Topological structures and syntenic conservation in sea anemone genomes.

There is currently little information about the evolution of gene clusters, genome architectures and karyotypes in early branching animals. Slowly evolving anthozoan cnidarians can be particularly informative about the evolution of these genome features. Here we report chromosome-level genome assemblies of two related anthozoans, the sea anemones Nematostella vectensis and Scolanthus callimorphus. We find a robust set of 15 chromosomes with a clear one-to-one correspondence between the two species. Both genomes show chromosomal conservation, allowing us to reconstruct ancestral cnidarian and metazoan chromosomal blocks, consisting of at least 19 and 16 ancestral linkage groups, respectively. We show that, in contrast to Bilateria, the Hox and NK clusters of investigated cnidarians are largely disintegrated, despite the presence of staggered hox/gbx expression in Nematostella. This loss of microsynteny conservation may be facilitated by shorter distances between cis-regulatory sequences and their cognate transcriptional start sites. We find no clear evidence for topologically associated domains, suggesting fundamental differences in long-range gene regulation compared to vertebrates. These data suggest that large sets of ancestral metazoan genes have been retained in ancestral linkage groups of some extant lineages; yet, higher order gene regulation with associated 3D architecture may have evolved only after the cnidarian-bilaterian split.

Spatial transcriptomics reveals a cnidarian segment polarity program in Nematostella vectensis.

During early animal evolution, the emergence of axially polarized segments was central to the diversification of complex bilaterian body plans. Nevertheless, precisely how and when segment polarity pathways arose remains obscure. Here, we demonstrate the molecular basis for segment polarization in developing larvae of the sea anemone Nematostella vectensis. Utilizing spatial transcriptomics, we first constructed a 3D gene expression atlas of developing larval segments. Capitalizing on accurate in silico predictions, we identified Lbx and Uncx, conserved homeodomain-containing genes that occupy opposing subsegmental domains under the control of both bone morphogenetic protein (BMP) signaling and the Hox-Gbx cascade. Functionally, Lbx mutagenesis eliminated all molecular evidence of segment polarization at the larval stage and caused an aberrant mirror-symmetric pattern of retractor muscles (RMs) in primary polyps. These results demonstrate the molecular basis for segment polarity in a non-bilaterian animal, suggesting that polarized metameric structures were present in the Cnidaria-Bilateria common ancestor over 600 million years ago.

Spatial transcriptomics reveals a conserved segment polarity program that governs muscle patterning in Nematostella vectensis.

During early animal evolution, the emergence of axially-polarized segments was central to the diversification of complex bilaterian body plans. Nevertheless, precisely how and when segment polarity pathways arose remains obscure. Here we demonstrate the molecular basis for segment polarization in developing larvae of the pre-bilaterian sea anemone Nematostella vectensis . Utilizing spatial transcriptomics, we first constructed a 3-D gene expression atlas of developing larval segments. Capitalizing on accurate in silico predictions, we identified Lbx and Uncx, conserved homeodomain-containing genes that occupy opposing subsegmental domains under the control of both BMP signaling and the Hox-Gbx cascade. Functionally, Lbx mutagenesis eliminated all molecular evidence of segment polarization at larval stage and caused an aberrant mirror-symmetric pattern of retractor muscles in primary polyps. These results demonstrate the molecular basis for segment polarity in a pre-bilaterian animal, suggesting that polarized metameric structures were present in the Cnidaria-Bilateria common ancestor over 600 million years ago. Highlights: Nematostella endomesodermal tissue forms metameric segments and displays a transcriptomic profile similar to that observed in bilaterian mesoderm Construction of a comprehensive 3-D gene expression atlas enables systematic dissection of segmental identity in endomesoderm Lbx and Uncx , two conserved homeobox-containing genes, establish segment polarity in Nematostella The Cnidarian-Bilaterian common ancestor likely possessed the genetic toolkit to generate polarized metameric structures.

Manipulation of Gene Activity in the Regenerative Model Sea Anemone, Nematostella vectensis.

With a surprisingly complex genome and an ever-expanding genetic toolkit, the sea anemone Nematostella vectensis has become a powerful model system for the study of both development and whole-body regeneration. Here we provide the most current protocols for short-hairpin RNA (shRNA )-mediated gene knockdown and CRISPR/Cas9-targeted mutagenesis in this system. We further show that a simple Klenow reaction followed by in vitro transcription allows for the production of gene-specific shRNAs and single guide RNAs (sgRNAs) in a fast, affordable, and readily scalable manner. Together, shRNA knockdown and CRISPR/Cas9-targeted mutagenesis allow for rapid screens of gene function as well as the production of stable mutant lines that enable functional genetic analysis throughout the Nematostella life cycle.

Cnidofest 2018: the future is bright for cnidarian research.

The 2018 Cnidarian Model Systems Meeting (Cnidofest) was held September 6-9th at the University of Florida Whitney Laboratory for Marine Bioscience in St. Augustine, FL. Cnidofest 2018, which built upon the momentum of Hydroidfest 2016, brought together research communities working on a broad spectrum of cnidarian organisms from North America and around the world. Meeting talks covered diverse aspects of cnidarian biology, with sessions focused on genomics, development, neurobiology, immunology, symbiosis, ecology, and evolution. In addition to interesting biology, Cnidofest also emphasized the advancement of modern research techniques. Invited technology speakers showcased the power of microfluidics and single-cell transcriptomics and demonstrated their application in cnidarian models. In this report, we provide an overview of the exciting research that was presented at the meeting and discuss opportunities for future research.

Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis.

A mechanistic understanding of evolutionary developmental biology requires the development of novel techniques for the manipulation of gene function in phylogenetically diverse organismal systems. Recently, gene-specific knockdown by microinjection of short hairpin RNA (shRNA) was applied in the sea anemone Nematostella vectensis, demonstrating that the shRNA approach can be used for efficient and robust sequence-specific knockdown of a gene of interest. However, the time- and labor-intensive process of microinjection limits access to this technique and its application in large scale experiments. To address this issue, here we present an electroporation protocol for shRNA delivery into Nematostella eggs. This method leverages the speed and simplicity of electroporation, enabling users to manipulate gene expression in hundreds of eggs or embryos within minutes. We provide a detailed description of the experimental procedure, including reagents, electroporation conditions, preparation of Nematostella eggs, and follow-up care of experimental animals. Finally, we demonstrate the knockdown of several endogenous and exogenous genes with known phenotypes and discuss the potential applications of this method.

An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis.

Hox genes encode conserved developmental transcription factors that govern anterior-posterior (A-P) pattering in diverse bilaterian animals, which display bilateral symmetry. Although Hox genes are also present within Cnidaria, these simple animals lack a definitive A-P axis, leaving it unclear how and when a functionally integrated Hox code arose during evolution. We used short hairpin RNA (shRNA)-mediated knockdown and CRISPR-Cas9 mutagenesis to demonstrate that a Hox-Gbx network controls radial segmentation of the larval endoderm during development of the sea anemone Nematostella vectensis. Loss of Hox-Gbx activity also elicits marked defects in tentacle patterning along the directive (orthogonal) axis of primary polyps. On the basis of our results, we propose that an axial Hox code may have controlled body patterning and tissue segmentation before the evolution of the bilaterian A-P axis.

An adaptable chromosome preparation methodology for use in invertebrate research organisms.

BACKGROUND: The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed. RESULTS: We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. CONCLUSIONS: Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.

Capicua controls Toll/IL-1 signaling targets independently of RTK regulation.

The HMG-box protein Capicua (Cic) is a conserved transcriptional repressor that functions downstream of receptor tyrosine kinase (RTK) signaling pathways in a relatively simple switch: In the absence of signaling, Cic represses RTK-responsive genes by binding to nearly invariant sites in DNA, whereas activation of RTK signaling down-regulates Cic activity, leading to derepression of its targets. This mechanism controls gene expression in both Drosophila and mammals, but whether Cic can also function via other regulatory mechanisms remains unknown. Here, we characterize an RTK-independent role of Cic in regulating spatially restricted expression of Toll/IL-1 signaling targets in Drosophila embryogenesis. We show that Cic represses those targets by binding to suboptimal DNA sites of lower affinity than its known consensus sites. This binding depends on Dorsal/NF-κB, which translocates into the nucleus upon Toll activation and binds next to the Cic sites. As a result, Cic binds to and represses Toll targets only in regions with nuclear Dorsal. These results reveal a mode of Cic regulation unrelated to the well-established RTK/Cic depression axis and implicate cooperative binding in conjunction with low-affinity binding sites as an important mechanism of enhancer regulation. Given that Cic plays a role in many developmental and pathological processes in mammals, our results raise the possibility that some of these Cic functions are independent of RTK regulation and may depend on cofactor-assisted DNA binding.

Non-model model organisms.

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future.

Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish.

The T-box transcription factor Tbx5 (Tbx5a in zebrafish) plays a crucial role in the formation of cardiac chambers in a dose-dependent manner. Its deregulation leads to congenital heart disease. However, little is known regarding its regulation. Here we isolate a zebrafish mutant with heart malformations, called 34c. The affected gene is identified as kctd10, a member of the potassium channel tetramerization domain (KCTD)-containing family. In the mutant, the expressions of the atrioventricular canal marker genes, such as tbx2b, hyaluronan synthase 2 (has2), notch1b and bmp4, are changed. The knockdown of tbx5 rescues the ectopic expression of has2, and knockdown of either tbx5a or has2 alleviates the heart defects. We show that Kctd10 directly binds to Tbx5 to repress its transcriptional activity. Our results reveal a new essential factor for cardiac development and suggest that KCTD10 could be considered as a new causative gene of congenital heart disease.