Achsenbildung in Cnidariern: Die Rolle der Wnt- und BMP/Chordin Signaltransduktionswege

Ein entscheidender Schritt in der Evolution der Tiere war die Entwicklung von Körperbauplänen mit zwei Achsen aus solchen mit nur einer Achse. Um diesen Schritt zu verstehen, ist es notwendig, die molekularen Prozesse die die Achsenbildung steuern, sowohl in bilateralsymmetrischen (zwei Achsen) als auch in radiärsymmetrischen Tieren zu kennen. Da diese molekularen Mechanismen in Bilateriern hoch konserviert sind, versuchten wir zu erforschen, ob homologe molekulare Signalwege bereits in dem radiärsymmetrischen Süßwasserpolypen Hydra (Phylum Cnidaria) existieren und dort an Achsenbildungsprozessen beteiligt sind. Wir haben gefunden, dass Moleküle der konservierten Wnt- und BMP/Chordin-Signaltransduktionswege in Hydra existieren und zeigen, dass ihre Expressionsmuster während regulärer und experimentell induzierter Musterbildungsprozesse eine Funktion in der Achsenbildung der Polypen nahe legen. Implikationen für die Evolution der Körperbaupläne werden diskutiert

Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis

Bilaterian neurogenesis is characterized by the generation of diverse neural cell types from dedicated neural stem/progenitor cells (NPCs). However, the evolutionary origin of NPCs is unclear, as neurogenesis in representatives of the bilaterian sister group, the Cnidaria, occurs via interstitial stem cells that also possess broader, non-neural, developmental potential. We address this question by analysing neurogenesis in an anthozoan cnidarian, Nematostella vectensis. Using a transgenic reporter line, we show that NvSoxB(2) – an orthologue of bilaterian SoxB genes that have conserved roles in neurogenesis – is expressed in a cell population that gives rise to sensory neurons, ganglion neurons and nematocytes: the three primary neural cell types of cnidarians. EdU labelling together with in situ hybridization, and within the NvSoxB(2)::mOrange transgenic line, demonstrates that cells express NvSoxB(2) before mitosis and identifies asymmetric behaviours of sibling cells within NvSoxB(2)+ lineages. Morpholino-mediated gene knockdown of NvSoxB(2) blocks the formation of all three neural cell types, thereby identifying NvSoxB(2) as an essential positive regulator of nervous system development. Our results demonstrate that diverse neural cell types derive from an NvSoxB(2)-expressing population of mitotic cells in Nematostella and that SoxB genes are ancient components of a neurogenic program. To our knowledge this is the first description of a lineage-restricted, multipotent cell population outside the Bilateria and we propose that neurogenesis via dedicated, SoxB-expressing NPCs predates the split between cnidarians and bilaterians

Histone demethylase Lsd1 is required for the differentiation of neural cells in Nematostella vectensis

Chromatin regulation is a key process in development but its contribution to the evolution of animals is largely unexplored. Chromatin is regulated by a diverse set of proteins, which themselves are tightly regulated in a cell/tissue-specific manner. Using the cnidarian Nematostella vectensis as a basal metazoan model, we explore the function of one such chromatin regulator, Lysine specific demethylase 1 (Lsd1). We generated an endogenously tagged allele and show that NvLsd1 expression is developmentally regulated and higher in differentiated neural cells than their progenitors. We further show, using a CRISPR/Cas9 generated mutant that loss of NvLsd1 leads to developmental abnormalities. This includes the almost complete loss of differentiated cnidocytes, cnidarian-specific neural cells, as a result of a cell-autonomous requirement for NvLsd1. Together this suggests that the integration of chromatin modifying proteins into developmental regulation predates the split of the cnidarian and bilaterian lineages and constitutes an ancient feature of animal development.publishedVersio

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.publishedVersio

NvPOU4/Brain3 Functions as a Terminal Selector Gene in the Nervous System of the Cnidarian Nematostella vectensis

Terminal selectors are transcription factors that control the morphological, physiological, and molecular features that characterize distinct cell types. Here, we show that, in the sea anemone Nematostella vectensis, NvPOU4 is expressed in post-mitotic cells that give rise to a diverse set of neural cell types, including cnidocytes and NvElav1-expressing neurons. Morphological analyses of NvPOU4 mutants crossed to transgenic reporter lines show that the loss of NvPOU4 does not affect the initial specification of neural cells. Transcriptomes derived from the mutants and from different neural cell populations reveal that NvPOU4 is required for the execution of the terminal differentiation program of these neural cells. These findings suggest that POU4 genes have ancient functions as terminal selectors for morphologically and functionally disparate types of neurons and they provide experimental support for the relevance of terminal selectors for understanding the evolution of cell types.publishedVersio

Cnidarian microRNAs frequently regulate targets by cleavage

In bilaterians, which comprise most of extant animals, microRNAs (miRNAs) regulate the majority of messenger RNAs (mRNAs) via base-pairing of a short sequence (the miRNA seed ) to the target, subsequently promoting translational inhibition and transcript instability. In plants, many miRNAs guide endonucleolytic cleavage of highly complementary targets. Because little is known about miRNA function in nonbilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovers scores of novel miRNAs in Nematostella, increasing the total miRNA gene count to 87. Yet only a handful are conserved in corals and hydras, suggesting that microRNA gene turnover in Cnidaria greatly exceeds that of other metazoan groups. We further show that Nematostella miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity. This mode of action resembles that of small interfering RNAs (siRNAs) and plant miRNAs. It appears to be common in Cnidaria, as several of the miRNA target sites are conserved among distantly related anemone species, and we also detected miRNA-directed cleavage in Hydra. Unlike in bilaterians, Nematostella miRNAs are commonly coexpressed with their target transcripts. In light of these findings, we propose that post-transcriptional regulation by miRNAs functions differently in Cnidaria and Bilateria. The similar, siRNA-like mode of action of miRNAs in Cnidaria and plants suggests that this may be an ancestral state

Generating transgenic reporter lines for studying nervous system development in the cnidarian nematostella vectensis

Neurons often display complex morphologies with long and fine processes that can be difficult to visualize, in particular in living animals. Transgenic reporter lines in which fluorescent proteins are expressed in defined populations of neurons are important tools that can overcome these difficulties. By using membrane-attached fluorescent proteins, such reporter transgenes can identify the complete outline of subsets of neurons or they can highlight the subcellular localization of fusion proteins, for example at pre- or postsynaptic sites. The relative stability of fluorescent proteins furthermore allows the tracing of the progeny of cells over time and can therefore provide information about potential roles of the gene whose regulatory elements are controlling the expression of the fluorescent protein. Here we describe the generation of transgenic reporter lines in the sea anemone Nematostella vectensis, a cnidarian model organism for studying the evolution of developmental processes. We also provide an overview of existing transgenic Nematostella lines that have been used to study conserved and derived aspects of nervous system development.acceptedVersio

Achsenbildung in Cnidariern: Die Rolle der Wnt- und BMP/Chordin Signaltransduktionswege

Ein entscheidender Schritt in der Evolution der Tiere war die Entwicklung von Körperbauplänen mit zwei Achsen aus solchen mit nur einer Achse. Um diesen Schritt zu verstehen, ist es notwendig, die molekularen Prozesse die die Achsenbildung steuern, sowohl in bilateralsymmetrischen (zwei Achsen) als auch in radiärsymmetrischen Tieren zu kennen. Da diese molekularen Mechanismen in Bilateriern hoch konserviert sind, versuchten wir zu erforschen, ob homologe molekulare Signalwege bereits in dem radiärsymmetrischen Süßwasserpolypen Hydra (Phylum Cnidaria) existieren und dort an Achsenbildungsprozessen beteiligt sind. Wir haben gefunden, dass Moleküle der konservierten Wnt- und BMP/Chordin-Signaltransduktionswege in Hydra existieren und zeigen, dass ihre Expressionsmuster während regulärer und experimentell induzierter Musterbildungsprozesse eine Funktion in der Achsenbildung der Polypen nahe legen. Implikationen für die Evolution der Körperbaupläne werden diskutiert

RGM Regulates BMP-Mediated Secondary Axis Formation in the Sea Anemone Nematostella vectensis

Summary: Patterning of the metazoan dorsoventral axis is mediated by a complex interplay of BMP signaling regulators. Repulsive guidance molecule (RGM) is a conserved BMP coreceptor that has not been implicated in axis specification. We show that NvRGM is a key positive regulator of BMP signaling during secondary axis establishment in the cnidarian Nematostella vectensis. NvRGM regulates first the generation and later the shape of a BMP-dependent Smad1/5/8 gradient with peak activity on the side opposite the NvBMP/NvRGM/NvChordin expression domain. Full knockdown of Smad1/5/8 signaling blocks the formation of endodermal structures, the mesenteries, and the establishment of bilateral symmetry, while altering the gradient through partial NvRGM or NvBMP knockdown shifts the boundaries of asymmetric gene expression and the positioning of the mesenteries along the secondary axis. These findings provide insight into the diversification of axis specification mechanisms and identify a previously unrecognized role for RGM in BMP-mediated axial patterning. : Leclère and Rentzsch identify repulsive guidance molecule (RGM) as an essential regulator of the BMP morphogen gradient that controls the formation and patterning of the secondary body axis in the sea anemone Nematostella. The evolutionary conservation of RGM-BMP interactions indicates that this function might also be important for bilaterian embryogenesis

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