Employing BAC-reporter constructs in the sea anemone Nematostella vectensis

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Integrative and Comparative Biology 53 (2013): 832-846, doi:10.1093/icb/ict091.Changes in the expression and function of genes drive evolutionary change. Comparing how genes are regulated in different species is therefore becoming an important part of evo-devo studies. A key tool for investigating the regulation of genes is represented by Bacterial Artificial Chromosomes-reporter constructs (BAC). BACs are large insert libraries, often >>100 kb, which thus capture the genomic sequences surrounding a gene of interest, including all, or nearly all, of the elements underpinning regulation. Recombinant BACs, containing a reporter gene in place of the endogenous coding sequence of genes, can be utilized to drive the expression of reporter genes under the regulatory control of the gene of interest while still embedded within its genomic context. Systematic deletions within the BAC reporter construct can be used to identify the minimal reporter in an unbiased way, avoiding the risk of overlooking regulatory elements that may be many kilobases away from the transcription start-site. Nematostella vectensis (Edwardsiidae, Anthozoa, Cnidaria) has become an important model in regenerative biology, ecology, and especially in studies of evo-devo and gene-regulatory networks due to its interesting phylogenetic position and amenability to molecular techniques. The increasing interest in this rising model system also led to a demand for methods that can be used to study the regulation of genes in Nematostella. Here we present our progress in employing BAC reporter constructs to visualize gene-expression in Nematostella. Using a new Nematostella-specific recombination cassette, we made nine different BAC reporter constructs. Although five BAC recombinants gave variable effects, three constructs, namely Nv-bra:eGFP::L10 BAC, Nv-dpp:eGFP::L10 BAC, and Nv-grm:eGFP::L10 BAC, delivered promising results. We show that these three constructs express the reporter gene eGFP in 10.4% – 17.2% of all analyzed larvae, out of which 26.2 – 41.9% express GFP in a mosaic fashion within the expected domain. In addition to the expression within the known domains, we also observed cases of misexpression of eGFP and examples that could represent actual expression outside the described domain. Furthermore, we deep-sequenced and assembled five different BACs containing Nv-chordin, Nv-foxa, Nv-dpp, Nv-wnta, and Nvwnt1, to improve assembly around these genes. The use of BAC reporter constructs will foster cis-regulatory analyses in Nematostella and thus help to improve our understanding of the regulatory network in this cnidarian system. Ultimately, this will advance the comparison of gene-regulation across species and lead to a much better understanding of evolutionary changes and novelties.2014-08-1