SUMMARYAs a sister group to Bilateria, Cnidaria is important for understanding early nervous system evolution. Here we examine neural development in the anthozoan cnidarian Nematostella vectensis in order to better understand whether similar developmental mechanisms are utilized to establish the strikingly different overall organization of bilaterian and cnidarian nervous systems. We generated a neuron-specific transgenic NvElav1 reporter line of N. vectensis and used it in combination with immunohistochemistry against neuropeptides, in situ hybridization and confocal microscopy to analyze nervous system formation in this cnidarian model organism in detail. We show that the development of neurons commences in the ectoderm during gastrulation and involves interkinetic nuclear migration. Transplantation experiments reveal that sensory and ganglion cells are autonomously generated by the ectoderm. In contrast to bilaterians, neurons are also generated throughout the endoderm during planula stages. Morpholino-mediated gene knockdown shows that the development of a subset of ectodermal neurons requires NvElav1, the ortholog to bilaterian neural elav1 genes. The orientation of ectodermal neurites changes during planula development from longitudinal (in early-born neurons) to transverse (in late-born neurons), whereas endodermal neurites can grow in both orientations at any stage. Our findings imply that elav1-dependent ectodermal neurogenesis evolved prior to the divergence of Cnidaria and Bilateria. Moreover, they suggest that, in contrast to bilaterians, almost the entire ectoderm and endoderm of the body column of Nematostella planulae have neurogenic potential and that the establishment of connectivity in its seemingly simple nervous system involves multiple neurite guidance systems.

Despite considerable differences in morphology and complexity of body plans among animals, a great part of the gene set is shared among Bilateria and their basally branching sister group, the Cnidaria. This suggests that the common ancestor of eumetazoans already had a highly complex gene repertoire. At present it is therefore unclear how morphological diversification is encoded in the genome. Here we address the possibility that differences in gene regulation could contribute to the large morphological divergence between cnidarians and bilaterians. To this end, we generated the first genome-wide map of gene regulatory elements in a nonbilaterian animal, the sea anemone Nematostella vectensis. Using chromatin immunoprecipitation followed by deep sequencing of five chromatin modifications and a transcriptional cofactor, we identified over 5000 enhancers in the Nematostella genome and could validate 75% of the tested enhancers in vivo. We found that in Nematostella, but not in yeast, enhancers are characterized by the same combination of histone modifications as in bilaterians, and these enhancers preferentially target developmental regulatory genes. Surprisingly, the distribution and abundance of gene regulatory elements relative to these genes are shared between Nematostella and bilaterian model organisms. Our results suggest that complex gene regulation originated at least 600 million yr ago, predating the common ancestor of eumetazoans.

Recent molecular phylogenies support that the non-bilaterian Cnidaria is the sister group to bilaterians (4,5) and are therefore informative to reconstruct the early history of bilaterian homeobox gene complements. Earlier searches for cnidarian homeobox genes have revealed the presence of anterior-like Hox genes as well as Gsx, so that the ProtoHox cluster must have been duplicated prior to the cnidarian-bilaterian split. Earlier reports (6-13) have proposed that posterior Hox genes but not central genes or Hox3 are also present in cnidarians. We used publicly available high coverage genome shotgun sequence to identify the complete set of homeobox genes of two distantly related cnidarians, the freshwater polyp Hydra magnipapillata (Hydrozoa) and the sea anemone Nematostella vectensis (Anthozoa).Nematostella is particularly informative as it is considered to represent the basal group within the Cnidaria (14,15 (Fig. 1b and SOM). Instead, they formed an independent and strongly supported branch. Finally, one of the two divergent genes showed conflicting affinities in network analyses with Cdx and Xlox (Fig. 1b) and is therefore most likely a ParaHox gene. (Fig. 2c), and Gsx was the only ParaHox gene detected.As lineage-specific duplications in the Hox cluster are rare within bilaterians, we wanted to know whether or not those found in Nematostella are limited to the Hox genes. For this we examined potential linkages between all other homeobox genes. Including the nine Hox-like genes mentioned above, we found a total of 139 homeobox genes, a surprisingly high number compared to other invertebrates (Table 1 and SOM). Phylogenetic analyses allowed placing at least 87 of those into 58 known groups of homeobox genes, out of 76 groups known for 5 bilaterians. They also suggested that 42 unclassified genes have arisen through recent amplifications of maximally ten genes. By comparison, Hydra has a considerably smaller number of homeobox genes and gene groups. Since all Hydra homeobox gene groups have representatives in the Nematostella genome, we assume that the homeobox complement has been dramatically reduced in the Hydra lineage (Table 1). Comparisons between the extended homeobox sequences of Nematostella allowed the detection of an additional 13 physical clusters ( Fig. 2b and SOM). Four of these clusters may have a more ancient origin, either because they have been identified in the genomes of bilaterians or because they associate distantly related genes. The other nine clusters are undoubtedly the result of recent tandem duplications. We also did not detect obvious synteny conservation between the environments of well related but unlinked homeobox genes, as might be expected after whole genome duplication (not shown). Hence, the gene duplications observed among the Nematostella Hox genes represent a general phenomenon for the homeobox gene complement.From our data, the most parsimonious scenario for the evolution of Hox/ParaHox clusters is as follows (Fig. 3): two ProtoHox genes (P1/2 and P3) gave rise to the ...

The sea anemone, Nematostella vectensis , has become an attractive new model organism for comparative genomics and evolutionary developmental biology. Over the last few years, many genes have been isolated and their expression patterns studied to gain insight into their function. More recently, functional tools have been developed to manipulate gene function; however, most of these approaches rely on microinjection and are limited to early stages of development. Transgenic lines would significantly enhance the tractability of the system. In particular, the study of gene- or tissue-specific promoters would be most useful. Here we report the stable establishment of a transgenic line using the I-SceI meganuclease system to facilitate integration into the genome. We isolated a 1.6-kb fragment of the regulatory upstream region of the Myosin Heavy Chain1 ( MyHC1 ) gene and found that the transgene is specifically expressed in the retractor and tentacle muscles of Nematostella polyps, faithfully reproducing the expression of the endogenous MyHC1 gene. This demonstrates that the 1.6-kb fragment contains all of the regulatory elements necessary to drive correct expression and suggests that retractor and tentacle muscles in Nematostella are distinct from other myoepithelial cells. The transgene is transmitted through the germline at high frequency, and G 1 transgenic polyps have only one integration site. The relatively high frequency of transgenesis, in combination with gene- or tissue-specific promoters, will foster experimental possibilities for studying in vivo gene functions in gene regulatory networks and developmental processes in the nonbilaterian sea anemone, Nematostella vectensis .

The TGF-␤ molecules Dpp/BMP2/4/7 and their antagonist Sog/Chd play a conserved role in establishing the dorso-ventral (DV) axis in bilaterians. Homologues of BMPs and the antagonist, Chordin, have been isolated from Cnidaria and show a striking asymmetric expression pattern with respect to the primary oral-aboral (OA) axis. We used Morpholino knockdowns of Nematostella dpp (bmp2/4), bmp5-8, chordin, and tolloid to investigate their function during early development of the sea anemone Nematostella vectensis. Molecular analysis of the BMP Morpholino phenotypes revealed an upregulated and radialized expression of bmps and chordin in ectoderm and endoderm indicating a negative feedback loop. Our data further suggest that BMP signaling is required for symmetry breaking of bmp and chordin expression during gastrulation. While bmps and chordin marker genes of the ectodermal OA axis extended aborally, other ectodermal markers of the OA axis were not significantly affected. By contrast, expression of other endodermal marker genes marking both the OA and the directive axis were abolished. Our data suggest that the logic of BMP2/4 signaling and the BMP antagonist, Chordin, differs significantly between Cnidaria and Bilateria, yet the double negative feedback loop detected in Nematostella bears systemic similarities with part of the regulatory network of the DV axis patterning system in amphibians.axis formation ͉ BMP signaling ͉ Cnidaria ͉ evolution ͉ Nematostella

The sea anemone Nematostella vectensis is a model system used by a rapidly growing research community for comparative genomics, developmental biology and ecology. Here, we describe a microinjection procedure for creating stable transgenic lines in Nematostella based on meganuclease (I-SceI)-assisted integration of a transgenic cassette into the genome. The procedure describes the preparation of the reagents, microinjection of the transgenesis vector and the husbandry of transgenic animals. The microinjection setup differs from those of previously published protocols by the use of a holding capillary mounted on an inverted fluorescence microscope. In one session of injections, a single researcher can microinject up to 1,300 zygotes with a reporter construct digested with the meganuclease I-SceI. Under optimal conditions, fully transgenic heterozygous F1 animals can be obtained within 4-5 months of the injections, with a germ-line transmission efficiency of ∼3%. The method is versatile and, after a short training phase, can be carried out by any researcher with basic training in molecular biology. Flexibility of construct design enables this method to be used for numerous applications, including the functional dissection of cis-regulatory elements, subcellular localization of proteins, detection of protein-binding partners, ectopic expression of genes of interest, lineage tracing and cell-type-specific reporter gene expression.

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.

The olfactory system in both vertebrates and invertebrates can recognize and distinguish thousands of chemical signals. Olfactory receptors are responsible for the early molecular events in the detection of volatile compounds and the perception of smell. Recently, candidate olfactory receptor genes have been identified in several organisms, but their characterization is far from been completed due to the difficulty to functionally express them in heterologous systems. To circumvent such difficulty, we expressed a mammalian olfactory gene, rat I7, in the nematode. We generated transgenic worms expressing I7 in AWA or AWB chemosensory neurons and performed behavioural assays using different concentrations of the rat I7 receptor agonist octanal. Pure octanal was repellent for wild-type worms whereas a 1:10 dilution was attractant. Expression of I7 in AWB neurons counteracted the volatile attraction to diluted octanal observed in control wild-type worms. Furthermore, expression of I7 in AWA neurons counteracted the volatile avoidance to pure octanal observed in wild-type worms. These results indicate that it is possible to functionally express mammalian olfactory receptors in providing a research tool to efficiently search for specific olfactory receptor ligands and to extend our understanding of the molecular basis of olfaction.