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In bilaterians and cnidarians, epithelial cell-polarity is regulated by the interactions between Par proteins, Wnt/PCP signaling pathway, and cell-cell adhesion. Par proteins are highly conserved across Metazoa, including ctenophores. But strikingly, ctenophore genomes lack components of the Wnt/PCP pathway and cell-cell adhesion complexes raising the question if ctenophore cells are polarized by mechanisms involving Par proteins. Here, by using immunohistochemistry and live-cell imaging of specific mRNAs, we describe for the first time the subcellular localization of selected Par proteins in blastomeres and epithelial cells during the embryogenesis of the ctenophoreMnemiopsis leidyi. We show that these proteins distribute differently compared to what has been described for other animals, even though they segregate in a host-specific fashion when expressed in cnidarian embryos. This differential localization might be related to the emergence of different junctional complexes during metazoan evolution.

The maternal-zygotic transition (MZT) describes the developmental reprogramming of gene expression marked by the degradation of maternally supplied gene products and activation of the zygotic genome. While the timing and duration of the MZT vary among taxa, little is known about early stage transcriptional dynamics in the non-bilaterian phylum Ctenophora. We sought to better understand the extent of maternal mRNA loading and subsequent differential transcript abundance during the earliest stages of development by performing comprehensive RNA-sequencing-based analyses of mRNA abundance in single- and eight-cell-stage embryos in the lobate ctenophore Mnemiopsis leidyi. We found 1,908 contigs with significant differential abundance between single- and eight-cell stages, of which 1,208 contigs were more abundant at the single-cell stage and 700 contigs were more abundant at the eight-cell stage. Of the differentially abundant contigs, 267 were exclusively present in the eight-cell samples, providing strong evidence that both the MZT and zygotic genome activation (ZGA) have commenced by the eight-cell stage. Many highly abundant transcripts encode genes involved in molecular mechanisms critical to the MZT, such as maternal transcript degradation, serine/threonine kinase activity, and chromatin remodeling. Our results suggest that chromosomal restructuring, which is critical to ZGA and the initiation of transcriptional regulation necessary for normal development, begins by the third cleavage within 1.5 hours post-fertilization in Mnemiopsis leidyi. This article is protected by copyright. All rights reserved

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.

BackgroundThe ability to regenerate is a widely distributed but highly variable trait among metazoans. A variety of modes of regeneration has been described for different organisms; however, many questions regarding the origin and evolution of these strategies remain unanswered. Most species of ctenophore (or “comb jellies”), a clade of marine animals that branch off at the base of the animal tree of life, possess an outstanding capacity to regenerate. However, the cellular and molecular mechanisms underlying this ability are unknown. We have used the ctenophoreMnemiopsis leidyias a system to study wound healing and adult regeneration and provide some first-time insights of the cellular mechanisms involved in the regeneration of one of the most ancient extant group of multicellular animals.ResultsWe show that cell proliferation is activated at the wound site and is indispensable for whole-body regeneration. Wound healing occurs normally in the absence of cell proliferation forming a scar-less wound epithelium. No blastema-like structure is generated at the cut site, and pulse-chase experiments and surgical intervention show that cells originating in the main regions of cell proliferation (the tentacle bulbs) do not seem to contribute to the formation of new structures after surgical challenge, suggesting a local source of cells during regeneration. While exposure to cell-proliferation blocking treatment inhibits regeneration, the ability to regenerate is recovered when the treatment ends (days after the original cut), suggesting that ctenophore regenerative capabilities are constantly ready to be triggered and they are somehow separable of the wound healing process.ConclusionsCtenophore regeneration takes place through a process of cell proliferation-dependent non-blastemal-like regeneration and is temporally separable of the wound healing process. We propose that undifferentiated cells assume the correct location of missing structures and differentiate in place. The remarkable ability to replace missing tissue, the many favorable experimental features (e.g., optical clarity, high fecundity, rapid regenerative performance, stereotyped cell lineage, sequenced genome), and the early branching phylogenetic position in the animal tree, all point to the emergence of ctenophores as a new model system to study the evolution of animal regeneration.

The ß-catenin protein has two major known functions in animal cells. It keeps epithelial tissue homeostasis by its connection with Adherens Junctions (AJ), and it serves as a transcriptional cofactor along with Lef/Tcf to enter the nucleus and regulate target genes of the Wnt/ß-catenin (cWnt) signaling pathway. To assess the ancestral role of ß-catenin during development we examined its distribution and function in the ctenophoreMnemiopsis leidyi(one of the earliest branching animal phyla) by using ctenophore-specific antibodies and mRNA injection. We found that ß-catenin protein never localizes to cell-cell contacts during embryogenesis as it does in other metazoans, most likely because ctenophore-cadherins do not have the cytoplasmic domain required for interaction with the catenin proteins. Downregulation of zygoticMlß-catenin signaling led to the loss of endodermal and mesodermal tissues indicating that nuclear ß-catenin may have a deep role in germ-layer evolution. Our results indicate that the ancestral role for ß-catenin was in the cell-fate specification and not in cell adhesion and also further emphasizes the critical role of this protein in the evolution of tissue layers in metazoans.

BackgroundThe ability to regenerate is a widely distributed but highly variable trait among metazoans. A variety of modes of regeneration has been described for different organisms, however, many questions regarding the origin and evolution of these strategies remain unanswered. Most species of ctenophore (or “comb jellies”), a clade of marine animals that branch off at the base of the animal tree of life, possess an outstanding capacity to regenerate. However, the cellular and molecular mechanisms underlying this ability are unknown. We have used the ctenophoreMnemiopsis leidyias a system to study wound healing and adult regeneration and provide some first-time insights of the cellular mechanisms involved in the regeneration of one of the most ancient extant group of multicellular animals.ResultsWe show that cell proliferation is activated at the wound site and is indispensable for whole-body regeneration. Wound healing occurs normally in the absence of cell proliferation forming a scar-less wound epithelium. No blastema-like structure is generated at the cut site, rather undifferentiated cells assume the correct location of missing structures and differentiate in place. Pulse-chase experiments and surgical intervention show that cells originating in the main regions of cell proliferation (the tentacle bulbs) do not seem to contribute to the formation of new structures after surgical challenge, suggesting a local source of cells during regeneration. While exposure to cell-proliferation blocking treatment inhibits regeneration, the ability to regenerate is recovered when the treatment ends (days after the original cut), suggesting that ctenophore regenerative capabilities are constantly ready to be triggered and they are somehow separable of the wound healing process.ConclusionsCtenophore regeneration takes place through a process of cell proliferation-dependent non blastemal-like regeneration and is temporally separable of the wound healing process. The remarkable ability to replace missing tissue, the many favorable experimental features (e.g. optical clarity, high fecundity, rapid regenerative performance, stereotyped cell lineage, sequenced genome), and the early branching phylogenetic position in the animal tree, all point to the emergence of ctenophores as a new model system to study the evolution of animal regeneration.

In bilaterians and cnidarians, embryonic and epithelial cell-polarity are regulated by the interactions between Par proteins, Wnt/PCP signaling pathway, and cell-cell adhesion. Par proteins are highly conserved across Metazoa, including ctenophores. But strikingly, ctenophore genomes lack components of the Wnt/PCP pathway and cell-cell adhesion complexes; raising the question if ctenophore cells are polarized by mechanisms involving Par proteins. Here, by using immunohistochemistry and live-cell imaging overexpression of specific mRNAs, we describe for the first time the subcellular localization of selected Par proteins in blastomeres and epithelial cells during the embryogenesis of the ctenophore Mnemiopsis leidyi. We show that these proteins distribute differently compared to what has been described for other animals, even though they segregate in a host-specific fashion when expressed in cnidarian embryos. This differential localization might be related to the emergence of different junctional complexes during metazoan evolution. Data obtained here challenge the ancestry of the apicobasal cell polarity and raise questions about the homology of epithelial tissue across the Metazoa.

Neuropeptides are evolutionarily ancient peptide hormones of the nervous and neuroendocrine systems, and are thought to have regulated metamorphosis in early animal ancestors. In particular, the deeply conserved Wamide family of neuropeptides—shared across Bilateria (e.g. insects and worms) and its sister group Cnidaria (e.g. jellyfishes and corals)—has been implicated in mediating life-cycle transitions, yet their endogenous roles remain poorly understood. By using CRISPR-Cas9-mediated reverse genetics, we show that cnidarian Wamide—referred to as GLWamide—regulates the timing of life cycle transition in the sea anemone cnidarian Nematostella vectensis. We find that mutant planula larvae lacking GLWamides transform into morphologically normal polyps at a rate slower than that of the wildtype control larvae. Treatment of GLWamide null mutant larvae with synthetic GLWamide peptides is sufficient to restore a normal rate of metamorphosis. These results demonstrate that GLWamide plays a dispensable, modulatory role in accelerating metamorphosis in a sea anemone.

In triploblastic animals, Par-proteins regulate cell-polarity and adherens junctions of both ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella vectensis, Par-proteins are degraded in all cells in the bifunctional gastrodermal epithelium. Using immunohistochemistry, CRISPR/Cas9 mutagenesis, and mRNA overexpression, we describe the functional association between Par-proteins, ß-catenin, and snail transcription factor genes in N. vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of ß-catenin in the ectoderm by stabilizing its role in cell-adhesion, and that endomesodermal epithelial cells are organized by a different cell-adhesion system than overlying ectoderm. We also show that ectopic expression of snail genes, which are expressed in mesodermal derivatives in bilaterians, is sufficient to downregulate Par-proteins and translocate ß-catenin from the junctions to the cytoplasm in ectodermal cells. These data provide molecular insight into the evolution of epithelial structure and distinct cell behaviors in metazoan embryos., eLife digest Most animals – including birds, fish and mammals – have symmetrical left and right sides, and are known as bilaterians. During early life, the embryos of animals in this group develop three distinct layers of cells: the ectoderm (outer layer), the endoderm (inner layer), and the mesoderm (middle layer). These layers then go on to form the animal’s tissues and organs. The ectoderm produces external tissues, such as the skin and the nervous system; the endoderm forms internal tissues, like the gut; and the mesoderm creates all tissues in between, like muscles and blood. Another, smaller group of animals, called cnidarians, do not have left and right sides. Instead, they have a ‘radial symmetry’, meaning they have multiple identical parts arranged in a circle. These animals – which include corals, jellyfish and sea anemones – only develop two distinct layers of cells, equivalent to the outer and inner layers of bilaterians. Cnidarians evolved before bilaterians, but their genetic material is equally complex. So why did these two groups evolve to have different layers of cells? And how exactly do animal embryos develop these distinct layers? To address these questions, Salinas-Saavedra et al. studied embryos of the sea anemone Nematostella vectensis. Molecules called Par-proteins play an important role in controlling how cells behave and attach to one another (and therefore how they form layers). So, using a technique called immunohistochemistry to look inside cells, Salinas-Saavedra et al. examined these proteins in the two layers of cells in sea anemone embryos. The experiments found that in the sea anemones, Par-proteins are arranged differently in cells that form the ‘skin’ compared to cells that form the ‘gut’. In other words, cells in the outer layer attach to one another in a different way than cells in the inner layer, where the Par-proteins are degraded by ‘mesodermal’ genes. The findings also show that these sea anemones have all they need to form a third middle layer of cells. Like bilaterians, they could potentially move cells in and out of sheets that line surfaces inside the body – but they do not naturally do this. Understanding how animals form different layers of cells is important for scientists studying evolution and the development of embryos. However, it also has wider applications. For instance, some cells involved in developing the mesoderm are also involved in forming tumors. Future research in this area could help scientists learn more about how cancer-like cells form in animals.

The evolution of gastrulation, the embryonic formation of distinct tissue layers, was a pivotal event in the metazoan radiation, as it paved the way for diversification of animal body plans from a hollow, ciliated, radially symmetrical ancestor [1]. The position of the site of gastrulation (that segregates internal endomesodermal precursors from outer ectodermal tissue) has played a role in our understanding of patterns of body plan evolution and is tightly regulated during development. In bilaterians (a large clade of bilaterally symmetrical animals that represent over 99% of all extant species), the site of gastrulation is determined by a localized molecular asymmetry resulting from a differential distribution of maternal determinants [2] along the so-called animal-vegetal axis (A-V axis) where the animal pole is marked by the site of polar body release during meiosis [1,3]. In most bilaterians, the site of gastrulation occurs at the vegetal pole (the side opposite the animal pole); however, in cnidarians (corals, sea anemones, and jellyfish) [3], the sister group to all bilaterians and ctenophores (comb jellies), likely to be the earliest branching group of extant metazoans [3], gastrulation occurs at the animal pole [3,4]. Here we show that components of the canonical Wnt-β-catenin (cWnt) signaling pathway mediate endomesoderm formation and patterns the adult primary body axis.

In triploblastic animals, Par proteins regulate cell-polarity and adherens junctions of both ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella vectensis, Par proteins are eliminated altogether in the bifunctional endomesodermal epithelia. Using immunohistochemistry, CRISPR/Cas9, and overexpression of specific mRNAs, we describe the functional association between Par proteins, s- catenin, and snail genes in N. vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of s-catenin in the ectoderm by directing its role in cell adhesion, and that endomesodermal epithelia are organized by a different cell adhesion system. We also show that snail genes, which are mesodermal markers in bilaterians, are sufficient to downregulate Par proteins and translocate s-catenin from the junctions to the cytoplasm in N. vectensis. These data provide insight into the evolution of epithelial structure and the evolution of mesoderm in metazoan embryos.

In triploblastic animals, Par-proteins regulate cell-polarity and adherens junctions of both ectodermal and endodermal epithelia. But, in embryos of the diploblastic cnidarian Nematostella vectensis, Par-proteins are degraded in all cells in the bifunctional gastrodermal epithelium. Using immunohistochemistry, CRISPR/Cas9 mutagenesis, and overexpression of specific mRNAs, we describe the functional association between Par-proteins, ß-catenin, and snail transcription factor genes in N. vectensis embryos. We demonstrate that the aPKC/Par complex regulates the localization of ß-catenin in the ectoderm by stabilizing its role in cell-adhesion, and that endomesodermal epithelial cells are organized by a different cell-adhesion system than that of overlying ectoderm. We also show that ectopic expression of snail genes, which are expressed in mesodermal derivatives in bilaterians, are sufficient to downregulate Par-proteins and translocate ß-catenin from the junctions to the cytoplasm in ectodermal cells. These data provide molecular insight into the evolution of epithelial structure and distinct mesodermal tissue in metazoan embryos.

The major longitudinal body axis of cnidarians (e.g. sea anemones, corals, hydroids) is called the oral-aboral axis and in the anthozoan cnidarian Nematostella vectensis, this axis is established by the selective stabilization and nuclear localization of the protein Beta-catenin (a component of the canonical Wnt signaling pathway) in cells derived from the animal pole. These cells form the endomesoderm during gastrulation with the oral (anterior) pole forming at the animal pole. In bilaterian embryos the site of gastrulation occurs at the vegetal pole. We used a two-step experimental approach to mis-activate cWnt signaling in the developing cnidarian N. vectensis embryos by first erasing the endogenous oral-aboral axis by inhibiting the activation of cWnt signaling in the embryo and then, ectopically activating cWnt signaling at by blastomere injection at a site other than the animal pole. Using marker gene expression, we show that mis-activation of cWnt signaling in N. vectensis embryos result in a new oral-aboral axis. Our data show that the change in the site of gastrulation and endomesoderm specification is the result of moving localized components of the Wnt signaling pathways to the vegetal pole in the last common ancestor of all bilaterians, thus, providing a plausible mechanism for the change in the site of gastrulation from the animal pole in non-bilaterians to the vegetal pole in bilaterians. This change in the metazoan developmental program is arguably the largest developmental constraint in animal evolution as it restricts endomesodermal fates and neural fates to opposite poles of the embryo and paved the way for the rapid diversification (e.g. cephalization and mesoderm formation) of the bilaterian body plans.

International audience; Germ layer formation and axial patterning are biological processes that are tightly linked during embryonic development of most metazoans. In addition to canonical WNT, it has been proposed that ERK-MAPK signaling is involved in specifying oral as well as aboral territories in cnidarians. However, the effector and the molecular mechanism underlying latter phenomenon is unknown. By screening for potential effectors of ERK-MAPK signaling in both domains, we identified a member of the ETS family of transcription factors, Nverg that is bi-polarily expressed prior to gastrulation. We further describe the crucial role of NvERG for gastrulation, endomesoderm as well as apical domain formation. The molecular characterization of the obtained NvERG knock-down phenotype using previously described as well as novel potential downstream targets, provides evidence that a single transcription factor, NvERG, simultaneously controls expression of two different sets of downstream targets, leading to two different embryonic gene regulatory networks (GRNs) in opposite poles of the developing embryo. We also highlight the molecular interaction of cWNT and MEK/ERK/ERG signaling that provides novel insight into the embryonic axial organization of Nematostella, and show a cWNT repressive role of MEK/ERK/ERG signaling in segregating the endomesoderm in two sub-domains, while a common input of both pathways is required for proper apical domain formation. Taking together, we build the first blueprint for a global cnidarian embryonic GRN that is the foundation for additional gene specific studies addressing the evolution of embryonic and larval development.

Gastrulation was arguably the key evolutionary innovation that enabled metazoan diversification, leading to the formation of distinct germ layers and specialized tissues. Differential gene expression specifying cell fate is governed by the inputs of intracellular and/or extracellular signals. Beta-catenin/Tcf and the TGF-beta bone morphogenetic protein (BMP) provide critical molecular signaling inputs during germ layer specification in bilaterian metazoans, but there has been no direct experimental evidence for a specific role for BMP signaling during endomesoderm specification in the early branching metazoan Nematostella vectensis (an anthozoan cnidarian). Using forward transcriptomics, we show that beta-catenin/Tcf signaling and BMP2/4 signaling provide differential inputs into the cnidarian endomesodermal gene regulatory network (GRN) at the onset of gastrulation (24 h postfertilization) in N. vectensis. Surprisingly, beta-catenin/Tcf signaling and BMP2/4 signaling regulate a subset of common downstream target genes in the GRN in opposite ways, leading to the spatial and temporal differentiation of fields of cells in the developing embryo. Thus, we show that regulatory interactions between beta-catenin/Tcf signaling and BMP2/4 signaling are required for the specification and determination of different embryonic regions and the patterning of the oral–aboral axis in Nematostella. We also show functionally that the conserved “kernel” of the bilaterian heart mesoderm GRN is operational in N. vectensis, which reinforces the hypothesis that the endoderm and mesoderm in triploblastic bilaterians evolved from the bifunctional endomesoderm (gastrodermis) of a diploblastic ancestor, and that slow rhythmic contractions might have been one of the earliest functions of mesodermal tissue.

The mesoderm was a key novelty in animal evolution, though we understand little of how 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 patterning both ectodermal and endodermal derivatives along the primary body axis.

Bilaterian photoreceptor cells are characterized by the expression of opsins, signal transduction genes, and ion channels, which together facilitate behavioral responses to light. We have previously identified a ciliary opsin gene from the brachiopod Terebratalia transversa, whose expression in gastrula stage embryos coincides with a photoresponse behavior, suggesting the presence of a functional phototransduction system in these early embryos. To further evaluate the potential for light reception in these embryos, we surveyed transcriptome data to identify phototransduction genes and evaluated their expression. In addition to the previously described ciliary opsin gene, we have identified two Go-class opsins that are also expressed in gastrula stage embryos. Representative members from all classes of Gα-protein genes were also expressed, with a Gα12-class gene being localized in the same anterior ectodermal domain as the opsin transcripts. Both CNG-class and TRP-class ion channels were expressed in the gastrula stage embryos, as were GRK and arrestin genes, which are associated with inhibition of rhodopsin activity. Taken together, these data support the presence of a functional phototransduction system in the early brachiopod embryo.

Background: The nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood.Results: We show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages.Conclusions: Together, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.

achaete-scute homologs (ash) regulate neural development in all bilaterian model animals indicating that they represent a component of the ancestral neurogenic pathway. We test this by investigating four ash genes during development of a basal metazoan, the cnidarian sea anemone Nematostella vectensis. Spatiotemporal expression of ash genes in the early embryo and larval stages suggests that they regulate neurogenesis. More specifically, NvashA is co-expressed with neural genes in the embryonic ectoderm. Knockdown of NvashA results in decreased expression of eight neural markers, including the six novel neural targets identified here. Conversely, overexpression of NvashA induces increased expression of all eight genes, but only within their normal axial domains. Overexpression of NvashB-D differentially increases expression of NvashA targets. The expression patterns and differential ability of ash genes to regulate neural gene expression reveals surprising molecular complexity in these ‘simple’ animals. These data suggest that achaete-scute homologs functioned in the ancestral metazoan neurogenic pathway and provide a foundation to investigate further the evolution of neurogenesis and the origin of complex central nervous systems.

This study describes the early expression and function of β-catenin in the gastropod, Crepidula fornicata. In other bilaterians β-catenin functions in cell adhesion, gastrulation, and cell signaling, which is related to the establishment of the dorso-ventral axis and mesendoderm. Here, we studied the distribution of β-catenin mRNA and protein in C. fornicata via whole mount in situ hybridization and by expressing GFP-tagged β-catenin in vivo. During early cleavage, β-catenin mRNA and protein appear to be broadly localized to all cells in the early embryo. The mRNA tends to be concentrated at inter-phase centrosomes in these cells. At later stages, the mRNA is predominantly in the vegetal macromeres, and subsequently in the rudiment of the hindgut, stomodeum, and velar lobes. Expression of full-length GFP-tagged protein suggests that there is no active mechanism to degrade β-catenin within cells of the early embryos prior to the 25-cell stage. However, by the second day of development, when the fourth quartet micromeres have formed, β-catenin becomes selectively stabilized in the progeny of the 4d mesentoblast (e.g., ML and MR and their daughters) and is missing from most other blastomeres, including vegetal macromeres. Over the next 2 days of development, during subsequent divisions of 4d, β-catenin protein becomes progressively degraded, along the proximo-distal axes, within the progeny of the paired mesendodermal bands. The cells located at the tips of the mesodermal bands (2 mL² and 2 mR²) are the last to contain this protein, which is no longer detected after 4 days of development. In animals like C. fornicata, which undergo a spiral cleavage program (e.g., molluscs, annelids, nemerteans, and polyclad flatworms), the mesentoblast or 4d cell represents the progenitor of endomesoderm (forming hindgut, internal and external kidneys, and various muscles). Therefore, the selective stabilization of β-catenin in the progeny of 4d in C. fornicata is consistent with arguments that a basic, ancestral role of β-catenin lies in the formation of endomesodermal fates. Experiments using a truncated β-catenin clone show that the regions located in the C-terminus, distal to the 11th armadillo repeat, are required for normal stabilization/degradation of β-catenin protein within the embryo. Microinjection of translation blocking β-catenin morpholinos into zygotes led to the down-regulation of β-catenin expression. This resulted in the subsequent failure of gastrulation, but did not interfere with the formation and early cleavage of 4d, although there were no discernable differentiated cell fates in these defective embryos. These results are compared with those obtained in other metazoans.

The relationship between egg polarity and the adult body plan is well understood in many bilaterians. However, the evolutionary origins of embryonic polarity are not known. Insight into the evolution of polarity will come from understanding the ontogeny of polarity in non-bilaterian forms, such as cnidarians. We examined how the axial properties of the starlet sea anemone, Nematostella vectensis (Anthozoa, Cnidaria), are established during embryogenesis. Egg-cutting experiments and sperm localization show that Nematostella eggs are only fertilized at the animal pole. Vital marking experiments demonstrate that the egg animal pole corresponds to the sites of first cleavage and gastrulation, and the oral pole of the adult. Embryo separation experiments demonstrate an asymmetric segregation of developmental potential along the animal–vegetal axis prior to the 8-cell stage. We demonstrate that Dishevelled (Dsh) plays an important role in mediating this asymmetric segregation of developmental fate. Although NvDsh mRNA is ubiquitously expressed during embryogenesis, the protein is associated with the female pronucleus at the animal pole in the unfertilized egg, becomes associated with the unipolar first cleavage furrow, and remains enriched in animal pole blastomeres. Our results suggest that at least one mechanism for Dsh enrichment at the animal pole is through its degradation at the vegetal pole. Functional studies reveal that NvDsh is required for specifying embryonic polarity and endoderm by stabilizing β-catenin in the canonical Wnt signaling pathway. The localization of Dsh to the animal pole in Nematostella and two other anthozoan cnidarians (scleractinian corals) provides a possible explanation for how the site of gastrulation has changed in bilaterian evolution while other axial components of development have remained the same and demonstrates that modifications of the Wnt signaling pathway have been used to pattern a wide variety of metazoan embryos.

SUMMARY Reef-building scleractinian corals widely engage in symbiotic relationships with Symbiodinium dinoflagellates (zooxanthellae), which reside inside cells of the gastrodermis. In most cases, sexually produced larvae acquire their symbionts from the environment in the early developmental stages preceding settlement; however, some scleractinian corals maternally “seed” their oocytes with symbionts, and these symbionts are reported to be restricted to the gastrodermis at the time of its formation (gastrulation). A precise mechanism for how Symbiodinium are translocated to endoderm in these seeded species was previously unknown. In order to examine the process of endoderm formation and Symbiodinium localization during gastrulation, we have examined two species of “robust” clade scleractinians: Fungia scutaria (nonseeded) and Pocillopora meandrina (maternally seeded). We determined that both species, independent of whether or not they are seeded, undergo a “nutritive” stage before gastrulation, wherein lipid-rich cells (F. scutaria) or membrane-bound cellular fragments (P. meandrina) are passed to the blastocoel where they are subsequently taken up by the definitive endoderm. This emergent property of anthozoan development has been co-opted to facilitate the movement of Symbiodinium to the blastocoel (future site of endoderm), in the seeded species, where they are later phagocytosed by the newly formed definitive endoderm. Additionally, both species of robust clade scleractinians examined gastrulate by way of invagination, as do the majority of anthozoans. This invagination differs from the prawn chip-type gastrulation seen in the complex clade corals and provides evidence for a possible linkage between gastrulation type and phylogenetic history.

The littorinimorph gastropod Crepidula fornicata shows a spiralian cleavage pattern and has been the subject of studies in experimental embryology, cell lineage, and the organization of the larval nervous system. To investigate the contribution of early blastomeres to the veliger larva, we used intracellular cell lineage tracers in combination with high-resolution confocal imaging. This study corroborates many features derived from other spiralian fate maps (such as the origins of the hindgut and mesoderm from the 4d mesentoblast), but also yields new findings, particularly with respect to the origins of internal structures, such as the nervous system and musculature that have never been described in detail. The ectomesoderm in C. fornicata is mainly formed by micromeres of the 3rd quartet (principally 3a and 3b), which presumably represents a plesiomorphic condition for molluscs. The larval central nervous system is mainly formed by the micromeres of the 1st and 2nd quartet, of which 1a, 1c, and 1d form the anterior apical ganglion and nerve tracks to the foot and velum, and 2b and 2d form the visceral loop and the mantle cell. Our study shows that both first and second velar ciliary bands are generated by the same cells that form the prototroch in other spiralians and apparently bear no homology to the metatroch found in annelids.

Gastrulation is a central event in metazoan development, involving many cellular behaviors including invagination, delamination, and ingression. Understanding the cell biology underlying gastrulation in many different taxa will help clarify the evolution of gastrulation mechanisms. Gastrulation in the anthozoan cnidarian Nematostella vectensis has been described as a combination of invagination and unipolar ingression through epithelial to mesenchymal transitions (EMT), possibly controlled by snail genes, important regulators of EMT in other organisms. Our examination, however, fails to reveal evidence of ingressing cells. Rather, we observe that endodermal cells constrict their apices, adopting bottle-like morphologies especially pronounced adjacent to the blastopore lip. They retain apical projections extending to the archenteron throughout gastrulation. Basally, they form actin-rich protrusions, including interdigitating filopodia that may be important in pulling the ectodermal and endodermal cells together. Endodermal cells retain cell–cell junctions while invaginating, and are organized throughout development. Never is the blastocoel filled by a mass of mesenchyme. Additionally, injection of splice-blocking morpholinos to Nematostella snail genes does not result in a phenotype despite dramatically reducing wild-type transcript, and overexpression of Snail-GFP in different clonal domains has no effect on cell behavior. These data indicate that EMT is not a major factor during gastrulation in Nematostella.

Background The evolutionary origins of cell polarity in metazoan embryos are unclear. In most bilaterian animals, embryonic and cell polarity are set up during embryogenesis with the same molecules being utilized to regulate tissue polarity at different life stages. Atypical protein kinase C (aPKC), lethal giant larvae (Lgl), and Partitioning-defective (Par) proteins are conserved components of cellular polarization, and their role in establishing embryonic asymmetry and tissue polarity have been widely studied in model bilaterian groups. However, the deployment and role of these proteins in animals outside Bilateria has not been studied. We address this by characterizing the localization of different components of the Par system during early development of the sea anemone Nematostella vectensis, a member of the clade Cnidaria, the sister group to bilaterian animals. Results Immunostaining using specific N. vectensis antibodies and the overexpression of mRNA-reporter constructs show that components of the N. vectensis Par system (NvPar-1, NvPar-3, NvPar-6, NvaPKC, and NvLgl) distribute throughout the microtubule cytoskeleton of eggs and early embryos without clear polarization along any embryonic axis. However, they become asymmetrically distributed at later stages, when the embryo forms an ectodermal epithelial layer. NvLgl and NvPar-1 localize in the basolateral cortex, and NvaPKC, NvPar-6, and NvPar-3 at the apical zone of the cell in a manner seen in bilaterian animals. Conclusions The cnidarian N. vectensis exhibits clear polarity at all stages of early embryonic development, which appears to be established independent of the Par system reported in many bilaterian embryos. However, in N. vectensis, using multiple immunohistochemical and fluorescently labeled markers in vivo, components of this system are deployed to organize epithelial cell polarity at later stages of development. This suggests that Par system proteins were co-opted to organize early embryonic cell polarity at the base of the Bilateria and that, therefore, different molecular mechanisms operate in early cnidarian embryogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13227-015-0014-6) contains supplementary material, which is available to authorized users.

We examined the evolutionary origins of the ether-à-go-go (EAG) family of voltage-gated K+ channels, which have a strong influence on the excitability of neurons. The bilaterian EAG family comprises three gene subfamilies (Eag, Erg and Elk) distinguished by sequence conservation and functional properties. Searches of genome sequence indicate that EAG channels are metazoan specific, appearing first in ctenophores. However, phylogenetic analysis including two EAG family channels from the ctenophore Mnemiopsis leidyi indicates that the diversification of the Eag, Erg and Elk gene subfamilies occurred in a cnidarian/bilaterian ancestor after divergence from ctenophores. Erg channel function is highly conserved between cnidarians and mammals. Here we show that Eag and Elk channels from the sea anemone Nematostella vectensis (NvEag and NvElk) also share high functional conservation with mammalian channels. NvEag, like bilaterian Eag channels, has rapid kinetics, whereas NvElk activates at extremely hyperpolarized voltages, which is characteristic of Elk channels. Potent inhibition of voltage activation by extracellular protons is conserved between mammalian and Nematostella EAG channels. However, characteristic inhibition of voltage activation by Mg2+ in Eag channels and Ca2+ in Erg channels is reduced in Nematostella because of mutation of a highly conserved aspartate residue in the voltage sensor. This mutation may preserve sub-threshold activation of Nematostella Eag and Erg channels in a high divalent cation environment. mRNA in situ hybridization of EAG channels in Nematostella suggests that they are differentially expressed in distinct cell types. Most notable is the expression of NvEag in cnidocytes, a cnidarian-specific stinging cell thought to be a neuronal subtype.

Background Brachiopods undergo radial cleavage, which is distinct from the stereotyped development of closely related spiralian taxa. The mesoderm has been inferred to derive from the archenteron walls following gastrulation, and the primary mesoderm derivative in the larva is a complex musculature. To investigate the specification and differentiation of the mesoderm in the articulate brachiopod Terebratalia transversa, we have identified orthologs of genes involved in mesoderm development in other taxa and investigated their spatial and temporal expression during the embryonic and larval development of T. transversa. Results Orthologs of 17 developmental regulatory genes with roles in the development of the mesoderm in other bilaterian animals were found to be expressed in the developing mesoderm of T. transversa. Five genes, Tt.twist, Tt.GATA456, Tt.dachshund, Tt.mPrx, and Tt.NK1, were found to have expression throughout the archenteron wall at the radial gastrula stage, shortly after the initiation of gastrulation. Three additional genes, Tt.Pax1/9, Tt.MyoD, and Tt.Six1/2, showed expression at this stage in only a portion of the archenteron wall. Tt.eya, Tt.FoxC, Tt.FoxF, Tt.Mox, Tt.paraxis, Tt.Limpet, and Tt.Mef2 all showed initial mesodermal expression during later gastrula or early larval stages. At the late larval stage, Tt.dachshund, Tt.Limpet, and Tt.Mef2 showed expression in nearly all mesoderm cells, while all other genes were localized to specific regions of the mesoderm. Tt.FoxD and Tt.noggin both showed expression in the ventral mesoderm at the larval stages, with gastrula expression patterns in the archenteron roof and blastopore lip, respectively. Conclusions Expression analyses support conserved roles for developmental regulators in the specification and differentiation of the mesoderm during the development of T. transversa. Expression of multiple mesodermal factors in the archenteron wall during gastrulation supports previous morphological observations that this region gives rise to larval mesoderm. Localized expression domains during gastrulation and larval development evidence early regionalization of the mesoderm and provide a basis for hypotheses regarding the molecular regulation underlying the complex system of musculature observed in the larva. Electronic supplementary material The online version of this article (doi:10.1186/s13227-015-0004-8) contains supplementary material, which is available to authorized users.

A small polar lobe forms at the first and second cleavage divisions in the gastropod mollusc Crepidula fornicata. These lobes normally fuse with the blastomeres that give rise to the D quadrant at the two- and four-cell stages (cells ultimately generating the 4d mesentoblast and D quadrant organizer). Significantly, removal of the small polar lobe had no noticeable effect on subsequent development of the veliger larva. The behavior of the polar lobe and characteristic early cell shape changes involving protrusion of the 3D macromere at the 24-cell suggest that the D quadrant is specified prior to the sixth cleavage division. On the other hand, blastomere deletion experiments indicate that the D quadrant is not determined until the time of formation of the 4d blastomere (mesentoblast). In fact, embryos can undergo regulation to form normal-appearing larvae if the prospective D blastomere or 3D macromere is removed. Removal of the 4d mesentoblast leads to highly disorganized, radial development. Removal of the first quartet micromeres at the 8-cell stage also leads to the development of radialized larvae. These findings indicate that the embryos of C. fornicata follow the mode of development exhibited by equal-cleaving spiralians, which involves conditional specification of the D quadrant organizer via inductive interactions, presumably from the first quartet micromeres.

Summary Cnidarians (corals, sea anemones, hydroids, and jellyfish) are a basal taxon closely related to bilaterally symmetrical animals [1–3] 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 TGFβ 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 TGFβ ligands and their antagonist are colocalized at the onset of gastrulation but then segregate by germ layer as gastrulation proceeds. TGFβ 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 TGFβ signaling operates at these stages. NvChordin expression forms a boundary between TGFβ-expressing endodermal cells and aboral ectoderm. Manipulation of nuclear β-catenin localization affects TGFβ 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.

Polyplacophorans, or chitons, are an important group of molluscs, which are argued to have retained many plesiomorphic features of the molluscan body plan. Polyplacophoran trochophore larvae posses several features that are distinctly different from those of their sister trochozoan taxa, including modifications of the ciliated prototrochal cells, the postrochal position of the larval eyes or ocelli, epidermal calcareous spicules, and a collection of serially reiterated epidermal shell plates. Despite these differences, chitons demonstrate a canonical pattern of equal spiral cleavage shared by other spiralian phyla that permits the identification of homologous cells across this animal clade. Cell lineage analysis using intracellular labeling on one chiton species, Chaetopleura apiculata, shows that the ocelli are generated from different lineal precursors (second-quartet micromeres: 2a, 2c) compared to those in all other spiralians studied to date (first-quartet micromeres: 1a, 1c). This situation implies that significant changes have also occurred in terms of the inductive interactions that control eye development in the spiralians. Although radical departures from the spiralian developmental program are seen in some molluscs (i.e., cephalopods), the findings presented here indicate that important changes can occur even within the highly constrained framework of the spiral cleavage program. Among spiralians, variation has been reported for the origin of the anterior, sensory, apical organ, which arises from the 1c and 1d micromeres in C. apiculata. The prototroch of C. apiculata consists of two to three irregular rows of ciliated cells but arise from 1q and 2q daughters, similar to that of Ischnochiton rissoi, as well as the gastropod, Patella vulgata. Despite certain early claims, there is no supporting evidence that any of the shell plates arise pretrochally in C. apiculata. The first seven of eight definitive shell plates that arise in the larva originate from shell secreting grooves in the postrochal region (derived from 2c, 2d, 3d). Earlier descriptions indicate that the eighth plate arises later at metamorphosis, and as this is formed posteriorly, it too forms in the postrochal region. On the other hand, epidermal spicules originate from both pretrochal and postrochal cells (1a,1d, 2a, 2c, 3c, 3d). The significance of these observations is discussed in light of various hypotheses concerning the origin of the conchiferan shell. This study reveals conservation, as well as evolutionary novelty, in the assignment of specific cell fates in the spiralians.

The first description of the cleavage program of the palaeonemertean Carinoma tremaphoros (a member of a basal clade of the Nemertea) is illustrated by confocal microscopy and microinjection and compared to development of more derived nemerteans and other eutrochozoans (Annelida, Mollusca, Sipunculida and Echiurida). Lineage tracers were injected into individual blastomeres of C. tremaphoros at the 2-, 4-, 8- and 16-cell stage. Subsequent development was followed to the formation of simple (so-called planuliform) planktonic larvae to establish the ultimate fates of the blastomeres. Results of labeling experiments demonstrate that the development of C. tremaphoros bears closer similarity to other Eutrochozoa than development of a previously studied hoplonemertean (Nemertopsis bivittata) and a heteronemertean (Cerebratulus lacteus) in that the first cleavage plane bears an invariant relationship to the plane of bilateral symmetry of the larval body. Additionally, our cell-labeling experiments support the earlier suggestion that the transitory pre-oral belt of cells in the larvae of C. tremaphoros corresponds to the prototroch of other Eutrochozoa. A unique feature of development of C. tremaphoros includes the oblique orientation of the trochal lineages with respect to the anterior–posterior axis of the larva. The significance and application of cleavage characters such as presence of molluscan vs. annelid cross for phylogenetic analyses is reviewed. We argue that molluscan or annelid cross, neither of which are present in nemerteans, are merely two out of much greater variety of patterns created by the differences in the relative size and timing of formation of micromere quartets and none can be considered, by itself, as evidence of close phylogenetic relationship between phyla.

The human oncogene beta-catenin is a bifunctional protein with critical roles in both cell adhesion and transcriptional regulation in the Wnt pathway. Wnt/beta-catenin signalling has been implicated in developmental processes as diverse as elaboration of embryonic polarity, formation of germ layers, neural patterning, spindle orientation and gap junction communication, but the ancestral function of beta-catenin remains unclear. In many animal embryos, activation of beta-catenin signalling occurs in blastomeres that mark the site of gastrulation and endomesoderm formation, raising the possibility that asymmetric activation of beta-catenin signalling specified embryonic polarity and segregated germ layers in the common ancestor of bilaterally symmetrical animals. To test whether nuclear translocation of beta-catenin is involved in axial identity and/or germ layer formation in 'pre-bilaterians', we examined the in vivo distribution, stability and function of beta-catenin protein in embryos of the sea anemone Nematostella vectensis (Cnidaria, Anthozoa). Here we show that N. vectensis beta-catenin is differentially stabilized along the oral-aboral axis, translocated into nuclei in cells at the site of gastrulation and used to specify entoderm, indicating an evolutionarily ancient role for this protein in early pattern formation.

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.

Background The primary hormone of the vertebrate pineal gland, melatonin, has been identified broadly throughout the eukaryotes. While the role for melatonin in cyclic behavior via interactions with the circadian clock has only been reported in vertebrates, comparative research has shown that the transcription-translation loops of the animal circadian clock likely date to the cnidarian-bilaterian ancestor, leaving open significant questions about the evolutionary origin of melatonin signaling in circadian behavior by interacting with the molecular clock. Results Expression of melatonin in adult anemones showed peak expression at the end of light period (zeitgeber time (ZT) = 12) when cultured under diel conditions, coinciding with expression of genes and enzyme activity for members of the melatonin synthesis pathway (tryptophan hydroxylase and hydroxyindol-O-methyltransferase), which also showed rhythmic expression. During embryogenesis and juvenile stages, melatonin showed cyclic oscillations in concentration, peaking in midday. Spatial (in situ hybridization) and quantitative (real-time PCR) transcription of clock genes during development of N. vectensis showed these ‘clock’ genes are expressed early in the development, prior to rhythmic oscillations, suggesting functions independent of a function in the circadian clock. Finally, time-course studies revealed that animals transferred from diel conditions to constant darkness lose circadian expression for most of the clock genes within 4 days, which can be reset by melatonin supplementation. Conclusions Our results support an ancient role for melatonin in the circadian behavior of animals by showing cyclic expression of this hormone under diel conditions, light-dependent oscillations in genes in the melatonin synthesis pathway, and the function of melatonin in initiating expression of circadian clock genes in the cnidarian N. vectensis. The differences in expression melatonin and the circadian clock gene network in the adult stage when compared with developmental stages of N. vectensis suggests new research directions to characterize stage-specific mechanisms of circadian clock function in animals.

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K(+) channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for IKr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K(+) channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the IKr-like gating phenotype with mammalian Erg channels. Thus, the IKr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral IKr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.

Background Wound healing is the first stage of a series of cellular events that are necessary to initiate a regenerative response. Defective wound healing can block regeneration even in animals with a high regenerative capacity. Understanding how signals generated during wound healing promote regeneration of lost structures is highly important, considering that virtually all animals have the ability to heal but many lack the ability to regenerate missing structures. Cnidarians are the phylogenetic sister taxa to bilaterians and are highly regenerative animals. To gain a greater understanding of how early animals generate a regenerative response, we examined the cellular and molecular components involved during wound healing in the anthozoan cnidarian Nematostella vectensis. Results Pharmacological inhibition of extracellular signal-regulated kinases (ERK) signaling blocks regeneration and wound healing in Nematostella. We characterized early and late wound healing events through genome-wide microarray analysis, quantitative PCR, and in situ hybridization to identify potential wound healing targets. We identified a number of genes directly related to the wound healing response in other animals (metalloproteinases, growth factors, transcription factors) and suggest that glycoproteins (mucins and uromodulin) play a key role in early wound healing events. This study also identified a novel cnidarian-specific gene, for a thiamine biosynthesis enzyme (vitamin B synthesis), that may have been incorporated into the genome by lateral gene transfer from bacteria and now functions during wound healing. Lastly, we suggest that ERK signaling is a shared element of the early wound response for animals with a high regenerative capacity. Conclusions This research describes the temporal events involved during Nematostella wound healing, and provides a foundation for comparative analysis with other regenerative and non-regenerative species. We have shown that the same genes that heal puncture wounds are also activated after oral-aboral bisection, indicating a clear link with the initiation of regenerative healing. This study demonstrates the strength of using a forward approach (microarray) to characterize a developmental phenomenon (wound healing) at a phylogenetically important crossroad of animal evolution (cnidarian-bilaterian ancestor). Accumulation of data on the early wound healing events across numerous systems may provide clues as to why some animals have limited regenerative abilities.

Ctenophores possess eight longitudinally arrayed rows of comb plate cilia. Previous intracellular cell lineage analysis has shown that these comb rows are derived from two embryonic lineages, both daughters of the four e(1) micromeres (e(11) and e(12)) and a single daughter of the four m(1) micromeres (the m(12) micromeres). Although isolated e(1) micromeres will spontaneously generate comb plates, cell deletion experiments have shown that no comb plates appear during embryogenesis following the removal of e(1) descendents. Thus, the m(1) lineage requires the inductive interaction of the e(1) lineage to contribute to comb plate formation. Here we show that, although m(12) cells are normally the only m(1) derivatives to contribute to comb plate formation, m(11) cells are capable of generating comb plates in the absence m(12) cells. The reason that m(11) cells do not normally make comb rows may be attributable either to their more remote location relative to critical signaling centers (e.g., e(1) descendants) or to inhibitory signals that may be provided by other nearby cells such as sister cells m(12). In addition, we show that the signals provided by the e(1) lineage are not sufficient for m(1)-derived comb plate formation. Signals provided by endomesodermal progeny of either the E or the M lineages (the 3E or 2M macromeres) are also required.

Lobate ctenophores (tentaculates) generally exhibit a remarkable ability to regenerate missing structures as adults. On the other hand, their embryos exhibit a highly mosaic behavior when cut into halves or when specific cells are ablated. These deficient embryos do not exhibit embryonic regulation, and generate incomplete adult body plans. Under certain conditions, however, these deficient animals are subsequently able to replace the missing structures during the adult phase in a process referred to as “post-regeneration.” We have determined that successful post-regeneration can be predicted on the basis of a modified polar coordinate model, and the rules of intercalary regeneration, as defined by French et al. (V. French, P. J. Bryant, and S. V. Bryant, 1976, Science 193, 969–981.) The model makes certain assumptions about the organization of the ctenophore body plan that fit well with what we have determined on the basis of cell lineage fates maps, and their twofold rotational (“biradial”) symmetry. The results suggest that cells composing the ctenophore adult body plan possess positional information, which is utilized to reconstruct the adult body plan. More specifically, we have found that the progeny of three specific cell lineages are required to support post-regeneration of the comb rows (the e1, e2, and m1 micromeres). Furthermore, post-regeneration of the comb rows involves a suite of cell–cell inductive interactions, which are similar to those that take place during their embryonic formation. The significance of these findings is discussed in terms of the organization of the ctenophore body plan, and the mechanisms involved in cell fate specification. This situation is also contrasted with that of the atentaculate ctenophores, which are unable to undergo post-regeneration.

Recent molecular evidence suggests the turbellarian Platyhelminthes may represent the extant basal members of the Spiralia and therefore probably exhibit ancient features of the spiralian developmental program. The stereotypic quartet spiral cleavage pattern of the polyclad turbellarian embryo, among other features, indicates that this group may be closely related to the ancestral flatworm; however, polyclad embryos have been the subject of few experimental studies. Here we report the results of a cell lineage analysis of the embryo of the polycladHoploplana inquilinabased on microinjection of DiI into cleavage-stage blastomeres following formation of each of the four quartets of micromeres. The first quartet gives rise to most of the lateral and anterior ectoderm of the Müller's larva; the second quartet forms largely dorsal and ventral ectoderm as well as the circular muscles; the third quartet forms only small clones of ectoderm; and only the 4d cell of the fourth quartet contributes to larval structure, forming the longitudinal muscles, mesenchyme, and probably endoderm. Our results demonstrate a striking similarity between the cell lineages of polyclad and higher spiralian embryos, in which the four quadrants also bear the same relationships to the larval axes and give rise to comparable larval structures, including derivation of mesoderm from both ectodermal (2b) and endodermal precursors (4d).

Lineage tracers were injected into individual blastomeres in embryos of the indirect-developing nemerteanCerebratulus lacteusthrough the formation of the fourth quartet of micromeres. Subsequent development was followed to the formation of feeding pilidium larvae to establish their ultimate fates. Results showed that these blastomeres have unique fates, and their clones give rise to highly predictable regions of the larval body. As in other spiralians, four discrete cell quadrants can be identified. For the most part, their identities are homologous to the typical spiralian A, B, C, and D cell quadrants. In some respects their fates differ from the typical spiralian fate map; however, these can be understood in terms of simple modifications of the early cleavage program. Unlike most spiralians, the first quartet micromeres in the eight-celled embryo are larger than the corresponding vegetal macromeres, and generate most of the larval ectoderm. All four of these micromeres contribute to the apical organ and generate four bilaterally situated domains of ectoderm, where the progeny of the 1a and 1d micromeres lie to the left of the median plane while those of 1b and 1c lie to the right. Unlike the progeny of the first quartet, those of the second quartet are situated in left (2a), ventral (2b), right (2c), and dorsal (2d) positions. The third quartet micromeres generate clones situated in a bilaterally symmetrical fashion similar to those of the first quartet. The alternating axial relationships exhibited by successive micromere quartets are a characteristic of spiralian development. Unlike other spiralian larvae possessing a ciliary band, the pilidium larval ciliary band is formed by all blastomeres of the first and second micromere quartets, as well as 3c and 3d. Ectomesoderm is derived from two blastomeres (3a and 3b), which give rise to the extensive array of the larval muscle cells.C. lacteusalso possesses a true mesentoblast (4d) which gives rise to a pair of mesodermal bandlets, and scattered mesenchymal cells. The dual origin of the mesoderm, as both ectomesoderm and endomesoderm, appears to be a condition present in all spiralians. The gut is formed by all the fourth quartet micromeres as well as the vegetal macromeres (4A, 4B, 4C, 4D). Despite differences in the determination of axial properties and some modifications in quadrant fates, nemerteans appear to be constructed on the typical spiralian developmental platform.

Ctenophores are a phylum of diploblastic marine animals displaying biradial symmetry organized along an oral aboral axis. One of the apomorphic sets of adult structures in ctenophores are the eight external comb rows, which run along the oral-aboral axis. Comb rows consist of serial arrays of individual comb plates of cilia, which beat in a coordinated fashion for locomotory behavior. Classical cell lineage experiments using chalk particles indicated that comb rows are derived exclusively from the four e1 micromeres at the 16-cell stage. This conclusion was also supported by the fact that no ctene rows (or their underly ing endodermal canals) form when all four e1 micromeres were deleted. We have used intracellular diI cell lineage tracing to determine that, in addition to e1 micromeres, the four m1 micromeres also make significant contributions to the ctene rows. Thus, e1 micromere derivatives not only generate comb plates but are required for ctene row formation by m1 derivatives. These results demonstrate that inductive interactions are an important component of early development in ctenophores and indicate that e1 micromeres influence the development of adjacent cell lineages (both m1 and endodermal lineages) during ctenophore embryogenesis. In addition, intracellular labeling has revealed that there are subtle variations in the composition of clones derived from identified embryonic blastomeres. Together these findings reveal a picture of ctenophore embryogenesis, which is in marked contrast to the former rigid ‘mosaic’ reputation of ctenophore devel opment, and invite speculation as to the role of the cleavage program in embryonic patterning in the lower Metazoa.

The msx gene family is one of the most highly conserved of the nonclustered homeobox-containing genes. We have isolated an msx homolog (Le-msx) from the glossiphoniid leech, Helobdella robusta, and characterized its pattern of expression by whole mount in situ hybridization. In situ expression and reverse transcription polymerase chain reaction (RT-PCR) data results show that Le-msx is a maternal transcript initially uniformly distributed in the cortex of immature oocytes that becomes asymmetrically localized to the polar regions of the uncleaved zygote. This is the earliest reported expression for the msx gene family and the first maternally expressed homeodomain-containing transcription factor reported in annelids. During embryonic development, Le-msx is expressed in all 10 embryonic stem cells and their segmental founder cell descendants. At midembryonic stages, Le-msx is expressed in the expanding germinal plate. Le-msx is confined to the central nervous system and nephridia at late (stage 9) stages and subsequently disappears from nephridia. In addition, we present a phylogenetic hypothesis for the evolution of the msx gene family, including the identification of a putative C. elegans msx homolog and the realignment of the sponge msx homolog to the NK class of homeodomain genes.

Experiments were performed to determine whether the first cleavage division plays a role in setting up the dorsoventral axis in embryos of the equal-cleaving nemerteanCerebratulus lacteus.Fertilized eggs were compressed to change the orientation of the first cleavage spindle, and thus the plane of the first cleavage division. One cell of the resulting two-celled embryos was then injected with lineage tracer to determine whether the first cleavage plane always maintains its normal relationships to the median and frontal planes or whether new relationships (and thus, novel cell lineages) could be created. Many of these compressed embryos gave rise to normal-appearing pilidium larvae in which the first cleavage plane had taken on various oblique angular relationships relative to the plane of bilateral symmetry and the dorsoventral axis of the larva. These findings indicate that the first cleavage plane can be dissociated from its normal relationships to these axial properties. Thus, the first cleavage division is not causally involved in the establishment of the dorsoventral and bilateral axes. We argue that the dorsoventral axis is specified prior to the first cleavage division.

The annelids are a diverse phylum of metamerically segmented animals. They are of special interest embryologically because of their highly stereotyped pattern of development, much of which is shared by other spiralian phyla. They are also of interest phylogenetically for what they can tell us about the evolution of segmentation, and the relationships of coelomate protostomes in general. In this paper we review the embryology of the different annelid groups, showing considerable conservation of the basic character of segmentation, with modifications associated with evolving life history strategies. We also describe the current knowledge of molecular mechanisms of segmentation in annelids. The most is known at a cellular and molecular level about the phylogenetically derived leeches, while information is just emerging on the more basal annelids, the polychaetes and oligochaetes. This review is intended to provide a framework for comparison of the different annelid groups, and to suggest avenues for future research that might help to illuminate the evolution of annelid developmental patterns.

The nemerteans belong to a phylum of coelomate worms that display a highly conserved pattern of cell divisions referred to as spiral cleavage. It has recently been shown that the fates of the four embryonic cell quadrants in two species of nemerteans are not homologous to those in other spiralian embryos, such as the annelids and molluscs (Henry, J. Q. and Martindale, M. Q. (1994a) Develop. Genetics 15, 64-78). Equal-cleaving molluscs utilize inductive interactions to establish quadrant-specific cell fates and embryonic symmetry properties following fifth cleavage. In order to elucidate the manner in which cell fates are established in nemertean embryos, we have conducted cell isolation and deletion experiments to examine the developmental potential of the early cleavage blastomeres of two equal-cleaving nemerteans, Nemertopsis bivittata and Cerebratulus lacteus. These two species display different modes of development: N. bivittata develops directly via a non-feeding larvae, while C. lacteus develops to form a feeding pilidium larva which undergoes a radical metamorphosis to give rise to the juvenile worm. By examining the development of certain structures and cell types characteristic of quadrant-specific fates for each of these species, we have shown that isolated blastomeres of the indirect-developing nemertean, C. lacteus, are capable of generating cell fates that are not a consequence of that cell’s normal developmental program. For instance, dorsal blastomeres can form muscle fibers when cultured in isolation. In contrast, isolated blastomeres of the directdeveloping species, N. bivittata do not regulate their development to the same extent. Some cell fates are specified in a precocious manner in this species, such as those that give rise to the eyes. Thus, these findings indicate that equalcleaving spiralian embryos can utilize different mechanisms of cell fate and axis specification. The implications of these patterns of nemertean development are discussed in relation to experimental work in other spiralian embryos, and a model is presented that accounts for possible evolutionary changes in cell lineage and the process of cell fate specification amongst these protostome phyla.

Background The contribution of cell proliferation to regeneration varies greatly between different metazoan models. Planarians rely on pluripotent neoblasts and amphibian limb regeneration depends upon formation of a proliferative blastema, while regeneration in Hydra can occur in the absence of cell proliferation. Recently, the cnidarian Nematostella vectensis has shown potential as a model for studies of regeneration because of the ability to conduct comparative studies of patterning during embryonic development, asexual reproduction, and regeneration. The present study investigates the pattern of cell proliferation during the regeneration of oral structures and the role of cell proliferation in this process. Results In intact polyps, cell proliferation is observed in both ectodermal and endodermal tissues throughout the entire oral-aboral axis, including in the tentacles and physa. Following bisection, there is initially little change in proliferation at the wound site of the aboral fragment, however, beginning 18 to 24 hours after amputation there is a dramatic increase in cell proliferation at the wound site in the aboral fragment. This elevated level of proliferation is maintained throughout the course or regeneration of oral structures, including the tentacles, the mouth, and the pharynx. Treatments with the cell proliferation inhibitors hydroxyurea and nocodazole demonstrate that cell proliferation is indispensable for the regeneration of oral structures. Although inhibition of regeneration by nocodazole was generally irreversible, secondary amputation reinitiates cell proliferation and regeneration. Conclusions The study has found that high levels of cell proliferation characterize the regeneration of oral structures in Nematostella, and that this cell proliferation is necessary for the proper progression of regeneration. Thus, while cell proliferation contributes to regeneration of oral structures in both Nematostella and Hydra, Nematostella lacks the ability to undergo the compensatory morphallactic mode of regeneration that characterizes Hydra. Our results are consistent with amputation activating a quiescent population of mitotically competent stem cells in spatial proximity to the wound site, which form the regenerated structures.