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SUMMARYN6-methyldeoxyadenosine (6mA) is a chemical alteration of DNA, observed across all realms of life. The functions of 6mA are well understood in bacteria but its roles in animal genomes have been controversial. We show that 6mA randomly accumulates in early embryos of the cnidarianHydractinia symbiolongicarpus, with a peak at the 16-cell stage followed by clearance to background levels two cell cycles later, at the 64-cell stage – the embryonic stage at which zygotic genome activation occurs in this animal. Knocking downAlkbh1, a putative initiator of animal 6mA clearance, resulted in higher levels of 6mA at the 64-cell stage and a delay in the commencement of zygotic transcription. Our data are consistent with 6mA originating from recycled nucleotides of degraded m6A-marked maternal RNA post-fertilization. Therefore, while 6mA does not function as an epigenetic mark inHydractinia, its random incorporation into the early embryonic genome inhibits transcription. Alkbh1 functions as a genomic 6mA ‘cleaner’, facilitating timely zygotic genome activation. Given the random nature of genomic 6mA accumulation and its ability to interfere with gene expression, defects in 6mA clearance may represent a hitherto unknown cause of various pathologies.

SUMMARYCell fate stability is essential to maintaining ‘law and order’ in complex animals. However, high stability comes at the cost of reduced plasticity and, by extension, poor regenerative ability. This evolutionary trade-off has resulted in most modern animals being rather simple and regenerative or complex and non-regenerative. The mechanisms mediating cellular plasticity and allowing for regeneration remain unknown. We show that signals emitted by senescent cells can destabilize the differentiated state of neighboring somatic cells, reprogramming them into stem cells that are capable of driving whole-body regeneration in the cnidarian Hydractinia symbiolongicarpus. Pharmacological or genetic inhibition of senescence prevented reprogramming and regeneration. Conversely, induction of transient ectopic senescence in a regenerative context resulted in supernumerary stem cells and faster regeneration. We propose that senescence signaling is an ancient mechanism mediating cellular plasticity. Understanding the senescence environment that promotes cellular reprogramming could provide a new avenue to enhance 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.

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.

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.

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

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.

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.

From early dinosaurs with as many as nine wrist bones, modern birds evolved to develop only four ossifications. Their identity is uncertain, with different labels used in palaeontology and developmental biology. We examined embryos of several species and studied chicken embryos in detail through a new technique allowing whole-mount immunofluorescence of the embryonic cartilaginous skeleton. Beyond previous controversy, we establish that the proximal–anterior ossification develops from a composite radiale+intermedium cartilage, consistent with fusion of radiale and intermedium observed in some theropod dinosaurs. Despite previous claims that the development of the distal–anterior ossification does not support the dinosaur–bird link, we found its embryonic precursor shows two distinct regions of both collagen type II and collagen type IX expression, resembling the composite semilunate bone of bird-like dinosaurs (distal carpal 1+distal carpal 2). The distal–posterior ossification develops from a cartilage referred to as “element x,” but its position corresponds to distal carpal 3. The proximal–posterior ossification is perhaps most controversial: It is labelled as the ulnare in palaeontology, but we confirm the embryonic ulnare is lost during development. Re-examination of the fossil evidence reveals the ulnare was actually absent in bird-like dinosaurs. We confirm the proximal–posterior bone is a pisiform in terms of embryonic position and its development as a sesamoid associated to a tendon. However, the pisiform is absent in bird-like dinosaurs, which are known from several articulated specimens. The combined data provide compelling evidence of a remarkable evolutionary reversal: A large, ossified pisiform re-evolved in the lineage leading to birds, after a period in which it was either absent, nonossified, or very small, consistently escaping fossil preservation. The bird wrist provides a modern example of how developmental and paleontological data illuminate each other. Based on all available data, we introduce a new nomenclature for bird wrist ossifications.

Background The homology of the digits in the bird wing is a high-profile controversy in developmental and evolutionary biology. The embryonic position of the digits cartilages with respect to the primary axis (ulnare and ulna) corresponds to 2, 3, 4, but comparative-evolutionary morphology supports 1, 2, 3. A homeotic frameshift of digit identity in evolution could explain how cells in embryonic positions 2, 3, 4 began developing morphologies 1, 2, 3. Another alternative is that no re-patterning of cell fates occurred, and the primary axis shifted its position by some other mechanism. In the wing, only the anterior digit lacks expression of HoxD10 and HoxD12, resembling digit 1 of other limbs, as predicted by 1, 2, 3. However, upon loss of digit 1 in evolution, the most anterior digit 2 could have lost their expression, deceitfully resembling a digit 1. To test this notion, we observed HoxD10 and HoxD12 in a limb where digit 2 is the most anterior digit: The rabbit foot. We also explored whether early inhibition of Shh signalling in the embryonic wing bud induces an experimental homeotic frameshift, or an experimental axis shift. We tested these hypotheses using DiI injections to study the fate of cells in these experimental wings. Results We found strong transcription of HoxD10 and HoxD12 was present in the most anterior digit 2 of the rabbit foot. Thus, we found no evidence to question the use of HoxD expression as support for 1, 2, 3. When Shh signalling in early wing buds is inhibited, our fate maps demonstrate that an experimental homeotic frameshift is induced. Conclusion Along with comparative morphology, HoxD expression provides strong support for 1, 2, 3 identity of wing digits. As an explanation for the offset 2, 3, 4 embryological position, the homeotic frameshift hypothesis is consistent with known mechanisms of limb development, and further proven to be experimentally possible. In contrast, the underlying mechanisms and experimental plausibility of an axis shift remain unclear.

Background Cytasters (cytoplasmic asters) are centriole-based nucleation centers of microtubule polymerization that are observable in large numbers in the cortical cytoplasm of the egg and zygote of bilaterian organisms. In both protostome and deuterostome taxa, cytasters have been described to develop during oogenesis from vesicles of nuclear membrane that move to the cortical cytoplasm. They become associated with several cytoplasmic components, and participate in the reorganization of cortical cytoplasm after fertilization, patterning the antero-posterior and dorso-ventral body axes. Presentation of the hypothesis The specific resemblances in the development of cytasters in both protostome and deuterostome taxa suggest that an independent evolutionary origin is unlikely. An assessment of published data confirms that cytasters are present in several protostome and deuterostome phyla, but are absent in the non-bilaterian phyla Cnidaria and Ctenophora. We hypothesize that cytasters evolved in the lineage leading to Bilateria and were already present in the most recent common ancestor shared by protostomes and deuterostomes. Thus, cytasters would be an ancient and highly conserved trait that is homologous across the different bilaterian phyla. The alternative possibility is homoplasy, that is cytasters have evolved independently in different lineages of Bilateria. Testing the hypothesis So far, available published information shows that appropriate observations have been made in eight different bilaterian phyla. All of them present cytasters. This is consistent with the hypothesis of homology and conservation. However, there are several important groups for which there are no currently available data. The hypothesis of homology predicts that cytasters should be present in these groups. Increasing the taxonomic sample using modern techniques uniformly will test for evolutionary patterns supporting homology, homoplasy, or secondary loss of cytasters. Implications of the hypothesis If cytasters are homologous and highly conserved across bilateria, their potential developmental and evolutionary relevance has been underestimated. The deep evolutionary origin of cytasters also becomes a legitimate topic of research. In Ctenophora, polyspermic fertilization occurs, with numerous sperm entering the egg. The centrosomes of sperm pronuclei associate with cytoplasmic components of the egg and reorganize the cortical cytoplasm, defining the oral-aboral axis. These resemblances lead us to suggest the possibility of a polyspermic ancestor in the lineage leading to Bilateria.