David Q. Matus

Broad phylogenomic sampling improves resolution of the animal tree of life.

Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.

Pre-bilaterian origins of the Hox cluster and the Hox code: evidence from the sea anemone, Nematostella vectensis.

Background Hox genes were critical to many morphological innovations of bilaterian animals. However, early Hox evolution remains obscure. Phylogenetic, developmental, and genomic analyses on the cnidarian sea anemone Nematostella vectensis challenge recent claims that the Hox code is a bilaterian invention and that no “true” Hox genes exist in the phylum Cnidaria. Methodology/Principal Findings Phylogenetic analyses of 18 Hox-related genes from Nematostella identify putative Hox1, Hox2, and Hox9+ genes. Statistical comparisons among competing hypotheses bolster these findings, including an explicit consideration of the gene losses implied by alternate topologies. In situ hybridization studies of 20 Hox-related genes reveal that multiple Hox genes are expressed in distinct regions along the primary body axis, supporting the existence of a pre-bilaterian Hox code. Additionally, several Hox genes are expressed in nested domains along the secondary body axis, suggesting a role in “dorsoventral” patterning. Conclusions/Significance A cluster of anterior and posterior Hox genes, as well as ParaHox cluster of genes evolved prior to the cnidarian-bilaterian split. There is evidence to suggest that these clusters were formed from a series of tandem gene duplication events and played a role in patterning both the primary and secondary body axes in a bilaterally symmetrical common ancestor. Cnidarians and bilaterians shared a common ancestor some 570 to 700 million years ago, and as such, are derived from a common body plan. Our work reveals several conserved genetic components that are found in both of these diverse lineages. This finding is consistent with the hypothesis that a set of developmental rules established in the common ancestor of cnidarians and bilaterians is still at work today.

vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms

Summary Most bilaterians specify primordial germ cells (PGCs) during early embryogenesis using either inherited cytoplasmic germ line determinants (preformation) or induction of germ cell fate through signaling pathways (epigenesis). However, data from nonbilaterian animals suggest that ancestral metazoans may have specified germ cells very differently from most extant bilaterians. Cnidarians and sponges have been reported to generate germ cells continuously throughout reproductive life, but previous studies on members of these basal phyla have not examined embryonic germ cell origin. To try to define the embryonic origin of PGCs in the sea anemone Nematostella vectensis, we examined the expression of members of the vasa and nanos gene families, which are critical genes in bilaterian germ cell specification and development. We found that vasa and nanos family genes are expressed not only in presumptive PGCs late in embryonic development, but also in multiple somatic cell types during early embryogenesis. These results suggest one way in which preformation in germ cell development might have evolved from the ancestral epigenetic mechanism that was probably used by a metazoan ancestor.

Dorso/Ventral Genes Are Asymmetrically Expressed and Involved in Germ-Layer Demarcation during Cnidarian Gastrulation

Cnidarians (corals, sea anemones, hydroids, and jellyfish) are a basal taxon closely related to bilaterally symmetrical animals and have been characterized as diploblastic and as radially symmetrical around their longitudinal axis. We show that some orthologs of key bilaterian dorso/ventral (D/V) patterning genes, including the TGFbeta signaling molecules NvDpp and NvBMP5-8 and their antagonist NvChordin, are initially expressed asymmetrically at the onset of gastrulation in the anthozoan sea anemone Nematostella vectensis. Surprisingly, unlike flies and vertebrates, the TGFbeta ligands and their antagonist are colocalized at the onset of gastrulation but then segregate by germ layer as gastrulation proceeds. TGFbeta ligands, their extracellular enhancer, NvTolloid, and components of their downstream signaling pathway (NvSmad1/5 and NvSmad4) are all coexpressed in presumptive endoderm, indicating that only planar TGFbeta signaling operates at these stages. NvChordin expression forms a boundary between TGFbeta-expressing endodermal cells and aboral ectoderm. Manipulation of nuclear beta-catenin localization affects TGFbeta ligand and antagonist expression, suggesting that the ancestral role of the dpp/chordin antagonism during gastrulation may have been in germ-layer segregation and/or epithelial patterning rather than dorsal/ventral patterning.

The evolutionary origin of hedgehog proteins

Summary Animal development is orchestrated largely by diffusible ligands of the Wnt, TGF- β , hedgehog (Hh) and FGF signaling pathways, as well as cell-surface molecules, such as Notch, cadherins, integrins and the immunoglobulin-like proteins [1,2]. Here, we show that Hh proteins are likely to have evolved very early in metazoan evolution by domain shuffling. We identify in sponges and cnidarians a transmembrane protein, Hedgling, that contains the amino-terminal, signalling domain of Hh (hedge-domain), as well as cadherin, EGF and immunoglobulin domains. While Hedgling appears to have been lost in bilaterians, the likely capture of a hedge-domain by the more ancient, intein derived hog-domain may have given rise to the Hh proteins.

Broad taxon and gene sampling indicate that chaetognaths are protostomes

Despite advances in phylogenetic methods, there are still a number of enigmatic phyla whose affinities remain poorly resolved. One of the most recalcitrant of these is a group of small predatory marine invertebrates, the chaetognaths (arrow worms). Resolution of the phylogenetic position of the chaetognaths is key for reconstructing the evolutionary history of some of the most fundamental features of animals, including those that have been used to delineate two major clades of animals — the protostomes and deuterostomes.

Expression of Pax gene family members in the anthozoan cnidarian, Nematostella vectensis

SUMMARY Pax genes are a family of homeodomain transcription factors that have been isolated from protostomes (e.g., eight in Drosophilia) and deuterostomes (e.g., nine in vertebrates) as well as outside the Bilateria, from sponges, a placozoan, and several classes of cnidarians. The genome of an anthozoan cnidarian, the starlet sea anemone, Nematostella vectensis, has been surveyed by both degenerate polymerase chain reaction and in silico for the presence of Pax genes. N. vectensis possesses seven Pax genes, which are orthologous to cnidarian Pax genes (A,B,C, and D) previously identified in another anthozoan, a coral, Acropora millepora. Phylogenetic analyses including data from nonchordate deuterostomes indicates that there were five Pax gene classes in the protostome–deuterostome ancestor, but only three in the cnidarian‐bilaterian ancestor, with PaxD class genes lost in medusozoan cnidarians. Pax genes play diverse roles in bilaterians, including eye formation (e.g., Pax6), segmentation (e.g., Pax3/7 class genes), and neural patterning (e.g., Pox‐neuro, Pax2/5/8). We show the first expression data for members of all four Pax classes in a single species of cnidarian. N. vectensis Pax genes are expressed in both a cell‐type and region‐specific manner during embryogenesis, and likely play a role in patterning specific components of the cnidarian ectodermal nerve net. The results of these patterns are discussed with respect to Pax gene evolution in the Bilateria.

FGF signaling in gastrulation and neural development in Nematostella vectensis, an anthozoan cnidarian

The fibroblast growth factor (FGF) signal transduction pathway serves as one of the key regulators of early metazoan development, displaying conserved roles in the specification of endodermal, mesodermal, and neural fates during vertebrate development. FGF signals also regulate gastrulation, in part, by triggering epithelial to mesenchymal transitions in embryos of both vertebrates and invertebrates. Thus, FGF signals coordinate gastrulation movements across many different phyla. To help understand the breadth of FGF signaling deployment across the animal kingdom, we have examined the presence and expression of genes encoding FGF pathway components in the anthozoan cnidarian Nematostella vectensis. We isolated three FGF ligands (NvFGF8A, NvFGF8B, and NvFGF1A), two FGF receptors (NvFGFRa and NvFGFRb), and two orthologs of vertebrate FGF responsive genes, Sprouty (NvSprouty), an inhibitor of FGF signaling, and Churchill (NvChurchill), a Zn finger transcription factor. We found these FGF ligands, receptors, and response gene expressed asymmetrically along the oral/aboral axis during gastrulation and in a developing chemosensory structure of planula stages known as the apical tuft. These results suggest a conserved role for FGF signaling molecules in coordinating both gastrulation and neural induction that predates the Cambrian explosion and the origins of the Bilateria.

Genomic Organization and Expression Demonstrate Spatial and Temporal Hox Gene Colinearity in the Lophotrochozoan Capitella sp. I

Hox genes define regional identities along the anterior–posterior axis in many animals. In a number of species, Hox genes are clustered in the genome, and the relative order of genes corresponds with position of expression in the body. Previous Hox gene studies in lophotrochozoans have reported expression for only a subset of the Hox gene complement and/or lack detailed genomic organization information, limiting interpretations of spatial and temporal colinearity in this diverse animal clade. We studied expression and genomic organization of the single Hox gene complement in the segmented polychaete annelid Capitella sp. I. Total genome searches identified 11 Hox genes in Capitella, representing 11 distinct paralog groups thought to represent the ancestral lophotrochozoan complement. At least 8 of the 11 Capitella Hox genes are genomically linked in a single cluster, have the same transcriptional orientation, and lack interspersed non-Hox genes. Studying their expression by situ hybridization, we find that the 11 Capitella Hox genes generally exhibit spatial and temporal colinearity. With the exception of CapI-Post1, Capitella Hox genes are all expressed in broad ectodermal domains during larval development, consistent with providing positional information along the anterior–posterior axis. The anterior genes CapI-lab, CapI-pb, and CapI-Hox3 initiate expression prior to the appearance of segments, while more posterior genes appear at or soon after segments appear. Many of the Capitella Hox genes have either an anterior or posterior expression boundary coinciding with the thoracic–abdomen transition, a major body tagma boundary. Following metamorphosis, several expression patterns change, including appearance of distinct posterior boundaries and restriction to the central nervous system. Capitella Hox genes have maintained a clustered organization, are expressed in the canonical anterior–posterior order found in other metazoans, and exhibit spatial and temporal colinearity, reflecting Hox gene characteristics that likely existed in the protostome–deuterostome ancestor.

In vivo identification of regulators of cell invasion across basement membranes.

A Caenorhabditis elegans screen provides insight into cell invasion and metastasis. Crossing the Basement Membrane The basement membrane is a fibrous, sheet-like layer of the extracellular matrix located underneath epithelial or endothelial cell layers. Cell invasion through basement membranes is required during development and also during metastasis, when tumor cells leave their tissue of origin and enter lymphatic or blood vessels to migrate to secondary sites. During development of the nematode Caenorhabditis elegans, a specialized gonadal cell known as the anchor cell invades the gonadal and ventral epidermal basement membranes to initiate the formation of the female reproductive tract. Matus et al. identified and characterized genes encoding factors that promoted the ability of the anchor cell to invade basement membranes in C. elegans, most of which had not been previously implicated in cell invasion. Two of these genes, cct-5 (encoding a member of a chaperonin complex) and lit-1 (encoding a NEMO-like kinase), have human orthologs that, when knocked down in breast or colon carcinoma cells, prevented basement membrane invasion in an ex vivo system. Thus, the pro-invasive genes identified in this nematode screen could be therapeutically targeted in the treatment of metastatic cancer. Cell invasion through basement membranes during development, immune surveillance, and metastasis remains poorly understood. To gain further insight into this key cellular behavior, we performed an in vivo screen for regulators of cell invasion through basement membranes, using the simple model of Caenorhabditis elegans anchor cell invasion, and identified 99 genes that promote invasion, including the genes encoding the chaperonin complex cct. Notably, most of these genes have not been previously implicated in invasive cell behavior. We characterized members of the cct complex and 11 other gene products, determining the distinct aspects of the invasive cascade that they regulate, including formation of a specialized invasive cell membrane and its ability to breach the basement membrane. RNA interference–mediated knockdown of the human orthologs of cct-5 and lit-1, which had not previously been implicated in cell invasion, reduced the invasiveness of metastatic carcinoma cells, suggesting that a conserved genetic program underlies cell invasion. These results increase our understanding of the genetic underpinnings of cell invasion and also provide new potential therapeutic targets to limit this behavior.