The Ci-Dll-A and Ci-Dll-B genes of Ciona intestinalis are arranged in a convergently transcribed gene cluster. This genomic arrangement is similar to that of the multiple bigene clusters of the Dlx homologs in vertebrates. Analysis of whole genome sequences showed that linkage to the Hox cluster is conserved with the vertebrate clusters. Phylogenetic analysis supports gene trees consistent with homology of the ascidian and vertebrate Dlx clusters, and in combination with the apparent conservation of genomic arrangement, it is concluded that the ascidian cluster is most likely homologous with the vertebrate clusters. Using whole-mount in situ hybridization, Ci-Dll-B transcripts were detected in all ectodermal lineages through gastrulation. Expression is radically downregulated in the neurula with detectable expression disappearing around the time that Ci-Dll-A expression appears in the anterior ectoderm. By the late tailbud stage Ci-Dll-Atranscripts were detected in the bilateral atrial primordia and persisted in the atrial rudiments to the larval stage, suggesting a role in development of these neural placode-like structures. This non-overlapping expression contradicts a common pattern seen in clustered genes, where as adjacent paralogs have largely overlapping expression domains. Enhancer sharing is often proposed as an explanation for the overlapping expression of gene cluster members. For this case of non-overlapping expression a model is proposed in which repressors acting at different stages override one or more shared enhancers. The enhancer sharing prevents breakup of the cluster while the independent temporal suppressors hide the presence of the shared enhancers.

The Ci-Dll-B gene is an early regulator of ectodermal development in the ascidian Ciona intestinalis (Imai et al., 2006). Ci-Dll-B is located in a convergently transcribed bigene cluster with a tandem duplicate, Ci-Dll-A. This clustered genomic arrangement is the same as those of the homologous vertebrate Dlx genes, which are also arranged in convergently transcribed bigene clusters. Sequence analysis of the C. intestinalis Dll-A-B cluster reveals a 378 bp region upstream of Ci-Dll-B, termed B1, which is highly conserved with the corresponding region from the congener Ciona savignyi. The B1 element is necessary and sufficient to drive expression of a lacZ reporter gene in a pattern mimicking the endogenous expression of Ci-Dll-B at gastrula stages. This expression pattern which is specific to the entire animal hemisphere is activated preferentially in posterior, or b-lineage, cells by a central portion of B1. Expression in anterior, or a-lineage cells, can be activated by this central portion in combination with the distal part of B1. Anterior expression can also be activated by the central part of B1 plus both the proximal part of B1 and non-conserved sequence upstream of B1. Thus, cis-regulation of early Ci-Dll-B expression is activated by a required submodule in the center of B1, driving posterior expression, which works in combination with redundant submodules that respond to differentially localized anterior factors to produce the total animal hemisphere expression pattern. Interestingly, the intergenic region of the cluster, which is important for expression of the Dlx genes in vertebrates, does not have a specific activating function in the reporter genes tested, but acts as an attenuator in combination with upstream sequences.

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