Early evolution of animal cell signaling and adhesion genes Scott A. Nichols*, William Dirks†, John S. Pearse‡, and Nicole King*†§¶ Departments of *Molecular and Cell Biology and §Integrative Biology and †Center for Integrative Genomics, University of California, Berkeley, CA 94720; and ‡Joseph M. Long Marine Laboratory, University of California, Santa Cruz, CA 95060 Edited by James W. Valentine, University of California, Berkeley, CA, and approved June 28, 2006 (received for review May 17, 2006) In stark contrast to the rapid morphological radiation of eumeta- underlying basement membrane (Fig. 1 c and d) and regularly zoans during the Cambrian explosion, the simple body plan of distributed cell–cell junctions (12, 13). The phylogenetic position sponges (Phylum Porifera) emerged from the Cambrian relatively of sponges and the cell biology of homoscleromorph sponges unchanged. Although the genetic and developmental underpin- suggest that homoscleromorphs may provide an unprecedented nings of these disparate evolutionary outcomes are unknown, window into the ancestry of genes important for eumetazoan comparisons between modern sponges and eumetazoans promise development and evolution. to reveal the extent to which critical genetic factors were present Two crucial classes of genes in this respect are the signaling in their common ancestors. Two particularly interesting classes of and adhesion genes that interact to coordinate complex mor- genes in this respect are those involved in cell signaling and phogenetic events in eumetazoans as diverse as flies, worms, and adhesion. These genes help guide development and morphogen- humans (14–16). Cell-signaling pathways allow cells to receive, esis in modern eumetazoans, but the timing and sequence of their relay, and interpret messages from the extracellular environment origins is unknown. Here, we demonstrate that the sponge Os- and are involved in developmental cell-fate specification and carella carmela, one of the earliest branching animals, expresses patterning (15). Classically, seven major signaling pathways have core components of the Wnt, transforming growth factor ␤, been identified as unique to, and ubiquitous among, eumetazo- receptor tyrosine kinase, Notch, Hedgehog, and Jak͞Stat signaling ans (15): Wnt, transforming growth factor ␤ (TGF␤), Hedgehog, pathways. Furthermore, we identify sponge homologs of nearly receptor tyrosine kinase (RTK), Jak͞STAT, Notch, and nuclear every major eumetazoan cell-adhesion gene family, including hormone receptor. Core elements from each of these pathways those that encode cell-surface receptors, cytoplasmic linkers, and are present in eumetazoans ranging from cnidarians to verte- extracellular-matrix proteins. From these data, we infer that key brates (17), where they have both retained their presumed signaling and adhesion genes were in place early in animal evo- ancestral roles, such as in axial patterning, and been coopted to lution, before the divergence of sponge and eumetazoan lineages. guide the development of derived features, such as wings and eyes. With the exception of several RTKs (18–23) and a Frizzled eumetazoa ͉ porifera ͉ Cambrian explosion ͉ homoscleromorpha ͉ receptor (part of the Wnt pathway) (24), it is not known whether Oscarella the core components of any of the remaining pathways exist in sponges. Because cell signaling sits at the top of key develop- mental regulatory networks (15, 25), one prediction is that he fossil record from Ϸ540 Mya documents the abrupt morphological novelties during the diversification of eumeta- Tappearance of fully diversified eumetazoan body plans dur- zoan body plans stemmed, in part, from evolutionary changes in ing the ‘‘Cambrian explosion.’’ In contrast, fossil evidence of Ϸ signaling pathways. sponges dates to 580 Mya and reveals that their simple body A second class of genes relevant to the search for intrinsic plan predates the Cambrian and has since remained relatively factors underlying the diversification of eumetazoan body plans unchanged (1, 2). Although extrinsic environmental factors have are the cell-adhesion genes, which are often downstream targets received much attention as the driving force behind the Cam- of signaling cascades. By helping to regulate the sorting and brian explosion (3), the disparate outcomes in lineages leading migration of different cell populations, cell-adhesion genes act to to modern sponges and eumetazoans point to intrinsic genetic effect pivotal morphogenetic changes during eumetazoan devel- differences. Intrinsic factors that potentially contributed to the opment (14, 16). Cell-adhesion proteins also play critical roles in selective morphological diversification of eumetazoans include eumetazoan epithelia to establish and maintain the transport the evolution of developmentally important gene families, sig- and occluding functions hypothesized to be prerequisites of naling pathways, or regulatory elements and linkages in their animal body-plan diversification (26). The functional properties ancestors. A key to illuminating how intrinsic factors contributed of epithelia laid the foundation for the evolution of differenti- to the Cambrian explosion will be to reconstruct the evolution- ated body compartments with unique physiological properties ary sequence of origination, elaboration, and assembly of de- (26) and, therefore, were likely to have served as an important velopmentally important gene families into interacting pathways template for morphological evolution during the Cambrian and networks. radiation. Despite intensive study of cell adhesion in sponges, few Modern sponges promise to offer critical insights into the sponge homologs of eumetazoan cell-adhesion gene families earliest events in animal genome evolution. The fossil record of have been identified. Instead, models of cell adhesion in sponges sponges predates that of all other animal groups (2), and largely emphasize the interactions between carbohydrate moi- phylogenetic analyses support sponges as an outgroup of all eumetazoan phyla (4). Indeed, recent studies indicate that sponges are paraphyletic (5–9) and that eumetazoans evolved Conflict of interest statement: No conflicts declared. from a sponge-like ancestor (9). Among the major sponge This paper was submitted directly (Track II) to the PNAS office. groups, the homoscleromorphs (Fig. 1b) have particular rele- Abbreviations: GO, Gene Ontology; NCBI, National Center for Biotechnology Information; vance to the study of eumetazoan origins and evolution. Al- nr, nonredundant. though of uncertain phylogenetic position (10, 11), homoscle- Data deposition: The sequences reported in this paper have been deposited in the GenBank romorphs are thought to be unique among sponges in their database (accession nos. EB741367–EB741549 and EC366551–EC377543). EVOLUTION possession of eumetazoan-like ultrastructural features, including ¶To whom correspondence should be addressed. E-mail: [email protected]. an epithelium characterized by closely apposed cells with an © 2006 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604065103 PNAS ͉ August 15, 2006 ͉ vol. 103 ͉ no. 33 ͉ 12451–12456 Downloaded by guest on September 26, 2021 Fig. 1. An introduction to the sponge, O. carmela. (a) Phylogenetic position of O. carmela (asterisk) relative to model bilaterians. O. carmela belongs to one of the earliest branching phyla of animals and may provide insight into the biology and genome of the first multicellular animals. (b) Macroscopic view of O. carmela overgrowing a standard brick (shown for scale). As an adult, O. carmela has no body axes and no differentiated structures other than epithelia. (c and d) Electron micrographs of the larval epithelium of O. carmela demonstrate that it has closely apposed cells (c) and a basement membrane (d, arrowheads), a feature thought to be exclusive to eumetazoans and the sponge clade containing Oscarella, the homoscleromorph sponges. Dd, D. discoideum; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Oc, O. carmela; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Gg, Gallus gallus; Mm, Mus musculus; Hs, Homo sapiens. (Scale bars, 500 nm.) eties of large extracellular proteoglycans (termed aggregation which complete genome sequences are available, both presence factors) that are hypothesized to bind to each other and to cell and absence data shown in Table 1 are meaningful. Notably, surface receptors that may interact with integrins (27, 28). pathway components that have been previously characterized To identify cell-signaling and adhesion genes that evolved only in eumetazoans [e.g., Wnt, Dickkopf, Patched, Notch, Delta, before the divergence of sponges and eumetazoans, we collected Mothers against decapentaplegic (Mad), and Janus kinase (Jak)] and annotated 11,520 ESTs from the homoscleromorph sponge are now known from sponges, thus extending the history of these Oscarella carmela (phylum Porifera; Fig. 1). Despite the mor- important genes back to some of the earliest animal ancestors. phological simplicity of sponges, we find that six of the seven An unexpected outcome from comparative genetic studies of major eumetazoan signaling pathways have homologs in O. early branching animals is that they sometimes have homologs of carmela. Furthermore, O. carmela expresses an unexpected genes previously thought to be derived within bilaterian clades. diversity of eumetazoan cell-adhesion gene homologs, repre- This was true of the discovery of Dickkopf in Cnidarians