Author Correction: The mid-developmental transition and the evolution of animal body plans.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ctenophores are direct developers that reproduce continuously beginning very early after hatching.

A substantial body of literature reports that ctenophores exhibit an apparently unique life history characterized by biphasic sexual reproduction, the first phase of which is called larval reproduction or dissogeny. Whether this strategy is plastically deployed or a typical part of these species' life history was unknown. In contrast to previous reports, we show that the ctenophore Mnemiopsis leidyi does not have separate phases of early and adult reproduction, regardless of the morphological transition to what has been considered the adult form. Rather, these ctenophores begin to reproduce at a small body size and spawn continuously from this point onward under adequate environmental conditions. They do not display a gap in productivity for metamorphosis or other physiological transition at a certain body size. Furthermore, nutritional and environmental constraints on fecundity are similar in both small and large animals. Our results provide critical parameters for understanding resource partitioning between growth and reproduction in this taxon, with implications for management of this species in its invaded range. Finally, we report an observation of similarly small-size spawning in a beroid ctenophore, which is morphologically, ecologically, and phylogenetically distinct from other ctenophores reported to spawn at small sizes. We conclude that spawning at small body size should be considered as the default, on-time developmental trajectory rather than as precocious, stress-induced, or otherwise unusual for ctenophores. The ancestral ctenophore was likely a direct developer, consistent with the hypothesis that multiphasic life cycles were introduced after the divergence of the ctenophore lineage.

A novel regulatory gene promotes novel cell fate by suppressing ancestral fate in the sea anemone Nematostella vectensis.

Cnidocytes (i.e., stinging cells) are an unequivocally novel cell type used by cnidarians (i.e., 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 develop 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 report a mechanism by which a truly novel regulatory gene (ZNF845) promotes the development of a truly novel cell type (cnidocyte) through duplication of an ancestral cell lineage (neuron) and inhibition of its ancestral identity (RFamide).

Cytoplasmic Polyadenylation Is an Ancestral Hallmark of Early Development in Animals.

Differential regulation of gene expression has produced the astonishing diversity of life on Earth. Understanding the origin and evolution of mechanistic innovations for control of gene expression is therefore integral to evolutionary and developmental biology. Cytoplasmic polyadenylation is the biochemical extension of polyadenosine at the 3'-end of cytoplasmic mRNAs. This process regulates the translation of specific maternal transcripts and is mediated by the Cytoplasmic Polyadenylation Element-Binding Protein family (CPEBs). Genes that code for CPEBs are amongst a very few that are present in animals but missing in nonanimal lineages. Whether cytoplasmic polyadenylation is present in non-bilaterian animals (i.e., sponges, ctenophores, placozoans, and cnidarians) remains unknown. We have conducted phylogenetic analyses of CPEBs, and our results show that CPEB1 and CPEB2 subfamilies originated in the animal stem lineage. Our assessment of expression in the sea anemone, Nematostella vectensis (Cnidaria), and the comb jelly, Mnemiopsis leidyi (Ctenophora), demonstrates that maternal expression of CPEB1 and the catalytic subunit of the cytoplasmic polyadenylation machinery (GLD2) is an ancient feature that is conserved across animals. Furthermore, our measurements of poly(A)-tail elongation reveal that key targets of cytoplasmic polyadenylation are shared between vertebrates, cnidarians, and ctenophores, indicating that this mechanism orchestrates a regulatory network that is conserved throughout animal evolution. We postulate that cytoplasmic polyadenylation through CPEBs was a fundamental innovation that contributed to animal evolution from unicellular life.

Independent Innexin Radiation Shaped Signaling in Ctenophores.

Innexins facilitate cell-cell communication by forming gap junctions or nonjunctional 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, and Beroe 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 the M. leidyi innexin cluster suggests that this cluster has been maintained due to constraints related to gene regulation. We describe the 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.

Cellular micromasonry: biofabrication with single cell precision.

In many tissues, cell type varies over single-cell length-scales, creating detailed heterogeneities fundamental to physiological function. To gain understanding of the relationship between tissue function and detailed structure, and eventually to engineer structurally and physiologically accurate tissues, we need the ability to assemble 3D cellular structures having the level of detail found in living tissue. Here we introduce a method of 3D cell assembly having a level of precision finer than the single-cell scale. With this method we create detailed cellular patterns, demonstrating that cell type can be varied over the single-cell scale and showing function after their assembly.

The mid-developmental transition and the evolution of animal body plans.

Animals are grouped into ~35 'phyla' based upon the notion of distinct body plans. Morphological and molecular analyses have revealed that a stage in the middle of development--known as the phylotypic period--is conserved among species within some phyla. Although these analyses provide evidence for their existence, phyla have also been criticized as lacking an objective definition, and consequently based on arbitrary groupings of animals. Here we compare the developmental transcriptomes of ten species, each annotated to a different phylum, with a wide range of life histories and embryonic forms. We find that in all ten species, development comprises the coupling of early and late phases of conserved gene expression. These phases are linked by a divergent 'mid-developmental transition' that uses species-specific suites of signalling pathways and transcription factors. This mid-developmental transition overlaps with the phylotypic period that has been defined previously for three of the ten phyla, suggesting that transcriptional circuits and signalling mechanisms active during this transition are crucial for defining the phyletic body plan and that the mid-developmental transition may be used to define phylotypic periods in other phyla. Placing these observations alongside the reported conservation of mid-development within phyla, we propose that a phylum may be defined as a collection of species whose gene expression at the mid-developmental transition is both highly conserved among them, yet divergent relative to other species.

Improved histological fixation of gelatinous marine invertebrates.

BACKGROUND: Gelatinous zooplankton can be difficult to preserve morphologically due to unique physical properties of their cellular and acellular components. The relatively large volume of mesoglea leads to distortion of the delicate morphology and poor sample integrity in specimens prepared with standard aldehyde or alcohol fixation techniques. Similar challenges have made it difficult to extend standard laboratory methods such as in situ hybridization to larger juvenile ctenophores, hampering studies of late development. RESULTS: We have found that a household water repellant glass treatment product commonly used in laboratories, Rain-X®, alone or in combination with standard aldehyde fixatives, greatly improves morphological preservation of such delicate samples. We present detailed methods for preservation of ctenophores of diverse sizes compatible with long-term storage or detection and localization of target molecules such as with immunohistochemistry and in situ hybridization and show that this fixation might be broadly useful for preservation of other delicate marine specimens. CONCLUSION: This new method will enable superior preservation of morphology in gelatinous specimens for a variety of downstream goals. Extending this method may improve the morphological fidelity and durability of museum and laboratory specimens for other delicate sample types.

"Dorsal-Ventral" Genes Are Part of an Ancient Axial Patterning System: Evidence from Trichoplax adhaerens (Placozoa).

Placozoa are a morphologically simplistic group of marine animals found globally in tropical and subtropical environments. They consist of two named species, Trichoplax adhaerens and more recently Hoilungia hongkongensis, both with roughly six morphologically distinct cell types. With a sequenced genome, a limited number of cell types, and a simple flattened morphology, Trichoplax is an ideal model organism from which to explore the biology of an animal with a cellular complexity analagous to that of the earliest animals. Using a new approach for identification of gene expression patterns, this research looks at the relationship of Chordin/TgfΒ signaling and the axial patterning system of Placozoa. Our results suggest that placozoans have an oral-aboral axis similar to cnidarians and that the parahoxozoan ancestor (common ancestor of Placozoa and Cnidaria) was likely radially symmetric.

Cas9-mediated excision of Nematostella brachyury disrupts endoderm development, pharynx formation and oral-aboral patterning.

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 vectensisNvbrachyury 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.

Antagonistic BMP-cWNT signaling in the cnidarian Nematostella vectensis reveals insight into the evolution of mesoderm.

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.

Regeneration in the ctenophore Mnemiopsis leidyi occurs in the absence of a blastema, requires cell division, and is temporally separable from wound healing.

BACKGROUND: The 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 ctenophore Mnemiopsis leidyi as 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. RESULTS: We 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. CONCLUSIONS: Ctenophore 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.

Integrating Embryonic Development and Evolutionary History to Characterize Tentacle-Specific Cell Types in a Ctenophore.

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.

A bipolar role of the transcription factor ERG for cnidarian germ layer formation and apical domain patterning.

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.

Nmf9 Encodes a Highly Conserved Protein Important to Neurological Function in Mice and Flies.

Many protein-coding genes identified by genome sequencing remain without functional annotation or biological context. Here we define a novel protein-coding gene, Nmf9, based on a forward genetic screen for neurological function. ENU-induced and genome-edited null mutations in mice produce deficits in vestibular function, fear learning and circadian behavior, which correlated with Nmf9 expression in inner ear, amygdala, and suprachiasmatic nuclei. Homologous genes from unicellular organisms and invertebrate animals predict interactions with small GTPases, but the corresponding domains are absent in mammalian Nmf9. Intriguingly, homozygotes for null mutations in the Drosophila homolog, CG45058, show profound locomotor defects and premature death, while heterozygotes show striking effects on sleep and activity phenotypes. These results link a novel gene orthology group to discrete neurological functions, and show conserved requirement across wide phylogenetic distance and domain level structural changes.

Major diversification of voltage-gated K+ channels occurred in ancestral parahoxozoans.

We examined the origins and functional evolution of the Shaker and KCNQ families of voltage-gated K(+) channels to better understand how neuronal excitability evolved. In bilaterians, the Shaker family consists of four functionally distinct gene families (Shaker, Shab, Shal, and Shaw) that share a subunit structure consisting of a voltage-gated K(+) channel motif coupled to a cytoplasmic domain that mediates subfamily-exclusive assembly (T1). We traced the origin of this unique Shaker subunit structure to a common ancestor of ctenophores and parahoxozoans (cnidarians, bilaterians, and placozoans). Thus, the Shaker family is metazoan specific but is likely to have evolved in a basal metazoan. Phylogenetic analysis suggested that the Shaker subfamily could predate the divergence of ctenophores and parahoxozoans, but that the Shab, Shal, and Shaw subfamilies are parahoxozoan specific. In support of this, putative ctenophore Shaker subfamily channel subunits coassembled with cnidarian and mouse Shaker subunits, but not with cnidarian Shab, Shal, or Shaw subunits. The KCNQ family, which has a distinct subunit structure, also appears solely within the parahoxozoan lineage. Functional analysis indicated that the characteristic properties of Shaker, Shab, Shal, Shaw, and KCNQ currents evolved before the divergence of cnidarians and bilaterians. These results show that a major diversification of voltage-gated K(+) channels occurred in ancestral parahoxozoans and imply that many fundamental mechanisms for the regulation of action potential propagation evolved at this time. Our results further suggest that there are likely to be substantial differences in the regulation of neuronal excitability between ctenophores and parahoxozoans.

Precision wildlife medicine: applications of the human-centred precision medicine revolution to species conservation.

The current species extinction crisis is being exacerbated by an increased rate of emergence of epizootic disease. Human-induced factors including habitat degradation, loss of biodiversity and wildlife population reductions resulting in reduced genetic variation are accelerating disease emergence. Novel, efficient and effective approaches are required to combat these epizootic events. Here, we present the case for the application of human precision medicine approaches to wildlife medicine in order to enhance species conservation efforts. We consider how the precision medicine revolution, coupled with the advances made in genomics, may provide a powerful and feasible approach to identifying and treating wildlife diseases in a targeted, effective and streamlined manner. A number of case studies of threatened species are presented which demonstrate the applicability of precision medicine to wildlife conservation, including sea turtles, amphibians and Tasmanian devils. These examples show how species conservation could be improved by using precision medicine techniques to determine novel treatments and management strategies for the specific medical conditions hampering efforts to restore population levels. Additionally, a precision medicine approach to wildlife health has in turn the potential to provide deeper insights into human health and the possibility of stemming and alleviating the impacts of zoonotic diseases. The integration of the currently emerging Precision Medicine Initiative with the concepts of EcoHealth (aiming for sustainable health of people, animals and ecosystems through transdisciplinary action research) and One Health (recognizing the intimate connection of humans, animal and ecosystem health and addressing a wide range of risks at the animal-human-ecosystem interface through a coordinated, collaborative, interdisciplinary approach) has great potential to deliver a deeper and broader interdisciplinary-based understanding of both wildlife and human diseases.

The maternal-zygotic transition and zygotic activation of the Mnemiopsis leidyi genome occurs within the first three cleavage cycles.

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 hr post-fertilization in M. leidyi.

Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr.

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.