Comparative single cell analysis reveals complex patterns of cell type and cell signaling innovations at the fetal-maternal interface.

The decidual-placental interface is one of the most diverse and rapidly evolving tissues in mammals. Its origin as a chimeric fetal-maternal tissue poses a unique evolutionary puzzle. We present single-cell RNA sequencing atlases from the fetal-maternal interfaces of the opossum, a marsupial, the Malagasy common tenrec, an afrotherian with primitive reproductive features, and mouse, guinea pig, and human. Invasive trophoblast shares a common transcriptomic signature across eutherians, which we argue represents a cell type family that radiated following the evolution of hemochorial placentation. We find evidence that the eutherian decidual stromal cell evolved stepwise from a predecidual state retained in Tenrec , followed by a second decidual cell type originating in Boreoeutheria with endocrine characteristics. We reconstruct ligand-receptor signaling to test evolutionary hypotheses at scale. Novel trophoblast and decidual cell types display strong integration into signaling networks compared to other cells. Additionally, we find consistent disambiguation between fetal and maternal signaling. Using phylogenetic analysis, we infer the cell-cell signaling network of the Placental common ancestor, and identify increased rates of signaling evolution in Euarchontoglires. Together, our findings reveal novel cell type identities and cell signaling dynamics at the mammalian fetal-maternal interface.

Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems.

Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs) - the most common receptors of bilaterian neuropeptides - but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by 1 to 3 of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively connected by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.

Updated single cell reference atlas for the starlet anemone Nematostella vectensis.

BACKGROUND: The recent combination of genomics and single cell transcriptomics has allowed to assess a variety of non-conventional model organisms in much more depth. Single cell transcriptomes can uncover hidden cellular complexity and cell lineage relationships within organisms. The recent developmental cell atlases of the sea anemone Nematostella vectensis, a representative of the basally branching Cnidaria, has provided new insights into the development of all cell types (Steger et al Cell Rep 40(12):111370, 2022; Sebé-Pedrós et al. Cell 173(6):1520-1534.e20). However, the mapping of the single cell reads still suffers from relatively poor gene annotations and a draft genome consisting of many scaffolds. RESULTS: Here we present a new wildtype resource of the developmental single cell atlas, by re-mapping of sequence data first published in Steger et al. (2022) and Cole et al. (Nat Commun 14(1):1747, 2023), to the new chromosome-level genome assembly and corresponding gene models in Zimmermann et al. (Nat Commun 14, 8270 (2023). https://doi.org/10.1038/s41467-023-44080-7 ). We expand the pre-existing dataset through the incorporation of additional sequence data derived from the capture and sequencing of cell suspensions from four additional samples: 24 h gastrula, 2d planula, an inter-parietal region of the bodywall from a young unsexed animal, and another adult mesentery from a mature male animal. CONCLUSION: Our analyses of the full cell-state inventory provide transcriptomic signatures for 127 distinct cell states, of which 47 correspond to neuroglandular subtypes. We also identify two distinct putatively immune-related transcriptomic profiles that segregate between the inner and outer cell layers. Furthermore, the new gene annotation Nv2 has markedly improved the mapping on the single cell transcriptome data and will therefore be of great value for the community and anyone using the dataset.

Gene regulatory patterning codes in early cell fate specification of the C. elegans embryo.

Pattern formation originates during embryogenesis by a series of symmetry-breaking steps throughout an expanding cell lineage. In Drosophila, classic work has shown that segmentation in the embryo is established by morphogens within a syncytium, and the subsequent action of the gap, pair-rule, and segment polarity genes. This classic model however does not translate directly to species that lack a syncytium - such as Caenorhabditis elegans - where cell fate is specified by cell-autonomous cell lineage programs and their inter-signaling. Previous single-cell RNA-Seq studies in C. elegans have analyzed cells from a mixed suspension of cells from many embryos to study late differentiation stages, or individual early stage embryos to study early gene expression in the embryo. To study the intermediate stages of early and late gastrulation (28- to 102-cells stages) missed by these approaches, here we determine the transcriptomes of the 1- to 102-cell stage to identify 119 embryonic cell states during cell fate specification, including 'equivalence-group' cell identities. We find that gene expression programs are modular according to the sub-cell lineages, each establishing a set of stripes by combinations of transcription factor gene expression across the anterior-posterior axis. In particular, expression of the homeodomain genes establishes a comprehensive lineage-specific positioning system throughout the embryo beginning at the 28-cell stage. Moreover, we find that genes that segment the entire embryo in Drosophila have orthologs in C. elegans that exhibit sub-lineage-specific expression. These results suggest that the C. elegans embryo is patterned by a juxtaposition of distinct lineage-specific gene regulatory programs each with a unique encoding of cell location and fate. This use of homologous gene regulatory patterning codes suggests a deep homology of cell fate specification programs across diverse modes of development.

Muscle cell-type diversification is driven by bHLH transcription factor expansion and extensive effector gene duplications.

Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the emergence of new cell types remain unclear. Here we address the origin and diversification of muscle cells in the non-bilaterian, diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations, which differ by extensive sets of paralogous structural protein genes. We find that the regulatory gene set of the slow cnidarian muscles is remarkably similar to the bilaterian cardiac muscle, while the two fast muscles differ substantially from each other in terms of transcription factor profiles, though driving the same set of structural protein genes and having similar physiological characteristics. We show that anthozoan-specific paralogs of Paraxis/Twist/Hand-related bHLH transcription factors are involved in the formation of fast and slow muscles. Our data suggest that the subsequent recruitment of an entire effector gene set from the inner cell layer into the neural ectoderm contributes to the evolution of a novel muscle cell type. Thus, we conclude that extensive transcription factor gene duplications and co-option of effector modules act as an evolutionary mechanism underlying cell type diversification during metazoan evolution.

Single-cell transcriptomics identifies conserved regulators of neuroglandular lineages.

Communication in bilaterian nervous systems is mediated by electrical and secreted signals; however, the evolutionary origin and relation of neurons to other secretory cell types has not been elucidated. Here, we use developmental single-cell RNA sequencing in the cnidarian Nematostella vectensis, representing an early evolutionary lineage with a simple nervous system. Validated by transgenics, we demonstrate that neurons, stinging cells, and gland cells arise from a common multipotent progenitor population. We identify the conserved transcription factor gene SoxC as a key upstream regulator of all neuroglandular lineages and demonstrate that SoxC knockdown eliminates both neuronal and secretory cell types. While in vertebrates and many other bilaterians neurogenesis is largely restricted to early developmental stages, we show that in the sea anemone, differentiation of neuroglandular cells is maintained throughout all life stages, and follows the same molecular trajectories from embryo to adulthood, ensuring lifelong homeostasis of neuroglandular cell lineages.

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.

The gene regulatory program of Acrobeloides nanus reveals conservation of phylum-specific expression.

The evolution of development has been studied through the lens of gene regulation by examining either closely related species or extremely distant animals of different phyla. In nematodes, detailed cell- and stage-specific expression analyses are focused on the model Caenorhabditis elegans, in part leading to the view that the developmental expression of gene cascades in this species is archetypic for the phylum. Here, we compared two species of an intermediate evolutionary distance: the nematodes C. elegans (clade V) and Acrobeloides nanus (clade IV). To examine A. nanus molecularly, we sequenced its genome and identified the expression profiles of all genes throughout embryogenesis. In comparison with C. elegans, A. nanus exhibits a much slower embryonic development and has a capacity for regulative compensation of missing early cells. We detected conserved stages between these species at the transcriptome level, as well as a prominent middevelopmental transition, at which point the two species converge in terms of their gene expression. Interestingly, we found that genes originating at the dawn of the Ecdysozoa supergroup show the least expression divergence between these two species. This led us to detect a correlation between the time of expression of a gene and its phylogenetic age: evolutionarily ancient and young genes are enriched for expression in early and late embryogenesis, respectively, whereas Ecdysozoa-specific genes are enriched for expression during the middevelopmental transition. Our results characterize the developmental constraints operating on each individual embryo in terms of developmental stages and genetic evolutionary history.

Analyses of Sox-B and Sox-E Family Genes in the Cephalopod Sepia officinalis: Revealing the Conserved and the Unusual.

Cephalopods provide an unprecedented opportunity for comparative studies of the developmental genetics of organ systems that are convergent with analogous vertebrate structures. The Sox-family of transcription factors is an important class of DNA-binding proteins that are known to be involved in many aspects of differentiation, but have been largely unstudied in lophotrochozoan systems. Using a degenerate primer strategy we have isolated coding sequence for three members of the Sox family of transcription factors from a cephalopod mollusk, the European cuttlefish Sepia officinalis: Sof-SoxE, Sof-SoxB1, and Sof-SoxB2. Analyses of their expression patterns during organogenesis reveals distinct spatial and temporal expression domains. Sof-SoxB1 shows early ectodermal expression throughout the developing epithelium, which is gradually restricted to presumptive sensory epithelia. Expression within the nervous system appears by mid-embryogenesis. Sof-SoxB2 expression is similar to Sof-SoxB1 within the developing epithelia in early embryogenesis, however appears in largely non-overlapping expression domains within the central nervous system and is not expressed in the maturing sensory epithelium. In contrast, Sof-SoxE is expressed throughout the presumptive mesodermal territories at the onset of organogenesis. As development proceeds, Sof-SoxE expression is elevated throughout the developing peripheral circulatory system. This expression disappears as the circulatory system matures, but expression is maintained within undifferentiated connective tissues throughout the animal, and appears within the nervous system near the end of embryogenesis. SoxB proteins are widely known for their role in neural specification in numerous phylogenetic lineages. Our data suggests that Sof-SoxB genes play similar roles in cephalopods. In contrast, Sof-SoxE appears to be involved in the early stages of vasculogenesis of the cephalopod closed circulatory system, a novel role for a member of this gene family.

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.

Characterization of homeobox genes reveals sophisticated regionalization of the central nervous system in the European cuttlefish Sepia officinalis.

Cephalopod mollusks possess a number of anatomical traits that often parallel vertebrates in morphological complexity, including a centralized nervous system with sophisticated cognitive functionality. Very little is known about the genetic mechanisms underlying patterning of the cephalopod embryo to arrive at this anatomical structure. Homeodomain (HD) genes are transcription factors that regulate transcription of downstream genes through DNA binding, and as such are integral parts of gene regulatory networks controlling the specification and patterning of body parts across lineages. We have used a degenerate primer strategy to isolate homeobox genes active during late-organogenesis from the European cuttlefish Sepia officinalis. With this approach we have isolated fourteen HD gene fragments and examine the expression profiles of five of these genes during late stage (E24-28) embryonic development (Sof-Gbx, Sof-Hox3, Sof-Arx, Sof-Lhx3/4, Sof-Vsx). All five genes are expressed within the developing central nervous system in spatially restricted and largely non-overlapping domains. Our data provide a first glimpse into the diversity of HD genes in one of the largest, yet least studied, metazoan clades and illustrate how HD gene expression patterns reflect the functional partitioning of the cephalopod brain.

Pattern and process during sea urchin gut morphogenesis: the regulatory landscape.

The development of the endoderm is a multistage process. From the initial specification of the endodermal domain in the embryo to the final regionalization of the gut, there are multiple stages that require the involvement of complex gene regulatory networks. In one concrete case, the sea urchin embryo, some of these stages and their genetic control are (relatively) well understood. Several studies have underscored the relevance of individual transcription factor activities in the process, but very few have focused the attention on gene interactions within specific gene regulatory networks (GRNs). Sea urchins offer an ideal system to study the different factors involved in the morphogenesis of the gut. Here we review the knowledge gained over the last 10 years on the process and its regulation, from the early specification of endodermal lineages to the late events linked to the patterning of functional domains in the gut. A lesson of remarkable importance has been learnt from comparison of the mechanisms involved in gut formation in different bilaterian animals; some of these genetic mechanisms are particularly well conserved. Patterning the gut seems to involve common molecular players and shared interactions, whether we look at mammals or echinoderms. This astounding degree of conservation reveals some key aspects of deep homology that are most probably shared by all bilaterian guts.

ZOONET: perspectives on the evolution of animal form. Meeting report.

What drives evolution? This was one of the main questions raised at the final ZOONET meeting in Budapest, Hungary, in November 2008. The meeting marked the conclusion of ZOONET, an EU-funded Marie-Curie Research Training Network comprising nine research groups from all over Europe (Max Telford, University College London; Michael Akam, University of Cambridge; Detlev Arendt, EMBL Heidelberg; Maria Ina Arnone, Stazione Zoologica Anton Dohrn Napoli; Michalis Averof, IMBB Heraklion; Graham Budd, Uppsala University; Richard Copley, University of Oxford; Wim Damen, University of Cologne; Ernst Wimmer, University of Göttingen). ZOONET meetings and practical courses held during the past four years provided researchers from diverse backgrounds--bioinformatics, phylogenetics, embryology, palaeontology, and developmental and molecular biology--the opportunity to discuss their work under a common umbrella of evolutionary developmental biology (Evo Devo). The Budapest meeting emphasized in-depth discussions of the key concepts defining Evo Devo, and bringing together ZOONET researchers with external speakers who were invited to present their views on the evolution of animal form. The discussion sessions addressed four main topics: the driving forces of evolution, segmentation, fossils and phylogeny, and the future of Evo Devo.

Two ParaHox genes, SpLox and SpCdx, interact to partition the posterior endoderm in the formation of a functional gut.

We report the characterization of the ortholog of the Xenopus XlHbox8 ParaHox gene from the sea urchin Strongylocentrotus purpuratus, SpLox. It is expressed during embryogenesis, first appearing at late gastrulation in the posterior-most region of the endodermal tube, becoming progressively restricted to the constriction between the mid- and hindgut. The physiological effects of the absence of the activity of this gene have been analyzed through knockdown experiments using gene-specific morpholino antisense oligonucleotides. We show that blocking the translation of the SpLox mRNA reduces the capacity of the digestive tract to process food, as well as eliminating the morphological constriction normally present between the mid- and hindgut. Genetic interactions of the SpLox gene are revealed by the analysis of the expression of a set of genes involved in endoderm specification. Two such interactions have been analyzed in more detail: one involving the midgut marker gene Endo16, and another involving the other endodermally expressed ParaHox gene, SpCdx. We find that SpLox is able to bind Endo16 cis-regulatory DNA, suggesting direct repression of Endo16 expression in presumptive hindgut territories. More significantly, we provide the first evidence of interaction between ParaHox genes in establishing hindgut identity, and present a model of gene regulation involving a negative-feedback loop.

Cartilage differentiation in cephalopod molluscs.

Amongst the various metazoan lineages that possess cartilage, tissues most closely resembling vertebrate hyaline cartilage in histological section are those of cephalopod molluscs. Although elements of the adult skeleton have been described, the development of these cartilages has not. Using serial histology of sequential developmental stages of the European cuttlefish, Sepia officinalis, we investigate these skeletal elements and offer the first description of the formation of any cellular invertebrate cartilage. Our data reveal that cuttlefish cartilage most often differentiates from uncondensed mesenchymal cells near the end of embryonic development, but that the earliest-forming cartilages differentiate from a cellular condensation which goes through a protocartilage stage in a manner typical of vertebrate primary cartilage formation. We further investigate the distribution and degree of differentiation of cartilages at the time of hatching in an additional four cephalopod species. We find that the timing of cartilage development varies between elements within a single species, as well as between species. We identify a tendency towards cartilage differentiation from uncondensed connective tissue in elements that form at the end of embryogenesis or after hatching. These data suggest a form of metaplasia from connective tissue is the ancestral mode of cartilage formation in this lineage.

Using ultrasound to understand vascular and mantle contributions to venous return in the cephalopod Sepia officinalis L.

Using ultrasound imaging, we investigated the roles of the potentially contractile veins and of the mantle (the powerful body wall that moves water over the gills, and also encloses the large veins and the hearts) in returning the blood of cuttlefish to its hearts. Ultrasound provided the first non-invasive observations of vascular function in an unanaesthetized, free-moving cephalopod. The large veins (anterior vena cava, lateral venae cavae and efferent branchial vessels) contracted in live, intact cuttlefish (Sepia officinalis L.). The anterior vena cava contracted at the same rate as the mantle, but it often expanded during mantle contraction. Furthermore, the anterior vena cava contracted peristaltically in vivo, suggesting that it actively aids venous return. The lateral venae cavae and efferent branchial vessels contracted at the same rate as the branchial and systemic hearts, but at a different rate from the mantle. A peristaltic wave appeared to travel along the lateral venae cavae to the branchial hearts, potentially aiding venous return. We found a muscular valve between the anterior and lateral venae cavae, which ensured that blood flowed only one way between these unsynchronized vessels. The mantle appears to have an unclear connection with cardiovascular function. We conclude that, when cuttlefish are at rest, the mantle does not compress any of the large veins that we imaged (including the anterior vena cava), and that peristaltic contractions of the large veins might be important in returning cephalopod blood to the hearts.

The central nervous system of the ascidian larva: mitotic history of cells forming the neural tube in late embryonic Ciona intestinalis.

Ascidian larvae develop after an invariant pattern of embryonic cleavage. Fewer than 400 cells constitute the larval central nervous system (CNS), which forms without either extensive migration or cell death. We catalogue the mitotic history of these cells in Ciona intestinalis, using confocal microscopy of whole-mount embryos at stages from neurulation until hatching. The positions of cells contributing to the CNS were reconstructed from confocal image stacks of embryonic nuclei, and maps of successive stages were used to chart the mitotic descent, thereby creating a cell lineage for each cell. The entire CNS is formed from 10th- to 14th-generation cells. Although minor differences exist in cell position, lineage is invariant in cells derived from A-line blastomeres, which form the caudal nerve cord and visceral ganglion. We document the lineage of five pairs of presumed motor neurons within the visceral ganglion: one pair arises from A/A 10.57, and four from progeny of A/A 9.30. The remaining cells of the visceral ganglion are in their 13th and 14th generations at hatching, with most mitotic activity ceasing around 85% of embryonic development. Of the approximately 330 larval cells previously reported in the CNS of Ciona, we document the lineage of 226 that derive predominantly from A-line blastomeres.

The nature and significance of invertebrate cartilages revisited: distribution and histology of cartilage and cartilage-like tissues within the Metazoa.

Tissues similar to vertebrate cartilage have been described throughout the Metazoa. Often the designation of tissues as cartilage within non-vertebrate lineages is based upon sparse supporting data. To be considered cartilage, a tissue should meet a number of histological criteria that include composition and organization of the extracellular matrix. To re-evaluate the distribution and structural properties of these tissues, we have re-investigated the histological properties of many of these tissues from fresh material, and review the existing literature on invertebrate cartilages. Chondroid connective tissue is common amongst invertebrates, and differs from invertebrate cartilage in the structure and organization of the cells that comprise it. Groups having extensive chondroid connective tissue include brachiopods, polychaetes, and urochordates. Cartilage is found within cephalopod mollusks, chelicerate arthropods and sabellid polychaetes. Skeletal tissues found within enteropneust hemichordates are unique in that the extracellular matrix shares many properties with vertebrate cartilage, yet these tissues are completely acellular. The possibility that this tissue may represent a new category of cartilage, acellular cartilage, is discussed. Immunoreactivity of some invertebrate cartilages with antibodies that recognize molecules specific to vertebrate bone suggests an intermediate phenotype between vertebrate cartilage and bone. Although cartilage is found within a number of invertebrate lineages, we find that not all tissues previously reported to be cartilage have the appropriate properties to merit their distinction as cartilage.

Regulation of early embryonic behavior by nitric oxide in the pond snail Helisoma trivolvis.

Helisoma trivolvis embryos display a cilia-driven rotational behavior that is regulated by a pair of serotonergic neurons named ENC1s. As these cilio-excitatory motor neurons contain an apical dendrite ending in a chemosensory dendritic knob at the embryonic surface, they probably function as sensorimotor neurons. Given that nitric oxide (NO) is often associated with sensory neurons in invertebrates, and has also been implicated in the control of ciliary activity, we examined the expression of NO synthase (NOS) activity and possible function of NO in regulating the rotational behavior in H. trivolvis embryos. NADPH diaphorase histochemistry on stage E25-E30 embryos revealed NOS expression in the protonephridia, buccal mass, dorsolateral ciliary cells and the sensory dendritic knobs of ENC1. At stages E35-40, the pedal ciliary cells and ENC1's soma, apical dendrite and proximal descending axon were also stained. In stage E25 embryos, optimal doses of the NO donors SNAP and SNP increased the rate of embryonic rotation by twofold, in contrast to the fourfold increase caused by 100 micro mol l(-1) serotonin. The NOS inhibitors L-NAME (10 mmol l(-1)) and 7-NI (100 micro mol l(-1)) decreased the rotation rate by approximately 50%, whereas co-addition of L-NAME and SNAP caused a twofold increase. In an analysis of the surge and inter-surge subcomponents of the rotational behavior, the NO donors increased the inter-surge rotation rate and the surge amplitude. In contrast, the NO inhibitors decreased the inter-surge rotation rate and the frequency of surges. These data suggest that the embryonic rotational behavior depends in part on the constitutive excitatory actions of NO on ENC1 and ciliary cells.