A predatory gastrula leads to symbiosis-independent settlement in Aiptasia.

The planula larvae of the sea anemone Aiptasia have so far not been reported to complete their life cycle by undergoing metamorphosis into adult forms. This has been a major obstacle in their use as a model for coral-dinoflagellate endosymbiosis. Here, we show that Aiptasia larvae actively feed on crustacean nauplii, displaying a preference for live prey. This feeding behavior relies on functional stinging cells, indicative of complex neuronal control. Regular feeding leads to significant size increase, morphological changes, and efficient settlement around 14 d postfertilization. Surprisingly, the presence of dinoflagellate endosymbionts does not affect larval growth or settlement dynamics but is crucial for sexual reproduction. Our findings finally close Aiptasia's life cycle and highlight the functional nature of its larvae, as in Haeckel's Gastrea postulate, yet reveal its active carnivory, thus contributing to our understanding of early metazoan evolution.

Phenotype-first hypotheses, spandrels and early metazoan evolution.

Against the neo-Darwinian assumption that genetic factors are the principal source of variation upon which natural selection operates, a phenotype-first hypothesis strikes us as revolutionary because development would seem to constitute an independent source of variability. Richard Watson and his co-authors have argued that developmental memory constitutes one such variety of phenotypic variability. While this version of the phenotype-first hypothesis is especially well-suited for the late metazoan context, where animals have a sufficient history of selection from which to draw, appeals to developmental memory seem less plausible in the evolutionary context of the early metazoans. I provide an interpretation of Stuart Newman's account of deep metazoan phylogenesis that suggests that spandrels are, in addition to developmental memory, an important reservoir of phenotypic variability. I conclude by arguing that Gerd Müller's "side-effect hypothesis" is an illuminating generalization of the proposed non-Watsonian version of the phenotype-first hypothesis.

Characterization of C. elegans Chondroitin Proteoglycans and Their Large Functional and Structural Heterogeneity; Evolutionary Aspects on Structural Differences Between Humans and the Nematode.

Proteoglycans regulate important cellular pathways in essentially all metazoan organisms. While considerable effort has been devoted to study structural and functional aspects of proteoglycans in vertebrates, the knowledge of the core proteins and proteoglycan-related functions in invertebrates is relatively scarce, even for C.elegans. This nematode produces a large amount of non-sulfated chondroitin in addition to small amount of low-sulfated chondroitin chains (Chn and CS chains, respectively). Until recently, 9 chondroitin core proteins (CPGs) had been identified in C.elegans, none of which showed any homology to vertebrate counterparts or to other invertebrate core proteins. By using a glycoproteomic approach, we recently characterized the chondroitin glycoproteome of C.elegans, resulting in the identification of 15 novel CPG core proteins in addition to the 9 previously established. Three of the novel core proteins displayed homology to human proteins, indicating that CPG and CSPG core proteins may be more conserved throughout evolution than previously perceived. Bioinformatic analysis of the primary amino acid sequences revealed that the core proteins contained a broad range of functional domains, indicating that specialization of proteoglycan-mediated functions may have evolved early in metazoan evolution. This review specifically discusses our recent data in relation to previous knowledge of core proteins and GAG-attachment sites in Chn and CS proteoglycans of C.elegans and humans, and point out both converging and diverging aspects of proteoglycan evolution.

Ultrafast epithelial contractions provide insights into contraction speed limits and tissue integrity.

By definition of multicellularity, all animals need to keep their cells attached and intact, despite internal and external forces. Cohesion between epithelial cells provides this key feature. To better understand fundamental limits of this cohesion, we study the epithelium mechanics of an ultrathin (∼25 µm) primitive marine animal Trichoplax adhaerens, composed essentially of two flat epithelial layers. With no known extracellular matrix and no nerves or muscles, T. adhaerens has been claimed to be the "simplest known living animal," yet is still capable of coordinated locomotion and behavior. Here we report the discovery of the fastest epithelial cellular contractions known in any metazoan, to be found in T. adhaerens dorsal epithelium (50% shrinkage of apical cell area within one second, at least an order of magnitude faster than other known examples). Live imaging reveals emergent contractile patterns that are mostly sporadic single-cell events, but also include propagating contraction waves across the tissue. We show that cell contraction speed can be explained by current models of nonmuscle actin-myosin bundles without load, while the tissue architecture and unique mechanical properties are softening the tissue, minimizing the load on a contracting cell. We propose a hypothesis, in which the physiological role of the contraction dynamics is to resist external stresses while avoiding tissue rupture ("active cohesion"), a concept that can be further applied to engineering of active materials.

Insights into the evolution of metazoan regenerative mechanisms: roles of TGF superfamily members in tissue regeneration of the marine sponge Chondrosia reniformis.

Tissue repair is an adaptive and widespread metazoan response. It is characterised by different cellular mechanisms and complex signalling networks that involve numerous growth factors and cytokines. In higher animals, transforming growth factor-ß (TGF-ß) signalling plays a fundamental role in wound healing. In order to evaluate the involvement of TGF superfamily members in lower invertebrate tissue regeneration, sequences for putative TGF ligands and receptors were isolated from the transcriptome of the marine sponge Chondrosia reniformis We identified seven transcripts that coded for TGF superfamily ligands and three for TGF superfamily receptors. Phylogenetically, C. reniformis TGF ligands were not grouped into any TGF superfamily clades and thus presumably evolved independently, whereas the TGF receptors clustered in the Type I receptor group. We performed gene expression profiling of these transcripts in sponge regenerating tissue explants. Data showed that three ligands (TGF1, TGF3 and TGF6) were mainly expressed during early regeneration and seemed to be involved in stem cell maintenance, whereas two others (TGF4 and TGF5) were strongly upregulated during late regeneration and thus were considered pro-differentiating factors. The presence of a strong TGF inhibitor, SB431542, blocked the restoration of the exopinacoderm layer in the sponge explants, confirming the functional involvement of the TGF pathway in tissue regeneration in these early evolved animals.

Wnt signaling and polarity in freshwater sponges.

BACKGROUND: The Wnt signaling pathway is uniquely metazoan and used in many processes during development, including the formation of polarity and body axes. In sponges, one of the earliest diverging animal groups, Wnt pathway genes have diverse expression patterns in different groups including along the anterior-posterior axis of two sponge larvae, and in the osculum and ostia of others. We studied the function of Wnt signaling and body polarity formation through expression, knockdown, and larval manipulation in several freshwater sponge species. RESULTS: Sponge Wnts fall into sponge-specific and sponge-class specific subfamilies of Wnt proteins. Notably Wnt genes were not found in transcriptomes of the glass sponge Aphrocallistes vastus. Wnt and its signaling genes were expressed in archaeocytes of the mesohyl throughout developing freshwater sponges. Osculum formation was enhanced by GSK3 knockdown, and Wnt antagonists inhibited both osculum development and regeneration. Using dye tracking we found that the posterior poles of freshwater sponge larvae give rise to tissue that will form the osculum following metamorphosis. CONCLUSIONS: Together the data indicate that while components of canonical Wnt signaling may be used in development and maintenance of osculum tissue, it is likely that Wnt signaling itself occurs between individual cells rather than whole tissues or structures in freshwater sponges.

Quantitative study of developmental biology confirms Dickinsonia as a metazoan.

The late Ediacaran soft-bodied macroorganism Dickinsonia (age range approx. 560-550 Ma) has often been interpreted as an early animal, and is increasingly invoked in debate on the evolutionary assembly of eumetazoan body plans. However, conclusive positive evidence in support of such a phylogenetic affinity has not been forthcoming. Here we subject a collection of Dickinsonia specimens interpreted to represent multiple ontogenetic stages to a novel, quantitative method for studying growth and development in organisms with an iterative body plan. Our study demonstrates that Dickinsonia grew via pre-terminal 'deltoidal' insertion and inflation of constructional units, followed by a later inflation-dominated phase of growth. This growth model is contrary to the widely held assumption that Dickinsonia grew via terminal addition of units at the end of the organism bearing the smallest units. When considered alongside morphological and behavioural attributes, our developmental data phylogenetically constrain Dickinsonia to the Metazoa, specifically the Eumetazoa plus Placozoa total group. Our findings have implications for the use of Dickinsonia in developmental debates surrounding the metazoan acquisition of axis specification and metamerism.

The evolutionary origin of epithelial cell-cell adhesion mechanisms.

A simple epithelium forms a barrier between the outside and the inside of an organism, and is the first organized multicellular tissue found in evolution. We examine the relationship between the evolution of epithelia and specialized cell-cell adhesion proteins comprising the classical cadherin/ß-catenin/α-catenin complex (CCC). A review of the divergent functional properties of the CCC in metazoans and non-metazoans, and an updated phylogenetic coverage of the CCC using recent genomic data reveal: (1) The core CCC likely originated before the last common ancestor of unikonts and their closest bikont sister taxa. (2) Formation of the CCC may have constrained sequence evolution of the classical cadherin cytoplasmic domain and ß-catenin in metazoa. (3) The α-catenin-binding domain in ß-catenin appears to be the favored mutation site for disrupting ß-catenin function in the CCC. (4) The ancestral function of the α/ß-catenin heterodimer appears to be an actin-binding module. In some metazoan groups, more complex functions of α-catenin were gained by sequence divergence in the non-actin-binding (N-, M-) domains. (5) Allosteric regulation of α-catenin may have evolved for more complex regulation of the actin cytoskeleton.

Dawn of complex animal food webs: A new predatory anthozoan (Cnidaria) from Cambrian.

Cnidarians diverged very early in animal evolution; therefore, investigations of the morphology and trophic levels of early fossil cnidarians may provide critical insights into the evolution of metazoans and the origin of modern marine food webs. However, there has been a lack of unambiguous anthozoan cnidarians from Ediacaran assemblages, and undoubted anthozoans from the Cambrian radiation of metazoans are very rare and lacking in ecological evidence. Here, we report a new polypoid cnidarian, Nailiana elegans gen. et sp. nov., represented by multiple solitary specimens from the early Cambrian Chengjiang biota (∼520 Ma) of South China. These specimens show eight unbranched tentacles surrounding a single opening into the gastric cavity, which may have born multiple mesenteries. Thus, N. elegans displays a level of organization similar to that of extant cnidarians. Phylogenetic analyses place N. elegans in the stem lineage of Anthozoa and suggest that the ancestral anthozoan was a soft-bodied, solitary polyp showing octoradial symmetry. Moreover, one specimen of the new polyp preserves evidence of predation on an epifaunal lingulid brachiopod. This case provides the oldest direct evidence of macrophagous predation, the advent of which may have triggered the emergence of complex trophic/ecological relationships in Cambrian marine communities and spurred the explosive radiation of animal body plans.

Two distinct compartments of a ctenophore comb plate provide structural and functional integrity for the motility of giant multicilia.

Comb plates are large ciliary structures uniquely seen in comb jellies (ctenophores).1,2,3 A comb plate is constructed from tens of thousands of cilia that are bundled together by structures called compartmenting lamellae (CLs).4,5,6 We previously reported the first component of the CL, CTENO64, and found that it was specifically found in ctenophores and was essential for the determination of ciliary orientation.3 However, CTENO64 is localized only in the proximal region of the CL; therefore, the molecular architecture of the CL over the entire length of a comb plate has not been elucidated. Here, we identified a second CL component, CTENO189. This ctenophore-specific protein was present in the distal region of comb plates, with a localization clearly segregated from CTENO64. Knockdown of the CTENO189 gene using morpholino antisense oligonucleotides resulted in complete loss of CLs in the distal region of comb plates but did not affect the formation of comb plates or the orientation of each cilium. However, the hexagonal distribution of cilia was disarranged, and the metachronal coordination of comb plates along a comb row was lost in the CTENO189 morphants. The morphant comb plate showed asymmetric ciliary-type movement in normal seawater, and in a high-viscosity solution, it could not maintain the normal waveforms but showed a symmetric flagellar-type movement. Our findings demonstrated two distinct compartments of a comb plate: the proximal CL as the building foundation that rigidly fixes the ciliary orientation, and the distal CL that reinforces the elastic connection among cilia to overcome the hydrodynamic drag of giant multiciliary plates.

The evolution of the metazoan Toll receptor family and its expression during protostome development.

BACKGROUND: Toll-like receptors (TLRs) play a crucial role in immunity and development. They contain leucine-rich repeat domains, one transmembrane domain, and one Toll/IL-1 receptor domain. TLRs have been classified into V-type/scc and P-type/mcc TLRs, based on differences in the leucine-rich repeat domain region. Although TLRs are widespread in animals, detailed phylogenetic studies of this gene family are lacking. Here we aim to uncover TLR evolution by conducting a survey and a phylogenetic analysis in species across Bilateria. To discriminate between their role in development and immunity we furthermore analyzed stage-specific transcriptomes of the ecdysozoans Priapulus caudatus and Hypsibius exemplaris, and the spiralians Crassostrea gigas and Terebratalia transversa. RESULTS: We detected a low number of TLRs in ecdysozoan species, and multiple independent radiations within the Spiralia. V-type/scc and P-type/mcc type-receptors are present in cnidarians, protostomes and deuterostomes, and therefore they emerged early in TLR evolution, followed by a loss in xenacoelomorphs. Our phylogenetic analysis shows that TLRs cluster into three major clades: clade α is present in cnidarians, ecdysozoans, and spiralians; clade ß in deuterostomes, ecdysozoans, and spiralians; and clade γ is only found in spiralians. Our stage-specific transcriptome and in situ hybridization analyses show that TLRs are expressed during development in all species analyzed, which indicates a broad role of TLRs during animal development. CONCLUSIONS: Our findings suggest that a clade α TLR gene (TLR-Ca) and a clade ß/γ TLR gene (TLR-Cß/γ) were already present in the cnidarian-bilaterian common ancestor. However, although TLR-Ca was conserved in cnidarians, TLR-Cß/γ was lost during the early evolution of these taxa. Moreover, TLR-Cß/γ duplicated to generate TLR-Cß and TLR-Cγ in the lineage to the last common protostome-deuterostome ancestor. TLR-Ca, TLR-Cß and TLR-Cγ further expanded generating the three major TLR clades. While all three clades radiated in several spiralian lineages, specific TLRs clades have been presumably lost in other lineages. Furthermore, the expression of the majority of these genes during protostome ontogeny suggests a likely role in development.

The fibrillar collagen family.

Collagens, or more precisely collagen-based extracellular matrices, are often considered as a metazoan hallmark. Among the collagens, fibrillar collagens are present from sponges to humans, and are involved in the formation of the well-known striated fibrils. In this review we discuss the different steps in the evolution of this protein family, from the formation of an ancestral fibrillar collagen gene to the formation of different clades. Genomic data from the choanoflagellate (sister group of Metazoa) Monosiga brevicollis, and from diploblast animals, have suggested that the formation of an ancestral alpha chain occurred before the metazoan radiation. Phylogenetic studies have suggested an early emergence of the three clades that were first described in mammals. Hence the duplication events leading to the formation of the A, B and C clades occurred before the eumetazoan radiation. Another important event has been the two rounds of "whole genome duplication" leading to the amplification of fibrillar collagen gene numbers, and the importance of this diversification in developmental processes. We will also discuss some other aspects of fibrillar collagen evolution such as the development of the molecular mechanisms involved in the formation of procollagen molecules and of striated fibrils.

Functional Diversification of SRSF Protein Kinase to Control Ubiquitin-Dependent Neurodevelopmental Signaling.

Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.

Notch-Mediated Cell Adhesion.

Notch family members are generally recognized as signaling molecules that control various cellular responses in metazoan organisms. Early fly studies and our mammalian studies demonstrated that Notch family members are also cell adhesion molecules; however, information on the physiological roles of this function and its origin is limited. In this review, we discuss the potential present and ancestral roles of Notch-mediated cell adhesion in order to explore its origin and the initial roles of Notch family members dating back to metazoan evolution. We hypothesize that Notch family members may have initially emerged as cell adhesion molecules in order to mediate multicellularity in the last common ancestor of metazoan organisms.

Molecular clocks and the early evolution of metazoan nervous systems.

The timing of early animal evolution remains poorly resolved, yet remains critical for understanding nervous system evolution. Methods for estimating divergence times from sequence data have improved considerably, providing a more refined understanding of key divergences. The best molecular estimates point to the origin of metazoans and bilaterians tens to hundreds of millions of years earlier than their first appearances in the fossil record. Both the molecular and fossil records are compatible, however, with the possibility of tiny, unskeletonized, low energy budget animals during the Proterozoic that had planktonic, benthic, or meiofaunal lifestyles. Such animals would likely have had relatively simple nervous systems equipped primarily to detect food, avoid inhospitable environments and locate mates. The appearance of the first macropredators during the Cambrian would have changed the selective landscape dramatically, likely driving the evolution of complex sense organs, sophisticated sensory processing systems, and diverse effector systems involved in capturing prey and avoiding predation.

The fifth element in animal Galpha protein evolution.

Heterotrimeric G proteins are key molecules regulating cellular responses to extracellular stimuli, and are composed of alpha, beta and gamma subunits. All alpha subunits in vertebrates belong to four major classes, Gs, Gi, Gq and G12, which are conserved throughout the animal kingdom. Unexpectedly, now a fifth class of Galpha protein, Gv, has been discovered. Gv is conserved across the animal kingdom and present in vertebrates, arthropods, mollusks, annelids and even sponges. Presumably, Gv has been missed so far, because it has been lost in many lineages in the major model organisms such as nematodes, fruit fly and mammals. On the other hand, gene gains are also observed for Gv, with at least two independent gene duplications, one in sponges and the other in the teleost lineage. Such frequent gene gains and losses fit to a birth-and-death mode of evolution, which is unusual for a well-conserved and ancient gene family like the Galpha proteins. The discovery of a novel major class of Galpha proteins provides new insights in the evolution of the Galpha protein family and opens new possibilities in G protein signaling research.

The Diploblast-Bilateria Sister hypothesis: parallel revolution of a nervous systems may have been a simple step.

For many familiar with metazoan relationships and body plans, the hypothesis of a sister group relationship between Diploblasta and Bilateria1 comes as a surprise. One of the consequences of this hypothesis-the independent evolution of a nervous system in Coelenterata and Bilateria-seems highly unlikely to many. However, to a small number of scientists working on Metazoa, the parallel evolution of the nervous system is not surprising at all and rather a confirmation of old morphological and new genetic knowledge.2-4 The controversial hypothesis that the Diploblasta and Bilateria are sister taxa is, therefore, tantamount to reconciling the parallel evolution of the nervous system in Coelenterata and Bilateria. In this addendum to Schierwater et al.1 we discuss two aspects critical to the controversy. First we discuss the strength of the inference of the proposed sister relationship of Diploblasta and Bilateria and second we discuss the implications for the evolution of nerve cells and nervous systems.