Meetings

Meetings Establishing a model organism: A report from the first annual Nematostella meeting

Introduction Moreover, this growing community has view of his lab’s efforts to address this developed a suite of molecular tech- limitation. He provided a brief overview The sea anemone Nematostella vecten- niques for Nematostella that mirror of a project with N-ethyl-N-nitrosourea sis, a member of the phylum Cnidaria, those developed for traditional model (ENU) mutagenesis coupled with restric- has rapidly developed into a corner- systems (Table 1). The overall growth tion site associated DNA (RAD) map- stone taxon in comparative studies of of this species as a laboratory model is ping, deep sequencing, and forward genome evolution and animal develop- reflected in the dramatic increase in genetics to identify mutations causing ment. Originally described in 1935 by publications that mention or feature abnormal phenotypes in Nematostella. T.A. Stephenson, Nematostella was Nematostella (Fig. 1). In response to this The Martindale lab has also developed a largely studied in the context of estuar- growing interest, we organized the first microarray for comparing gene expres- ine ecology and comparative taxonomy Nematostella meeting, which was held sion in response to chemical (e.g. lith- for the next 60 years. Beginning in the at the Woods Hole Oceanographic ium chloride and alsterpaullone for 1990s, descriptive work by Hand Institution on June 27, 2011, in Woods inhibition of glycogen synthase kinase and Uhlinger [1], as well as gene expres- Hole, Massachusetts, USA. The meeting 3, GSK-3), misexpression, and gene sion and comparative phylogenetics focused on developing strategies for knockdown manipulations of research by Finnerty and Martindale improving and expanding community development. [2], laid the foundation for the emer- resources, on expertise related to animal In the discussion following Mark’s gence of Nematostella as an informative culturing and experimental techniques, presentation, several participants laboratory model for cnidarian evol- as well as highlighting the diversity of brought up examples of widely-used ution and development [3, 4]. In 2007, Nematostella research topics. techniques that work well in other Nematostella became the first non-bilat- models, but perform poorly or in an erian animal with a sequenced genome unexpected manner in Nematostella. [5]. The availability of the genome Developments in tests For example, while morpholino-based quickly established Nematostella as a of gene expression and methodstosuppressexpressionof key invertebrate animal for both devel- protein function Nematostella genes have proven effec- opmental biology and comparative tive in some studies, several groups genomics. Indeed, one of the immediate Studies involving Nematostella have reported difficulties doing the same and major surprises was the realization been integral to our understanding of with RNA interference, despite this that, in many respects, the Nematostella how embryogenesis and animal evol- technique’s broad utility in another genome is more similar to the human ution are interconnected. In particular, cnidarian, Hydra.Basedonthisdis- genome than it is to two more commonly spatial and temporal characterizations cussion, we have listed some com- studied invertebrate models, Drosophila of gene expression have been useful in ments from meeting participants in melanogaster and Caenorhabditis ele- identifying likely roles for genes reference to particular techniques gans [6]. Since then, research on involved in axial patterning [7], germ (seeNotescolumninTable1).Wehope Nematostella has rapidly grown in layer development [8], and cell specifi- that by sharing the experiences of the prominence, extended into other areas cation [9], to name a few. However, far participants at this meeting we can of biology, and is continually being fewer studies have been conducted help researchers to avoid duplication adopted by labs throughout the world. involving techniques that directly test of effort, select the most appropriate gene function (see [10, 11]). Mark techniques, and identify areas where Martindale (Kewalo Marine Laboratory, additional method development is DOI 10.1002/bies.201100145 University of Hawaii) presented an over- needed.

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Table 1. Summary of experimental methods that have been successfully applied to Nematostella research

Representative Meetings Method Approach publication(s) Meeting notesa

In situ hybridization [8, 18, 19] Immunohistochemistry [11, 14] Visualization of broad spatial expression in whole mount and subcellular localization in thin sections (TDG) Gene knockdown Morpholino [10, 11] Morpholinos are typically injected, but animals can also be soaked for incorporation (MQM) RNAi [20] Participants at meeting suggested RNAi has been less reliable or ineffective (MQM) Heterologous protein expression Cell-free [21] Vertebrate cell lines [14] EMSA for DNA binding [14] Protein crystallography [22] Transgenics [23] Spawning and culturing [24, 25] Varied culturing conditions (salinity, temperature) may favor better cultures for some populations (AMR) We have also included a notes column with observations from participants from the meeting. a Initials indicate source for observation from meeting. MQM: Mark Q. Martindale, AMR: Adam M. Reitzel, TDG: Thomas D. Gilmore.

The recent emergence of inexpensive In this context, Casey reported results ing. Experiments describing protein- sequencing technologies has presented from detailed comparisons of different protein interactions and protein func- new opportunities to study the transcrip- sequencing platforms (e.g. Illumina, tion in the regulation of downstream tional changes associated with develop- SOLiD, and Helicos). Additionally, genes will be crucial for understanding ment in unprecedented detail and to Casey described their protocol for assem- the evolution of metazoan gene function uncover the underlying molecular mech- bling transcriptomes using the OASIS and signaling networks. Tom Gilmore anisms. Casey Dunn (Brown University) assembly software. (Boston University) presented one of presented research utilizing next-gener- To date, much of the molecular the most comprehensive protein-based ation sequencing for comparative tran- research involving Nematostella has experimental efforts in Nematostella to scriptomic studies. The Dunn lab uses focused on gene transcripts. However, date, a detailed characterization of the Nematostella for establishing techniques it is clear that until we characterize the Nematostella NF-kB signal transduction that can be applied to other cnidarian protein products of these genes, our pathway. His lab has shown that the NF- species that are less amenable to labora- view of genetic networks will remain kB pathway was likely in place prior to tory study, like large siphonophores [12]. partial, and will sometimes be mislead- the last common ancestor of Metazoa [13]. Using a variety of in vitro and cell-based assays, they have shown 500 Nematostella NF-kB can bind to and 450 activate promoters containing canonical 400 kB binding motifs and that activities of NF-kB can be inhibited by two 350 Nematostella IkB-like inhibitors [14]. 300 Using a newly developed antibody, they 250 showed that expression of Nematostella

200 vectensis NF-kB (NvNF-kB) is restricted to a subset of ectodermal cells in both 150 juveniles and adults.

Number of Publications 100

50 0 Expanding comparative 1966 1968 1970 1972 1974 1976 1978 1980 1982 1984 1986 1988 1990 1992 1994 1996 1998 2000 2002 2004 2006 2008 2010 genomics in early diverging

Figure 1. The number of publications per year (1966–2010) containing the search term phyla ‘‘Nematostella’’ in the text. The search was conducted with Google Scholar on June 15, 2011. The Nematostella genome was published in 2007 and continues to be a strong con- Another theme of the meeting was the tributor to the increase in publications. context for comparative genomics that

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has developed around the Nematostella [15]. As a denizen of high marsh estua- laboration with Ann Tarrant (WHOI) to genome. Since the sea anemone’s ries, Nematostella occupies a highly var- describe transcriptional responses by genome was published in 2007, two iable habitat that has frequently been Nematostella to environmental stress. additional cnidarian genomes have impacted by human encroachment and been published: Hydra magnipapillata pollution. Little is known about how (2010) and Acropora digitifera (2011). Nematostella survives and thrives in Developing research The sequencing and comparative these environments and if populations analysis of other genomes will continue throughout the range show any pheno- integration and new to be instrumental in identifying critical typic variation, potentially consistent opportunities Meetings molecular events in the evolution of with local adaptation. cnidarian and other animal genomes. Janelle Thompson (Massachusetts Throughout the meeting, there were fre- John Finnerty (Boston University) Institute of Technology) spoke about quent discussions of how the described a comparative genomics effort her lab’s work on the microbiome of Nematostella community can create or to sequence the genomes of several Nematostella. Her lab has compared improve resources to facilitate future populations of Nematostella. The goal the microbial community associated research efforts. Some of these efforts of this effort is to characterize intraspe- with Nematostella from wild popu- are well underway or completed. cific variation and identify genomic lations and laboratory strains. To date, Mattias Ormestad and Eric Roettinger regions under selection. The Finnerty most comparisons have used 16S rRNA from the Martindale lab have developed lab has initiated efforts to sequence sequencing for identifying microbial a database of gene expression the genomes of two other species in species, but she has also identified (KahiKai.org) – a tool for querying the family Edwardsiidae (i.e. broader sequencing approaches. The expression patterns from Nematostella Edwardsiella lineata and Edwardsia ele- Thompson lab uses these studies to and other marine invertebrates [17]. gans) as well as other anthozoans. identify patterns in microbial diversity Synthesizing these data into one Despite a close relationship with within these populations and to deter- location will permit individuals to Nematostella, Edwardsiella lineata has mine if these associations are stable in develop hypotheses about potential evolved a parasitic life history, thus a laboratory culture. Upon identification gene interactions, given similar comparative genomics study will aim to of stable associates, Janelle will use temporal-spatial expression, and to understand the transition between para- experimental approaches to determine select genes to complement future sitic and non-parasitic life history effects of microbial associates on research efforts. The Finnerty lab stages. In the broader phylogenetic con- Nematostella physiology. Early results described ongoing efforts to overhaul text, Joseph Ryan (National Human suggest that there is considerable vari- current on-line resources (CnidBase, Genome Research Institute, National ation between the microbiomes of StellaBase, and Nematostella.org) and Institutes of Health) provided an over- the laboratory populations and field- to integrate these sites with new resour- view of the sequencing and annotation collected populations. ces (PdamBase and EdBase) to facilitate of the genome of the ctenophore Adam Reitzel (Woods Hole comparative genomics within the Mnemiopsis leidyi. This presentation Oceanographic Institution, WHOI) Cnidaria. focused on lessons learned from a de spoke about genetic and phenotypic One goal for this first Nematostella novo sequencing project based entirely variation in populations of meeting was to identify a specific area on next-generation sequencing technol- Nematostella collected throughout their where immediate efforts could have the ogies. The ctenophore genome will pro- known range. Using a suite of neutral largest impact on the community. We vide a necessary data source for markers (e.g. amplified fragment length suggest continuing this goal-oriented comparative genomics with other non- polymorphisms (AFLPs), and microsa- discussion as a priority in future meet- bilaterian phyla, such as Porifera and tellites), this work has shown extensive ings. This meeting demonstrated an Placozoa, to better assess the branching genetic variation within and between overwhelming interest in developing order at the base of the animal tree and populations. Additionally, Nematostella an improved genome annotation. To to identify shifts in gene complements in harbors many non-synonymous poly- facilitate this, numerous labs will be these early diverging phyla. morphisms in conserved domains that donating sequence data from transcrip- are restricted to particular geographic tional profiling and targeted cloning to locations [16]. These markers may pro- produce improved gene models. In total, vide good candidates for future studies these data amount to more than Studying Nematostella of functional variation between alleles 6 Â 1010 bp of sequence, which will in its natural environment that may underlie molecular mechan- result in more complete annotation of isms of local adaptation. He also transcripts and a catalog of splice var- The final major theme of the meeting reported significant phenotypic vari- iants. On this topic, there was a discus- was interactions between Nematostella ation in growth rate and temperature sion on how to update current and the environment. Nematostella has tolerance of individuals collected from annotations, which have largely a natural distribution along much of the populations spanning the thermocline remained static since the initial genome Atlantic coast of North America and has along the Atlantic coast of North release in 2007. Solutions centered on been introduced numerous times to America. Finally, Adam highlighted cur- generating an updated transcriptome, estuaries outside of the natural range rent and future work conducted in col- implementing a new genome browser,

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and creating wiki-based gene pages 2) Genome Technology Branch, National 13. Sullivan JC, Kalaitzidis D, Gilmore TD, Finnerty JR. 2007. Rel homology domain- (similar to those being generated for Human Genome Research Institute, containing transcription factors in the cnidar- the Mnemiopsis genome project; Ryan, National Institutes of Health, ian Nematostella vectensis. Dev Genes Evol Meetings personal communication) that could be Bethesda, MD, USA 217: 63–72. edited by the community researchers *E-mail: [email protected] 14. Wolenski FS, Garbati MR, Lubinski TJ, Traylor-Knowles N, et al. 2011. and easily linked to and from third party Characterization of the core elements of the resources. The overall goal is to have a NF-kB signaling pathway of the sea anemone single, well-annotated data source for Nematostella vectensis. Mol Cell Biol 31: References 1076–87. researchers using Nematostella. 15. Reitzel AM, Darling JA, Sullivan JC, The final topic of the meeting was a 1. Hand C, Uhlinger K. 1992. The culture, Finnerty JR. 2008. Global population genetic plan for future annual meetings with sexual and asexual reproduction, and growth structure of the starlet anemone Nematostella broader representation. Researchers in the sea anemone Nematostella vectensis. vectensis: multiple introductions and implica- Biol Bull 182: 169–76. tions for conservation policy. Biol Invasions throughout the world are continually 2. Finnerty JR, Martindale MQ. 1999. Ancient 10: 1197–213. adopting Nematostella to explore a origins of axial patterning genes: Hox genes 16. Reitzel AM, Sullivan JC, Finnerty JR. 2010. broad range of biological fields and it and ParaHox genes in the Cnidaria. Evol Dev Discovering SNPs in protein coding regions 1: 16–23. with StellaSNP: illustrating the characteriz- was agreed that future meetings should 3. Darling JA, Reitzel AM, Burton PM, Mazza ation and geographic distribution of polymor- capitalize on this geographic and inter- ME, et al. 2005. Rising starlet: the starlet sea phisms in the estuarine anemone disciplinary diversity. Attendees anemone, Nematostella vectensis. BioEssays Nematostella vectensis. Estuar Coast 33: 27: 211–21. 930–43. suggested holding meetings that alter- 4. Technau U, Steele RE. 2011. Evolutionary 17. Ormestad M, Martindale MQ, Rottinger E. nate between the USA and Europe. The crossroads in developmental biology: 2011. A comparative gene expression data- next proposed meeting is scheduled for Cnidaria. Development 138: 1447–58. base for invertebrates. Evodevo 2: 17. the summer of 2012 at the Marine 5. Putnam N, Srivastava M, Hellsten U, Dirks 18. Genikhovich G, Technau U. 2009. In situ B, et al. 2007. Sea anemone genome reveals hybridization of starlet sea anemone Biological Laboratory (Woods Hole, ancestral eumetazoan gene repertoire and (Nematostella vectensis) embryos, larvae, MA), and will be organized by Joel genomic organization. Science 317: 86–94. and polyps. Cold Spring Harbor Protocols Smith (MBL), Ann Tarrant (WHOI), 6. Sullivan JC, Reitzel AM, Finnerty JR. 2006. 2009: pdb.prot5282. A high percentage of introns in human genes 19. Finnerty JR, Paulson D, Burton P, Pang K, Adam Reitzel (WHOI), and Joseph were present early in animal evolution: evi- et al. 2003. Early evolution of a homeobox Ryan (Sars International Centre for dence from the basal metazoan Nematostella gene: the ParaHox gene Gsx in the Cnidaria Marine Molecular Biology). For inter- vectensis. Genome Inform 17:219–29. and the Bilateria. Evol Dev 5: 331–45. ested individuals, we have created a 7. Finnerty JR, Pang K, Burton P, Paulson D, 20. Pankow S, Bamberger C. 2007. The p53 et al. 2004. Origins of bilateral symmetry: Hox tumor suppressor-like protein nvp63 medi- website that will publish updated infor- and dpp expression in a sea anemone. ates selective germ cell death in the sea ane- mation (www.nematostellameeting.- Science 304: 1335–7. mone Nematostella vectensis. PLoS ONE 2: com). Future meetings will have 8. Martindale M, Pang K, Finnerty JR. 2004. e782. Investigating the origins of triploblasty: ‘‘mes- 21. Reitzel AM, Behrendt L, Tarrant AM. 2010. workshops that will focus on emerging odermal’’ gene expression in a diploblastic Light entrained rhythmic gene expression in experimental techniques, Nematostella- animal, the sea anemone, Nematostella vec- the sea anemone Nematostella vectensis: the centric bioinformatic applications, and tensis (Phylum Cnidaria; Class Anthozoa). evolution of the animal circadian clock. PLoS Development 131: 2463–74. ONE 5: e12805. field collections. Together, efforts to 9. Marlow HQ, Srivastava M, Matus DQ, 22. Murai MJ, Chruszcz M, Reddy G, increase geographic representation, Rokhsar D, et al. 2009. Anatomy and devel- Grembecka J, et al. 2011. Crystal structure broaden the disciplinary scope, and tar- opment of the nervous system of of menin reveals binding site for mixed lineage get learning opportunities will continue Nematostella vectensis, an anthozoan cnidar- leukemia (MLL) protein. J Biol Chem 286: ian. Dev Neurobiol 69: 235–54. 31742–8. to develop Nematostella as a powerful 10. Magie C, Daly M, Martindale MQ. 2007. 23. Renfer E, Amon-Hassenzahl A, Steinmetz model in comparative biology. Gastrulation in the cnidarian Nematostella PRH, Technau U. 2010. A muscle-specific vectensis occurs via invagination not ingres- transgenic reporter line of the sea anemone, sion. Dev Biol 305: 483–97. Nematostella vectensis. Proc Natl Acad Sci 11. Rentzsch F, Fritzenwanker J, Scholz C, USA 107: 104–8. Adam M. Reitzel1)Ã Technau U. 2008. FGF signalling controls 24. Fritzenwanker J, Technau U. 2002. Joseph F. Ryan2) formation of the apical sensory organ in the Induction of gametogenesis in the basal cni- 1) cnidarian Nematostella vectensis. darian Nematostella vectensis (Anthozoa). Ann M. Tarrant Development 135: 1761–9. Dev Genes Evol 212: 99–103. 12. Siebert S, Robinson MD, Tintori SC, Goetz 25. Genikhovich G, Technau U. 2009. Induction 1) Biology Department, Woods Hole F, et al. 2011. Differential gene expression in of spawning in the starlet sea anemone the siphonophore Nanomia bijuga (Cnidaria) Nematostella vectensis, in vitro fertilization Oceanographic Institution, Woods assessed with multiple next-generation of gametes, and dejellying of zygotes. Cold Hole, MA, USA sequencing workflows. PLoS ONE 6: e22953. Spring Harbor Protocols 2009: pdb.prot5281.

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