The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.

One of the striking findings of comparative developmental genetics was that expression patterns of core transcription factors are extraordinarily conserved in bilaterians. However, it remains unclear whether cis-regulatory elements of their target genes also exhibit common signatures associated with conserved embryonic fields. To address this question, we focused on genes that are active in the anterior neuroectoderm and non-neural ectoderm of the ascidian Ciona intestinalis. Following the dissection of a prototypic anterior placodal enhancer, we searched all genomic conserved non-coding elements for duplicated motifs around genes showing anterior neuroectodermal expression. Strikingly, we identified an over-represented pentamer motif corresponding to the binding site of the homeodomain protein OTX, which plays a pivotal role in the anterior development of all bilaterian species. Using an in vivo reporter gene assay, we observed that 10 of 23 candidate cis-regulatory elements containing duplicated OTX motifs are active in the anterior neuroectoderm, thus showing that this cis-regulatory signature is predictive of neuroectodermal enhancers. These results show that a common cis-regulatory signature corresponding to K50-Paired homeodomain transcription factors is found in non-coding sequences flanking anterior neuroectodermal genes in chordate embryos. Thus, field-specific selector genes impose architectural constraints in the form of combinations of short tags on their target enhancers. This could account for the strong evolutionary conservation of the regulatory elements controlling field-specific selector genes responsible for body plan formation.

Author Summary

Regional identity in embryos is defined by a few specific transcription factors that activate a large number of target genes through binding to common tags in regulatory sequences. In chordates it is unclear if such tags can be identified in the cis-regulatory regions of regionally expressed genes. To address this question we focused on the anterior nervous system where Otx codes for a transcription factor that triggers expression of many other head-specific genes. We analyzed an element that is active in the region bordering the anterior nervous system in the marine invertebrate Ciona intestinalis. We found that the crucial binding sites have to be duplicated and close enough. One of the pairs is bound by OTX. We showed that anterior nervous system genes are often flanked by duplicated OTX binding sites. We confirmed by transgenic assays that about half of these genomic sequences are active and drive expression anteriorly. This study unravels a simple regulatory logic in the anterior enhancers. It indicates that although there are major changes in the organization of the binding sites at short evolutionary range, conserved expression patterns are partly generated by a duplicated organization of conserved binding sites for region-specific transcription factors.