Most genes belong to gene families, which have emerged through consecutive cycles of gene duplications during evolution 1. With the availability of entire genome sequences, much progress has been made towards the understanding of gene duplication dynamics 23 and the evolutionary forces responsible for the retention of a proportion of duplicate genes, such as neo-functionalization 4 and sub-functionalization 5, both at the level of gene expression patterns 67 and protein sequence evolution 8. However, further investigation is required to understand how genomic processes, such as gene duplications and losses, result in higher-level co-ordinated molecular events, such as the emergence of novel signal transduction pathways, which in turn give rise to phenotypic innovations, such as novel organs, developmental phases, or body plans.

To approach this question from a comparative genomics viewpoint, we focus herein on the emergence and evolution of the transforming growth factor-β (TGF-β) pathway within the animal kingdom. This pathway has been recognized as one of the fundamental and versatile metazoan signal transduction engines, with central roles in development, organogenesis, stem-cell control, immunity, and cancer 91011. A concise description of the human pathway has been deposited by the authors of this article in the Reactome 12 knowledge base 13.

The cellular core of all TGF-β superfamily pathways consists of cell surface receptors, called type I and type II serine-threonine kinase receptors, and intracellular Smad proteins 14. The latter constitute the actual signal transduction engine of the pathway 15. There are eight known Smads in the human genome, classified as: two TGF-β sensu stricto (Smad2,3) and three bone morphogenetic protein (BMP)-type (Smad1,5,8) receptor-activated Smads (R-Smads), one common mediator Smad (Co-Smad; Smad4), and two inhibitory Smads (I-Smads; Smad6,7). These eight genes are highly similar in sequence and are evidently results of multiple gene duplications of unknown origins. While the functional differences between the three biochemical classes of Smads are well known, their evolutionary history, the characteristics of the ancestral Smad molecule, and the selection forces behind the retention of multiple subtypes of R- and I-Smads are poorly understood.

In humans, we encounter five distinct type II receptors and seven distinct type I receptors 16. The functional receptor unit is a hetero-tetramer of two type II receptors with two type I receptors, in which upon binding of the ligand the type II receptors phosphorylate the type I receptors, while the latter phosphorylate and activate R-Smads. Analysis of the receptor genes, so far has been limited to a few species, namely humans, rodents, African clawed frog (Xenopus laevis), fruit fly (Drosophila melanogaster) and the free-living roundworm Caenorhabditis elegans 17.

Mammalian genomes encode up to 33 TGF-β related ligands, D. Melanogaster seven and C. elegans five (out of which only two are functionally characterised) 18. However, we do not focus on TGF-β related proteins in this study, as these sequences are rather diverged (and similarity is mostly confined to the carboxyterminal polypeptide of the much larger precursor proteins) rendering them difficult to analyse in multiple genomes using a unified computational pipeline. We refer the interested reader to a review by Herpin et al 19. The most prevalent mode of extracellular modulation of TGF-β signalling is by means of soluble antagonists, called ligand traps, such of those of the chordin and gremlin family 20. BAMBI is another important negative regulator of TGF-β signalling, related to TGF-β family type I receptors but lacking an intracellular kinase domain 21.

BMP signalling gradients, modulated by chordin, have been found to induce dorsoventral axis formation in the Spemann organizer 22. Thus, traditionally, the TGF-β pathway had been thought to have evolved in the context of dorsoventral patterning, and thus be present only in Bilateria. This view has been recently challenged by the discovery of the functional pathway in multiple cnidarians 23242526272829. Furthermore, the origin of animals themselves is only now being understood (for reviews see 3031). On the basis of mitochondrial DNA sequence comparison, the choanoflagellates have been identified as the closest single-celled animal relatives 3233 while the Placozoan Trichoplax adhaerens, the so-called tablet animal 3435, has been placed at the root of animal phylogeny 3436. However, some authors regard sponges as earlier diverging than Placozoans 3738. Regardless of the relative position of Placozoans and sponges, the critical step of transformation to multicellulararity must have been accompanied by the development of adhesion molecules, extracellular matrix proteins (such as collagen), and cell-to-cell communication. It is essential to identify the critical signalling pathways, in particular those involved in control of development, cellular differentiation and body plan formation 31. Such comparisons will not only shed light on metazoan origins, and advance the field of evo-devo, but will also help us understand the fundamental functional motifs that underlie interwoven signal transduction networks of higher animals, with impact on human health.

It was reported previously that atypical dauer pathway Smads could be found in free-living bacterial feeding nematodes of the genus Rhabditis (Rhabditoid nematodes) 39. The dauer (German for resting) is a survival and dissemination form, formed by all Rhabditoid nematodes, an alternative to the active third stage larvae (L3). Dauers are induced by environmental stress factors, such as lack of food, overcrowding, or high temperature. The dauer pathway (which also includes insulin pathway-like and guanyl cyclase pathway-like genes) is of high general interest, as it has been linked with aging 40, biodiversity 41 and the development of parasitism in nematodes 42. However, the origins of the dauer pathway Smads had been previously unknown.

The growing number of sequenced genomes provides a relatively wide coverage of the animal genome space. This makes it possible to reconstruct ancestral developmental signalling pathways, and to retrace the ancient evolutionary events which led to their emergence and modulation, in particular gene duplications, instances of sub- and neo-functionalization, and gene losses. Herein, we focus on the gene set constituting the fundamental building blocks of a major component of the animal developmental toolkit – the TGF-β pathway.

We have examined in detail the gene content of the TGF-β pathway in extant genomes of different metazoan phyla, where high-coverage genomic data are available (Table 1). Smads are of particular interest, as they constitute the core engine of the TGF-β signal transduction machinery. We have estimated the origin of different types of Smads by examining extant genomes and inferring ancestral genes (Point of Divergence analysis – Figure 1 summarises Figure S1 [see Additional file 1]). We justify somewhat anthropocentric approach of the POD analysis by the high significance of the TGF-β pathway in human health and disease, which drives substantial proportion of research in the field. On the lineage of PODs leading to human, the Smads clearly appear to have gone through a major wave of duplications, fitting well with the 2R hypothesis of two-fold genome duplication at the base of vertebrates 47484950. Additional duplications occurred along the teleost fish lineage, in congruence with the hypothesis of a teleost fish-specific genome duplication – FSGD 5152. Diversification of type I and type II receptors has also followed the course agreeable with the 2R hypothesis, with multiple additional duplications in teleost fishes (Figure S2, [see Additional file 2]).

POD analysis (Figure 1) shows that the core pathway (both receptors and Smads) expanded dramatically and permanently at the base of vertebrates. Table 1 demonstrates that this expansion correlates well with the increase of complexity of regulatory networks associated with the extended pathway, such as ligand traps of the chordin and gremlin family. The same is true of many transcriptional co-activators, and target genes – particularly those in the concurrently developed active immune system, as well as the endocytic regulators that control Smad signalling, SARA and endofin, which emerged through the duplication of a single ancestral gene (data not shown).

Analysis of the C. elegans genome revealed atypical Smads belonging to Sma/Mab and dauer pathways. Our phylogenetic tree indicates that daf-8 is an R-Smad, daf-3 a Co-Smad and Tag-68 an I-Smad (Figure 2). Sma-2 and sma-3 are likely duplicates of the ancestral BMP R-Smad, as they both contain the characteristic RQDVTS motif of the L3 loop. Conversely, daf-8 and daf-14 might be duplicates of the ancestral TGF-β R-Smad, although daf-14 is too divergent to allow firm conclusions. Sma-4 and daf-3 share a similar pattern of multiple splice variants, which together with the tree topology suggests that they derive from the ancestral Co-Smad via a gene duplication event. Comparative analysis revealed that Sma/Mab and dauer pathway content is identical between C. elegans and C. briggsae, with strong conservation of the overall gene structure and synteny (Table 3). This proves that all the relevant genes already existed in the last common ancestor of the two Rhabditoid species. Although similar in morphology, C. briggsae and C. elegans are rather distant relatives in evolutionary time: the two species split roughly 100 million years ago 53. Analysis of lengths of protein branches (Figure 2) is indicative of accelerated evolution of daf-3, 8 and 14. Additionally, analysis of Ka/Ks ratios between pairs of orthologs in C. briggsae and C. elegans suggests that the dauer pathway evolved faster since the two species diverged (Table 3). The average Ka/Ks ratio for dauer pathway orthologs is 0.72 versus 0.16 for non-dauer TGF-β pathway genes. Thus, the initial duplications and neo-functionalization occurred early in nematode evolution, but have been followed by further change in separate Rhabditoid lineages, as different species experienced slightly different selection pressures for entry and persistence in their dauer forms. For example, C. elegans, unlike C. briggsae, is strongly induced to form dauers at temperatures higher than 26°C 54. Overall, the dauer pathway represents an interesting example of rapidly evolving pathway neo-functionalization, developed as a lineage-specific adaptation towards the colonization of the environmental niche of the soil.

The crucial question about the taxonomic origin of the TGF-β pathway has not been categorically answered yet. Herein, we have identified TGF-β pathway components in T. adhaerens, the representative of the early diverging phylum Placozoa, and the demosponge Amphimedon queenslandica 55. Choanoflagellata are the closest unicellular relatives of animals 32 and possess some genes linked to metazoan development, for example a receptor tyrosine kinase – MBRTK1 56. However, we have not been able to detect Smads, TGF-β receptors, ligands, SARA, chordin or gremlin in the genome of the unicellular choanoflagellate M. brevicollis 33, or more distantly related protozoans Volvox carteri and Naegleria gruberi. This indicates that the appearance of the TGF-β pathway was intrinsically linked to the emergence of earliest animals, and the pathway may thus be regarded as a key feature of the metazoan life forms. It is also rather striking that such an early diverging animal as Trichoplax already possesses the complete functional pathway, including multiple Smads, receptors, and ligands, as well as orthologs of chordin, gremlin and SARA.

We hypothesize that the single primeval common mediator/receptor activated Smad functioned as a homo-dimer (or homo-trimer), and possessed the universal functionality of R-Smads and the Co-Smad; i.e. it could be phosphorylated by the receptor/ligand complex, shuttle to the nucleus, interact with transcriptional co-activators via the MH2 domain and bind DNA via the MH1 domain. As the number of ligands and receptors grew, the primeval Smad duplicated and, through sub-functionalization, gave rise to two separate R-Smads which from then on interact with non-overlapping sets of receptors (Figure 4: ancestral metazoan duplication – AMD 1; Table 4). One of the R-Smads duplicated again (Figure 4: AMD 2) giving rise to a Co-Smad which enhanced the set of regulatory protein interactions, and possibly provided a way of integrating the signals from the two R-Smad channels through competition for the available pool of Co-Smad molecules. The critical role of Co-Smad bioavailability is also suggested by its low duplicability; in the great majority of species there is only one Co-Smad (Figure 1). Xenopus laevis is the notable exception having two genes XSMad4a and XSmad4b 5758, but they are differentially expressed both in embryos and adult tissues. The fast diverging I-Smad was the last addition to the pathway (Figure 4: AMD 3). It neofunctionalized to create a controlling negative feedback loop; I-Smad transcription is induced by the pathway, the protein can bind the activated receptor complex, but lacking a terminal phosphorylation motif it does not propagate the signal. Since it is not being used, over time the MH1 of the I-Smad converted to a vestigial domain. It will be interesting to see if future genome projects of basal animals and closest extant unicellular relatives of animals 59 will provide a proof of our single Smad hypothesis.

The emergence of the TGF-β pathway coincided with appearance of the first animal species, and was most likely linked with duplications of the single primeval common mediator/receptor activated Smad. This resulted in the creation of the ancestral eumetazoan repertoire of four Smads, forming the basis of the pathway in the Placozoa, the Cnidaria, the Arthropoda, and in the Lophotrochozoa. After application of a formal speciation and duplication inference algorithm, we conclude that the diversification of Smads and receptors in chordates is parsimoniously explained according to the 2R model, with additional rounds of duplications in teleost fishes. The Nematoda posses a heavily modified pathway which evolution has been marked by accelerated sequence change.

Our multi-genome comparison and ancestral inference approach has implications extending beyond the TGF-β pathway. Origins of other developmental signalling pathways, for example Hox and hedgehog, are also being investigated using phylogenomic approaches 6061. Results obtained for all developmental signalling pathways should be integrated and compared with paleontological records and molecular clock data, to identify the molecular nature and timing of all major changes in the shared animal developmental toolkit 62, including those which gave rise to vertebrate innovations 63.

LH gathered the data, designed and performed all the analyses, and wrote the manuscript. AM and CHH participated in the study design, provided feedback on results, and contributed to writing the manuscript. LG, SF and CO prepared a pilot version of Figure 1 and were involved in drafting the manuscript. All authors read and approved the final manuscript.