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Innexins facilitate cell-cell communication by forming gap junctions or non-junctional hemichannels, which play important roles in metabolic, chemical, ionic, and electrical coupling. The lack of knowledge regarding the evolution and role of these channels in ctenophores (comb jellies), the likely sister group to the rest of animals, represents a substantial gap in our understanding of the evolution of intercellular communication in animals. Here we identify and phylogenetically characterize the complete set of innexins of four ctenophores:Mnemiopsis leidyi, Hormiphora californensis, Pleurobrachia bachei, andBeroe ovata. Our phylogenetic analyses suggest that ctenophore innexins diversified independently from those of other animals and were established early in the emergence of ctenophores. We identified a four-innexin genomic cluster, which was present in the last common ancestor of these four species and has been largely maintained in these lineages. Evidence from correlated spatial and temporal gene expression of theM. leidyiinnexin cluster suggest that this cluster has been maintained due to constraints related to gene regulation. We describe basic electrophysiological properties of putative ctenophore hemichannels from muscle cells using intracellular recording techniques, showing substantial overlap with the properties of bilaterian innexin channels. Together, our results suggest that the last common ancestor of animals had gap junctional channels also capable of forming functional innexin hemichannels, and that innexin genes have independently evolved in major lineages throughout Metazoa.

Cnidocytes are the explosive stinging cells found only in cnidarians (corals, jellyfish, etc). Specialized for prey capture and defense, cnidocytes are morphologically complex and vary widely in form and function across taxa; how such diversity evolved is unknown. Using CRISPR/Cas9-mediated genome editing in the burrowing sea anemoneNematostella vectensis, we show that a single transcription factor (NvSox2) acts as a binary switch between two alternative cnidocyte fates. Knockout ofNvSox2caused a complete transformation of nematocytes (piercing cells) into spirocytes (ensnaring cells). The type of spirocyte induced byNvSox2knockout (robust spirocyte) is not normally found inN. vectensisbut is common in sea anemones from other habitats. Homeotic control of cell fate provides a mechanistic explanation for the discontinuous distribution of cnidocyte types across cnidarians and demonstrates how simple counts of cell types can underestimate biodiversity.

Cnidocytes (“stinging cells”) are an unequivocally novel cell type used by cnidarians (corals, jellyfish, and their kin) to immobilize prey. Although they are known to share a common evolutionary origin with neurons, the developmental program that promoted the emergence of cnidocyte fate is not known. Using functional genomics in the sea anemone, Nematostella vectensis, we show that cnidocytes evolved by suppression of neural fate in a subset of neurons expressing RFamide. We further show that a single regulatory gene, a C2H2-type zinc finger transcription factor (ZNF845), coordinates both the gain of novel (cnidocyte-specific) traits and the inhibition of ancestral (neural) traits during cnidocyte development and that this gene arose by domain shuffling in the stem cnidarian. Thus, we uncover a mechanism by which a truly novel regulatory gene (ZNF845) promoted the origin of a truly novel cell type (cnidocyte) through duplication of an ancestral cell lineage (neuron) and inhibition of its ancestral identity (RFamide).SignificanceIn this study, we demonstrate how new cell types can arise in animals through duplication of an ancestral (old) cell type followed by functional divergence of the new daughter cell. Specifically, we show that stinging cells in cnidarians (jellyfish and corals) evolved by duplication of an ancestral neuron followed by inhibition of the RFamide neuropeptide it once secreted. This is the first evidence that stinging cells evolved from a specific subtype of neurons and suggests some neurons may be easier to co-opt for novel functions than others.

Background Pax genes are a family of conserved transcription factors that regulate many aspects of developmental morphogenesis, notably the development of ectodermal sensory structures including eyes. Nematostella vectensis, the starlet sea anemone, has numerous Pax orthologs, many of which are expressed early during embryogenesis. The function of Pax genes in this eyeless cnidarian is unknown. Results Here, we show that PaxA, but not PaxC, plays a critical role in the development of cnidocytes in N. vectensis. Knockdown of PaxA results in a loss of developing cnidocytes and downregulation of numerous cnidocyte-specific genes, including a variant of the transcription factor Mef2. We also demonstrate that the co-expression of Mef2 in a subset of the PaxA-expressing cells is associated with the development with a second lineage of cnidocytes and show that knockdown of the neural progenitor gene SoxB2 results in downregulation of expression of PaxA, Mef2, and several cnidocyte-specific genes. Because PaxA is not co-expressed with SoxB2 at any time during cnidocyte development, we propose a simple model for cnidogenesis whereby a SoxB2-expressing progenitor cell population undergoes division to give rise to PaxA-expressing cnidocytes, some of which also express Mef2. Discussion The role of PaxA in cnidocyte development among hydrozoans has not been studied, but the conserved role of SoxB2 in regulating the fate of a progenitor cell that gives rise to neurons and cnidocytes in Nematostella and Hydractinia echinata suggests that this SoxB2/PaxA pathway may well be conserved across cnidarians.

The origin of novel traits can promote expansion into new niches and drive speciation. Ctenophores (comb jellies) are unified by their possession of a novel cell type: the colloblast, an adhesive cell found only in the tentacles. Although colloblast-laden tentacles are fundamental for prey capture among ctenophores, some species have tentacles lacking colloblasts and others have lost their tentacles completely. We used transcriptomes from 36 ctenophore species to identify gene losses that occurred specifically in lineages lacking colloblasts and tentacles. We cross-referenced these colloblast- and tentacle-specific candidate genes with temporal RNA-Seq during embryogenesis in Mnemiopsis leidyi and found that both sets of candidates are preferentially expressed during tentacle morphogenesis. We also demonstrate significant upregulation of candidates from both data sets in the tentacle bulb of adults. Both sets of candidates were enriched for an N-terminal signal peptide and protein domains associated with secretion; among tentacle candidates we also identified orthologs of cnidarian toxin proteins, presenting tantalizing evidence that ctenophore tentacles may secrete toxins along with their adhesive. Finally, using cell lineage tracing, we demonstrate that colloblasts and neurons share a common progenitor, suggesting the evolution of colloblasts involved co-option of a neurosecretory gene regulatory network. Together these data offer an initial glimpse into the genetic architecture underlying ctenophore cell-type diversity.

Understanding how new cell types arise is critical for understanding the evolution of organismal complexity. Questions of this nature, however, can be difficult to answer due to the challenge associated with defining the identity of a truly novel cell. Cnidarians (anemones, jellies, and their allies) provide a unique opportunity to investigate the molecular regulation and development of cell-novelty because they possess a cell that is unique to the cnidarian lineage and that also has a very well-characterized phenotype: the cnidocyte (stinging cell). Because cnidocytes are thought to differentiate from the cell lineage that also gives rise to neurons, cnidocytes can be expected to express many of the same genes expressed in their neural “sister” cells. Conversely, only cnidocytes posses a cnidocyst (the explosive organelle that gives cnidocytes their sting); therefore, those genes or gene-regulatory relationships required for the development of the cnidocyst can be expected to be expressed uniquely (or in unique combination) in cnidocytes. This system provides an important opportunity to: (1) construct the gene-regulatory network (GRN) underlying the differentiation of cnidocytes, (2) assess the relative contributions of both conserved and derived genes in the cnidocyte GRN, and (3) test hypotheses about the role of novel regulatory relationships in the generation of novel cell types. In this review, we summarize common challenges to studying the evolution of novelty, introduce the utility of cnidocyte differentiation in the model cnidarian, Nematostella vectensis, as a means of overcoming these challenges, and describe an experimental approach that leverages comparative tissue-specific transcriptomics to generate hypotheses about the GRNs underlying the acquisition of the cnidocyte identity.

The mesoderm is a key novelty in animal evolution, although we understand little of how the mesoderm arose. brachyury, the founding member of the T-box gene family, is a key gene in chordate mesoderm development. However, the brachyury gene was present in the common ancestor of fungi and animals long before mesoderm appeared. To explore ancestral roles of brachyury prior to the evolution of definitive mesoderm, we excised the gene using CRISPR/Cas9 in the diploblastic cnidarian Nematostella vectensis. Nvbrachyury is normally expressed in precursors of the pharynx, which separates endoderm from ectoderm. In knockout embryos, the pharynx does not form, embryos fail to elongate, and endoderm organization, ectodermal cell polarity and patterning along the oral-aboral axis are disrupted. Expression of many genes both inside and outside the Nvbrachyury expression domain is affected, including downregulation of Wnt genes at the oral pole. Our results point to an ancient role for brachyury in morphogenesis, cell polarity and the patterning of both ectodermal and endodermal derivatives along the primary body axis., Summary: Brachyury is a key regulator of mesoderm, but is also found in diploblasts, which lack this tissue layer. Knockout experiments reveal its widespread effects on the endomesodermal gene regulatory network.

Since their discovery in 1958, the function of specialized salt-secreting glands in tetrapods has been studied in great detail, and such studies continue to contribute to a general understanding of transport mechanisms of epithelial water and ions. Interestingly, during that same time period, there have been only few attempts to understand the convergent evolution of this tissue, likely as a result of the paucity of taxonomic, embryological, and molecular data available. In this review, we synthesize the available data regarding the distribution of salt glands across extant and extinct tetrapod lineages and the anatomical position of the salt gland in each taxon. Further, we use these data to develop hypotheses about the various factors that have influenced the convergent evolution of salt glands across taxa with special focus on the variation in the anatomical position of the glands and on the molecular mechanisms that may have facilitated the development of a salt gland by co-option of a nonsalt-secreting ancestral gland. It is our hope that this review will stimulate renewed interest in the topic of the convergent evolution of salt glands and inspire future empirical studies aimed at evaluating the hypotheses we lay out herein.

SUMMARY To understand renal responses to salinity change in aquatic reptiles, we examined the structure and function of the kidney in three species of snake: a marine species with a salt gland (Laticauda semifasciata), a marine species without a salt gland (Nerodia clarkii clarkii) and a freshwater species without a salt gland (Nerodia fasciata). Both marine species maintained relatively constant plasma ions, even after acclimation to saltwater. By contrast, both plasma Cl– and mortality increased with salinity in the freshwater species. To investigate putative renal ion regulatory mechanisms, we examined the distribution and abundance of Na+/K+-ATPase (NKA) and the Na+/K+/2Cl– cotransporter (NKCC2). In all species, NKA localized to the basolateral membranes of the distal tubule and the connecting segments and collecting ducts only; there was no effect of salinity on the distribution of NKA or on the abundance of NKA mRNA in any species. NKCC2 protein was undetectable in the kidney of any of the species and there was no effect of salinity on NKCC2 mRNA abundance. We also examined the distribution and abundance of aquaporin 3 (AQP3) in the kidney of these species; although putative AQP3 localized to the basolateral membranes of the connecting segments and collecting ducts of all three species, there was no effect of salinity on the localization of the protein or the abundance of the transcript. Interestingly, we found very few differences across species, suggesting that the snake kidney may play a trivial role in limiting habitat use.

Communication among cells was paramount to the evolutionary increase in cell type diversity and, ultimately, the origin of large body size. Across the diversity of Metazoa, there are only few conserved cell signalling pathways known to orchestrate the complex cell and tissue interactions regulating development; thus, modification to these few pathways has been responsible for generating diversity during the evolution of animals. Here, we summarize evidence for the origin and putative function of the intracellular, membrane-bound and secreted components of seven metazoan cell signalling pathways with a special focus on early branching metazoans (ctenophores, poriferans, placozoans and cnidarians) and basal unikonts (amoebozoans, fungi, filastereans and choanoflagellates). We highlight the modular incorporation of intra- and extracellular components in each signalling pathway and suggest that increases in the complexity of the extracellular matrix may have further promoted the modulation of cell signalling during metazoan evolution. Most importantly, this updated view of metazoan signalling pathways highlights the need for explicit study of canonical signalling pathway components in taxa that do not operate a complete signalling pathway. Studies like these are critical for developing a deeper understanding of the evolution of cell signalling. This article is part of the themed issue ‘Evo-devo in the genomics era, and the origins of morphological diversity’.

Background Cnidarians are the closest living relatives to bilaterians and have been instrumental to studying the evolution of bilaterian properties. The cnidarian model, Nematostella vectensis, is a unique system in which embryology and regeneration are both studied, making it an ideal candidate to develop in vivo imaging techniques. Live imaging is the most direct way for quantitative and qualitative assessment of biological phenomena. Actin and tubulin are cytoskeletal proteins universally important for regulating many embryological processes but so far studies in Nematostella primarily focused on the localization of these proteins in fixed embryos. Results We used fluorescent probes expressed in vivo to investigate the dynamics of Nematostella development. Lifeact-mTurquoise2, a fluorescent cyan F-actin probe, can be visualized within microvilli along the cellular surface throughout embryonic development and is stable for two months after injection. Co-expression of Lifeact-mTurquoise2 with End-Binding protein1 (EB1) fused to mVenus or tdTomato-NLS allows for the visualization of cell-cycle properties in real time. Utilizing fluorescent probes in vivo helped to identify a concentrated ‘flash’ of Lifeact-mTurquoise2 around the nucleus, immediately prior to cytokinesis in developing embryos. Moreover, Lifeact-mTurquoise2 expression in adult animals allowed the identification of various cell types as well as cellular boundaries. Conclusion The methods developed in this manuscript provide an alternative protocol to investigate Nematostella development through in vivo cellular analysis. This study is the first to utilize the highly photo-stable florescent protein mTurquoise2 as a marker for live imaging. Finally, we present a clear methodology for the visualization of minute temporal events during cnidarian development. Electronic supplementary material The online version of this article (doi:10.1186/s12860-014-0044-2) contains supplementary material, which is available to authorized users.

Dehydration and procurement of water are key problems for vertebrates that have secondarily invaded marine environments. Sea snakes and other marine reptiles are thought to remain in water balance without consuming freshwater, owing to the ability of extrarenal salt glands to excrete excess salts obtained either from prey or from drinking seawater directly. Contrary to this long-standing dogma, we report that three species of sea snake actually dehydrate in marine environments. We investigated dehydration and drinking behaviors in three species of amphibious sea kraits (Laticauda spp.) representing a range of habits from semiterrestrial to very highly marine. Snakes that we dehydrated either in air or in seawater refused to drink seawater but drank freshwater or very dilute brackish water (10%-30% seawater) to remain in water balance. We further show that Laticauda spp. can dehydrate severely in the wild and are far more abundant at sites where there are sources of freshwater. A more global examination of all sea snakes demonstrates that species richness correlates positively with mean annual precipitation within the Indo-West Pacific tropical region. The dependence of Laticauda spp. on freshwater might explain the characteristically patchy distributions of these reptiles and is relevant to understanding patterns of extinctions and possible future responses to changes in precipitation related to global warming. In particular, metapopulation dynamics of the Laticauda group of sea snakes are expected to change in relation to projected reductions of tropical dry-season precipitation.