Doublecortin-like kinase is required for cnidocyte development in Nematostella vectensis.

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.

Fluorescence In Situ Hybridization as a Tool for Studying the Specification and Differentiation of Cell Types in Nematostella vectensis.

The sea anemone Nematostella vectensis is a genetically tractable cnidarian species that has become a model organism for studying the evolution of developmental processes and genome regulation, resilience to fluctuations in environmental conditions, and the response to pollutants. Gene expression analyses are central to many of these studies, and in situ hybridization has been an important method for obtaining spatial information, in particular during embryonic development. Like other cnidarians, Nematostella embryos are of comparably low morphological complexity, but they possess many cell types that are dispersed throughout the tissue and originate from broad and overlapping areas. These features have made two-color fluorescence in situ hybridization an important method to determine potential co-expression of genes and to generate hypotheses for their functions in cell fate specification. We here share protocols for single and double fluorescence in situ hybridization in Nematostella and for the combination of fluorescence in situ hybridization and immunofluorescence.

NvPrdm14d-expressing neural progenitor cells contribute to non-ectodermal neurogenesis in Nematostella vectensis.

Neurogenesis has been studied extensively in the ectoderm, from which most animals generate the majority of their neurons. Neurogenesis from non-ectodermal tissue is, in contrast, poorly understood. Here we use the cnidarian Nematostella vectensis as a model to provide new insights into the molecular regulation of non-ectodermal neurogenesis. We show that the transcription factor NvPrdm14d is expressed in a subpopulation of NvSoxB(2)-expressing endodermal progenitor cells and their NvPOU4-expressing progeny. Using a new transgenic reporter line, we show that NvPrdm14d-expressing cells give rise to neurons in the body wall and in close vicinity of the longitudinal retractor muscles. RNA-sequencing of NvPrdm14d::GFP-expressing cells and gene knockdown experiments provide candidate genes for the development and function of these neurons. Together, the identification of a population of endoderm-specific neural progenitor cells and of previously undescribed putative motoneurons in Nematostella provide new insights into the regulation of non-ectodermal neurogenesis.

Insm1-expressing neurons and secretory cells develop from a common pool of progenitors in the sea anemone Nematostella vectensis.

Neurons are highly specialized cells present in nearly all animals, but their evolutionary origin and relationship to other cell types are not well understood. We use here the sea anemone Nematostella vectensis as a model system for early-branching animals to gain fresh insights into the evolutionary history of neurons. We generated a transgenic reporter line to show that the transcription factor NvInsm1 is expressed in postmitotic cells that give rise to various types of neurons and secretory cells. Expression analyses, double transgenics, and gene knockdown experiments show that the NvInsm1-expressing neurons and secretory cells derive from a common pool of NvSoxB(2)-positive progenitor cells. These findings, together with the requirement for Insm1 for the development of neurons and endocrine cells in vertebrates, support a close evolutionary relationship of neurons and secretory cells.

Evidence for a centrosome-attracting body like structure in germ-soma segregation during early development, in the urochordate Oikopleura dioica.

BACKGROUND: Germ cell formation has been investigated in sessile forms of tunicates. This process involves the release of a subset of maternal transcripts from the centrosome-attracting body (CAB) in the progenitor cells of the germ line. When germ-soma segregation is completed, CAB structures are missing from the newly formed primordial germ cells (PGCs). In free-swimming tunicates, knowledge about germ cell formation is lacking. In this investigation, comparative gene expression and electron microscopy studies were used to address germ cell formation in Oikopleura dioica (O. dioica). RESULTS: We found that the RNA localization pattern of pumilio (pum1) is similar to the pattern described for a subset of maternal transcripts marking the posterior end of ascidian embryos. Transcripts marking the posterior end are called postplasmic or posterior-end mark (PEM) transcripts. We found no localization of vasa (vas) transcripts to any sub-region within the germ-line precursor cells. Expression of vas4 was detected in the newly formed PGCs. Electron microscopy studies confirmed the presence of structures with similar morphology to CAB. In the same cytoplasmic compartment, we also identified pum1 transcripts and an epitope recognized by an antibody to histone H3 phosphorylated on serine 28. CONCLUSIONS: Our findings support that a CAB-like structure participates in the segregation of maternal pum1 transcripts during germ-soma separation in O. dioica.

Evolutionary Proteomics Uncovers Ancient Associations of Cilia with Signaling Pathways.

Cilia are organelles specialized for movement and signaling. To infer when during evolution signaling pathways became associated with cilia, we characterized the proteomes of cilia from sea urchins, sea anemones, and choanoflagellates. We identified 437 high-confidence ciliary candidate proteins conserved in mammals and discovered that Hedgehog and G-protein-coupled receptor pathways were linked to cilia before the origin of bilateria and transient receptor potential (TRP) channels before the origin of animals. We demonstrated that candidates not previously implicated in ciliary biology localized to cilia and further investigated ENKUR, a TRP channel-interacting protein identified in the cilia of all three organisms. ENKUR localizes to motile cilia and is required for patterning the left-right axis in vertebrates. Moreover, mutation of ENKUR causes situs inversus in humans. Thus, proteomic profiling of cilia from diverse eukaryotes defines a conserved ciliary proteome, reveals ancient connections to signaling, and uncovers a ciliary protein that underlies development and human disease.

Unipotent progenitors contribute to the generation of sensory cell types in the nervous system of the cnidarian Nematostella vectensis.

Nervous systems often consist of a large number of different types of neurons which are generated from neural stem and progenitor cells by a series of symmetric and asymmetric divisions. The origin and early evolution of these neural progenitor systems is not well understood. Here we use a cnidarian model organism, Nematostella vectensis, to gain insight into the generation of neural cell type diversity in a non-bilaterian animal. We identify NvFoxQ2d as a transcription factor that is expressed in a population of spatially restricted, proliferating ectodermal cells that are derived from NvSoxB(2)-expressing neural progenitor cells. Using a transgenic reporter line we show that the NvFoxQ2d cells undergo a terminal, symmetric division to generate a morphologically homogeneous population of putative sensory cells. The abundance of these cells, but not their proliferation status is affected by treatment with the γ-secretase inhibitor DAPT, suggesting regulation by Notch signalling. Our data suggest that intermediate progenitor cells and symmetric divisions contribute to the formation of the seemingly simple nervous system of a sea anemone.

A cnidarian homologue of an insect gustatory receptor functions in developmental body patterning.

Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.

Molecular characterization of the apical organ of the anthozoan Nematostella vectensis.

Apical organs are sensory structures present in many marine invertebrate larvae where they are considered to be involved in their settlement, metamorphosis and locomotion. In bilaterians they are characterised by a tuft of long cilia and receptor cells and they are associated with groups of neurons, but their relatively low morphological complexity and dispersed phylogenetic distribution have left their evolutionary relationship unresolved. Moreover, since apical organs are not present in the standard model organisms, their development and function are not well understood. To provide a foundation for a better understanding of this structure we have characterised the molecular composition of the apical organ of the sea anemone Nematostella vectensis. In a microarray-based comparison of the gene expression profiles of planulae with either a wildtype or an experimentally expanded apical organ, we identified 78 evolutionarily conserved genes, which are predominantly or specifically expressed in the apical organ of Nematostella. This gene set comprises signalling molecules, transcription factors, structural and metabolic genes. The majority of these genes, including several conserved, but previously uncharacterized ones, are potentially involved in different aspects of the development or function of the long cilia of the apical organ. To demonstrate the utility of this gene set for comparative analyses, we further analysed the expression of a subset of previously uncharacterized putative orthologs in sea urchin larvae and detected expression for twelve out of eighteen of them in the apical domain. Our study provides a molecular characterization of the apical organ of Nematostella and represents an informative tool for future studies addressing the development, function and evolutionary history of apical organ cells.

The bilaterian head patterning gene six3/6 controls aboral domain development in a cnidarian.

The origin of the bilaterian head is a fundamental question for the evolution of animal body plans. The head of bilaterians develops at the anterior end of their primary body axis and is the site where the brain is located. Cnidarians, the sister group to bilaterians, lack brain-like structures and it is not clear whether the oral, the aboral, or none of the ends of the cnidarian primary body axis corresponds to the anterior domain of bilaterians. In order to understand the evolutionary origin of head development, we analysed the function of conserved genetic regulators of bilaterian anterior development in the sea anemone Nematostella vectensis. We show that orthologs of the bilaterian anterior developmental genes six3/6, foxQ2, and irx have dynamic expression patterns in the aboral region of Nematostella. Functional analyses reveal that NvSix3/6 acts upstream of NvFoxQ2a as a key regulator of the development of a broad aboral territory in Nematostella. NvSix3/6 initiates an autoregulatory feedback loop involving positive and negative regulators of FGF signalling, which subsequently results in the downregulation of NvSix3/6 and NvFoxQ2a in a small domain at the aboral pole, from which the apical organ develops. We show that signalling by NvFGFa1 is specifically required for the development of the apical organ, whereas NvSix3/6 has an earlier and broader function in the specification of the aboral territory. Our functional and gene expression data suggest that the head-forming region of bilaterians is derived from the aboral domain of the cnidarian-bilaterian ancestor.

The Xenopus doublesex-related gene Dmrt5 is required for olfactory placode neurogenesis.

The Dmrt (doublesex and mab-3 related transcription factor) genes encode a large family of evolutionarily conserved transcription factors whose function in sex specific differentiation has been well studied in all animal lineages. In vertebrates, their function is not restricted to the developing gonads. For example, Xenopus Dmrt4 is essential for neurogenesis in the olfactory system. Here we have isolated and characterized Xenopus Dmrt5 and found that it is coexpressed with Dmrt4 in the developing olfactory placodes. As Dmrt4, Dmrt5 is positively regulated in the ectoderm by neural inducers and negatively by proneural factors. Both Dmrt5 and Dmrt4 genes are also activated by the combined action of the transcription factor Otx2, broadly transcribed in the head ectoderm and of Notch signaling, activated in the anterior neural ridge. As for Dmrt4, knockdown of Dmrt5 impairs neurogenesis in the embryonic olfactory system and in neuralized animal caps. Conversely, its overexpression promotes neuronal differentiation in animal caps, a property that requires the conserved C-terminal DMA and DMB domains. We also found that the sea anenome Dmrt4/5 related gene NvDmrtb also induces neurogenesis in Xenopus animal caps and that conversely, its knockdown in Nematostella reduces elav-1 positive neurons. Together, our data identify Dmrt5 as a novel important regulator of neurogenesis whose function overlaps with that of Dmrt4 during Xenopus olfactory system development. They also suggest that Dmrt may have had a role in neurogenesis in the last common ancestor of cnidarians and bilaterians.