regulation as well as limited availability of experimental measurements for many of them. Here, we used a Bayesian approach to model diverse experimental regulatory data, leading to accurate predictions of both spatial and temporal aspects of gene expression. We integrated whole-embryo information on transcription factor recruitment to multiple cis-regulatory modules, insulator binding and histone modification status in the vicinity of individual gene loci, at a genome-wide scale during Drosophila development. The model uses Bayesian networks to represent the relation between transcription factor occupancy and enhancer activity in specific tissues and stages. All parameters are optimized in an Expectation Maximization procedure providing a model capable of predicting tissue- and stage-specific activity of new, previously unassayed genes. Performing the optimization with subsets of input data demonstrated that neither enhancer occupancy nor chromatin state alone can explain all gene expression patterns, but taken together allow for accurate predictions of spatio-temporal activity. Model predictions were validated using the expression patterns of more than 600 genes recently made available by the BDGP consortium, demonstrating an average 15-fold enrichment of genes expressed in the predicted tissue over a naïve model. We further validated the model by experimentally testing the expression of 20 predicted target genes of unknown expression, resulting in an accuracy of 95% for temporal predictions and 50% for spatial. While this is, to our knowledge, the first genome-wide approach to predict tissue-specific gene expression in metazoan development, our results suggest that integrative models of this type will become more prevalent in the future.

The impact of microRNAs on the evolution of metazoan complexity

MicroRNAs (miRNAs) are a novel class of ~22 nt non-coding small RNAs. As crucial post-transcriptional regulators, miRNAs are involved in comprehensive biological processes such as developmental timing, cell proliferation and differentiation, oncogenesis and viral defenses. In addition to the roles in ontogenic physiology, researches on the area of miRNA phylogenetic conservation and diversity suggested that miRNAs play important roles in animal evolution through driving phenotypic variations in development. It has been postulated that miRNAs have enormous impacts on phenotypic variation and developmental complexity. Here we reviewed recent advances in the studies on the roles of miRNA in animal evolution, from aspects of the rate of miRNA evolution, the spatio-temporal expression pattern, the variation of target sites, and miRNA gene dynamics. We gave evidence to support the hypothesis that innovations in miRNA-mediated regulations drive the increase of metazoan complexity.

Origin and evolution of ubiquitin-conjugating enzymes from Guillardia theta nucleomorph to hominoid

The origin of eukaryotic ubiquitin-conjugating enzymes (E2s) can be traced back to the Guillardia theta nucleomorph about 2500 million years ago (Mya). E2s are largely vertically inherited over eukaryotic evolution [Lespinet, O., Wolf, Y.I., Koonin, E.V., Aravind, L., 2002. The role of lineage-specific gene family expansion in the evolution of eukaryotes. Genome Res. 1048–1059], while mammal E2s experienced evolution of multigene families by gene duplications which have been accompanied by the increase in the species complexity. Because of alternatively splicing, primate-specific expansions of E2s happened once again at a transcriptional level. Both of them resulted in increasing genomic complexity and diversity of primate E2 proteomic function. The evolutionary processes of human E2 gene structure during expansions were accompanied by exon duplication and exonization of intronic sequences. Exonizations of Transposable Elements (TEs) in UBE2D3, UBE2L3 and UBE2V1 genes from primates indicate that exaptation of TEs also plays important roles in the structural innovation of primate-specific E2s and may create alternative splicing isoforms at a transcriptional level. Estimates for the ratio of dN/dS suggest that a strong purifying selection had acted upon protein-coding sequences of their orthologous UBE2D2, UBE2A, UBE2N, UBE2I and Rbx1 genes from animals, plants and fungi. The similar rates of synonymous substitutions are in accordance with the neutral mutation–random drift hypothesis of molecular evolution. Systematic detection of the origin and evolution of E2s, analyzing the evolution of E2 multigene families by gene duplications and the evolutionary processes of E2s during expansions, and testing its evolutionary force using E2s from distant phylogenetic lineages may advance our distinguishing of ancestral E2s from created E2s, and reveal previously unknown relationships between E2s and metazoan complexity. Analysis of these conserved proteins provides strong support for a close relationship between social amoeba and eukaryote, choanoflagellate and metazoan, and for the central roles of social amoeba and choanoflagellate in the origin and evolution of eukaryote and metazoan. Retracing the different stages of primate E2 exonization by monitoring genomic events over 63 Myr of primate evolution will advance our understanding of how TEs dynamically modified primate transcriptome and proteome in the past, and continue to do so.

Cycliophoran Dwarf Males Break the Rule: High Complexity with Low Cell Numbers

Complexity of metazoan bodyplans is commonly assumed to be correlated to the absolute number of cells and the number of cell types present in a species (1). Sexually mature individuals of the smallest free-living animals have a minimum of several hundred somatic cells, and only secondarily simplified parasitic or commensal species range below this threshold. Males of the two hitherto described representatives of the phylum Cycliophora (2), with a body length of about 40 microm, are among the smallest existing free-living metazoans, yet they exhibit an amazingly complex bodyplan. Herein, we show that only a few dozen cells account for this complexity. We conclude therefore that metazoan complexity is not obligatorily correlated with body size or with the overall cell number of an individual. Accordingly, the earliest multicellular animals on Earth, which most probably were small individuals, may have had more complex bodyplans than commonly assumed.

Evolution of an RNP assembly system: A minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2-8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction.

Evolution of an RNP assembly system: A minimal SMN‐complex facilitates formation of U snRNPs in Drosophila melanogaster

In vertebrates, assembly of spliceosomal UsnRNPs is mediated by the SMN‐complex, a macromolecular entity composed of the proteins SMN and Gemins 2–8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN‐complex has gained complexity in evolution by a block‐wise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a novel biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains a SMN‐complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents mis‐assembly onto non‐target RNAs. Our data suggest that only a minority of Gemins is required for the assembly reaction per se, whereas others may serve additional functions in the context of U snRNP biogenesis. The evolution of the SMN‐complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction.

A bichromatic fluorescent reporter for cell-based screens of alternative splicing.

Alternative splicing is the primary source of proteome complexity in metazoans and its regulation shapes the proteome in response to shifting physiological requirements. We developed a bichromatic splicing reporter that uses a peculiar feature of some fluorescent protein coding regions to express two different fluorescent proteins from a single alternative splicing event. The mutually exclusive expression of different fluorescent proteins from a single reporter provides a uniquely sensitive approach for high-throughput screening and analysis of cell-specific splicing events in mixed cell cultures and tissues of transgenic animals. This reporter is applicable to the majority of alternative splicing patterns and can be used to quantify alternative splicing within single cells and to select cells that express specific splicing patterns. The ability to perform quantitative single-cell analysis of alternative splicing and high-throughput screens will enhance progress toward understanding splicing regulatory networks and identifying compounds that reverse pathogenic splicing defects.

Regulating a Circadian Clock's Period, Phase and Amplitude by Phosphorylation: Insights from Drosophila

Much progress has been made in understanding the molecular underpinnings governing circadian ( approximately 24 h) rhythms. Despite the increased complexity in metazoans whereby inter-cellular networks form the basis for driving overt rhythms, such as wake-sleep cycles in animals, single isolated cells can exhibit all the formal properties of a circadian pacemaker. How do these cell-autonomous rhythm generators operate? Breakthrough studies in Drosophila melanogaster led to the realization that the molecular logic underlying circadian clocks are highly shared. Most notably, interconnected transcriptional-translational feedback loops produce coordinated rhythms in "clock" RNAs and proteins that are required for the daily progression of clocks, synchronization to local time and transducing temporal signals to downstream effector pathways. More recent findings indicate prominent roles for reversible phosphorylation of clock proteins in the core oscillatory mechanism. In this review we focus on findings in Drosophila to explore the multiple levels that reversible phosphorylation plays in clock function. Specific clock proteins in this system are subjected to different phosphorylation programs, which affect three key properties of a circadian oscillator, its period, amplitude and phase. The role of phosphorylation in clocks is of clear relevance to human health because mutations that affect the PERIOD (PER) phosphorylation program are associated with familial sleep disorders. In addition, the central role of phosphorylation in the assembly of a circadian oscillator was dramatically shown recently by the ability to reconstitute a circadian phosphorylation/dephosphorylation cycle in vitro, suggesting that the dynamics of clock protein phosphorylation are at the "heart" of circadian time-keeping.

Big, bad hearts: From flies to man

Genetic screens have revealed essential genes that guide metazoan complexity, from fates of individual cells, to patterning of cell arrays, to their assembly into organs (1–3).

Rat models of complex disease and evolution of metazoan complexity

We suggest a unified view that oxygen metabolism underlies mechanistically major aspects of the evolution of complexity, and thus by default, complex disease. This view is based upon connecting two generalities. First, geochemical and fossil records support the notion that atmospheric oxygen is unique for the evolution of metazoan complexity (Science 309, 2202). Second, clinical observations demonstrate a strong statistical association between aerobic capacity and all-cause mortality (N. Eng. J. Med, 346, 793). From these ideas, we hypothesized that artificial selection of rats for low and high aerobic exercise capacity would yield models that contrast for the divide between health and disease. In 1996 we started large-scale selective breeding to develop strains of rats that contrast for intrinsic (i.e., untrained) aerobic treadmill running capacity. After eleven generations of selection, the low capacity runners (LCR) and high capacity runners (HCR) differed by over 300% in aerobic running capacity. The LCR scored higher on cardiovascular risks and features of the metabolic syndrome, including higher blood pressure, insulin, random glucose,fasting glucose, free fatty acids, visceral fat, and triglycerides. The HCR were higher for health factors such as maximal oxygen consumption, heart function, nitric oxide formation in arteries, economy of oxygen use, and abundance of proteins required for mitochondrial function in skeletal muscle (Science 307, 418). These rats represent potentially useful models for unraveling the genetic and environmental causes of complex disease at all levels of biologic organization. Supported by NIH grants HL64270 and RR17718.

Genome complexity and repetitive DNA in metazoans from extreme marine environments

As genomics converges with ecology and evolution to identify the fundamental linkages between genome structure and function, genome and transcriptome complexity will need to be measured in organisms from more diverse habitats, most often in the absence of complete sequence data. Here, we describe the complexity of ten genomes measured by a novel, high-throughput fluorescence-based kinetic hybridization assay. We applied the Shannon information index, H, and a related, fluorescence-adjusted index, H f, as unique metrics of the hybridization kinetics to complement the conventional rate constant, k. A strong, positive relationship was present between H f, and the repetitive DNA content of five eukaryotic genomes previously determined by Cot kinetic analyses ( Onchorynchus keta, Ilyanassa obsoleta, Bos taurus, Limulus polyphemus, Saccharyomyces cerevisiae). This relationship was used to characterize the complexity of previously unstudied genomic samples in five metazoan taxa from three marine environments, including deep-sea hydrothermal vents ( Alvinella pompejana), the temperate subtidal ( Streblospio benedicti), and Antarctic coastal bays ( Sterechinus neumayeri, Odontaster validus, Tritonia antarctica). Contrary to the predictions of nucleotypic theory, Antarctic invertebrates consistently had the lowest quantities of repetitive DNA in conjunction with low metabolic rates and highly protracted rates of cell division and larval development. Conversely, hydrothermal vent species with rapid cell division and growth do not have significantly different genome sizes or particularly low amounts of repetitive DNA as compared to non-vent, deep-sea taxa. Furthermore, there appears to be a positive correlation between the temperature at which the most abundant repetitive sequence classes anneal and habitat thermal stability. Thus, our study reveals a potential shift in repetitive sequence representation between these extreme environments that may be related to genome function in species living at these different thermal regimes.

Simulation study on effects of signaling network structure on the developmental increase in complexity

The developmental increase in structural complexity in multicellular lifeforms depends on local, often non-periodic differences in gene expression. These, in turn, depend on a network of gene–gene interactions coded within the organismal genome. To see what architectural features of a network (size, connectivity, etc.) affect the likelihood of patterns with multiple cell types (i.e. patterns where cells express ⩾3 different combinations of genes), developmental pattern formation was simulated in virtual blastoderm embryos with small artificial genomes. Several basic properties of these genomic signaling networks, such as the number of genes, the distributions of positive (inductive) and negative (repressive) interactions, and the strengths of gene–gene interactions were tested. The results show that the frequencies of complex and/or stable patterns depended not only on the existence of negative interactions, but also on the distribution of regulatory interactions: for example, coregulation of signals and their intracellular effectors increased the likelihood of pattern formation compared to differential regulation of signaling pathway components. Interestingly, neither quantitative differences in strengths of signaling interactions nor multiple response thresholds to different levels of signal concentration (as in morphogen gradients) were essential for formation of multiple, spatially unique “cell types”. However, those combinations of architectural features that greatly increased the likelihood for pattern complexity tended to decrease the likelihoods for pattern stability and developmental robustness. Nevertheless, elements of complex patterns (e.g. genes, cell type order within the pattern) could differ in their developmental robustness, which may be important for the evolution of complexity. The results show that depending on the network structure, the same set of genes can produce patterns of different complexity, robustness and stability. Because of this, the evolution of metazoan complexity with a combinatorial code of gene regulation may have depended at least as much on selection for favorable distribution of connections between existing developmental regulatory genes as on the simple increase in numbers of regulatory genes.