Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

Vertebrate-like sex steroid hormones have been widely detected in mollusks, and numerous experiments have shown the importance of steroids in gonad development. Nevertheless, their signaling pathways in invertebrates have not been uncovered yet. Steroid receptors are an ancient class of transcription factors with multiple roles in not only vertebrates but also invertebrates. Estrogen signaling is thought to have major roles in mollusk physiology, but the full repertoire of estrogen receptors is unknown. We presented the successful cloning of two novel forms of estrogen receptor-like genes. These receptors are present in two closely related species of Mytilus: Mytilus edulis and Mytilus galloprovincialis, commonly known and widely distributed sentinel species. Our phylogenetic analysis revealed that one of these receptors is an estrogen receptor (ER) and the other one is an estrogen-related receptor (ERR). Studies of expression analysis showed that both receptor mRNAs were localized in the oocytes and follicle cells in contact with developing oocytes in the ovary and Sertoli cells in the testis, and in the ciliated cells of the gill. In addition, we have evidence that one (ER) of these may have a capacity to autoregulate its own expression in the gonadal cells by estrogen (E2) and that this gene is responsive to estrogenic compounds.

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Highlights d The neurotransmitter GABA is a key regulator of Ciona metamorphosis d Gonadotropin-releasing hormone (GnRH) is the downstream neuropeptide of GABA d GABA positively regulates secretion of GnRH through the metabotropic GABA receptor

Recent studies suggest that transcriptional activators and components of the pre-initiation complex (PIC) form higher order associations—clusters or condensates—at active loci. Considerably less is known about the distribution of repressor proteins responsible for gene silencing. Here, we develop an expression assay in living Ciona embryos that captures the liquid behavior of individual nucleoli undergoing dynamic fusion events. The assay is used to visualize puncta of Hes repressors, along with the Groucho/TLE corepressor. We observe that Hes.a/Gro puncta have the properties of viscous liquid droplets that undergo limited fusion events due to association with DNA. Hes.a mutants that are unable to bind DNA display hallmarks of liquid–liquid phase separation, including dynamic fusions of individual condensates to produce large droplets. We propose that the DNA template serves as a scaffold for the formation of Hes condensates, but limits the spread of transcriptional repressors to unwanted regions of the genome.