Cis-regulatory organization of the Pax6 gene in the ascidian Ciona intestinalis

Abstract

The Pax6 gene has attracted intense research interest due to its apparently important role in the development of eyes and the central nervous system (CNS) in many animal groups. Pax6 is also of interest for comparative genomics since it has not been duplicated in tetrapods, making for a direct orthology between the Ciona intestinalis gene CiPax6 and Pax6 in mammals. CiPax6 has been shown to be expressed in the anterior brain, caudal nerve cord, and in parts of the brain associated with the photoreceptive ocellus. This information was extended here using in-situ hybridization, and shows that CiPax6 transcripts mark the lateral regions of the nerve cord, remarkably similar to Pax6 expression in the mouse. As a means of dissecting the cis-regulation of CiPax6 we tested 8 kb of sequence using transient reporter transgene assays. Three separate regions were found that work together to drive the overall CiPax6 expression pattern. A 211 bp sequence 2 kb upstream of the first exon was found to be a major enhancer driving expression in the sensory vesicle (the anterior portion of the ascidian brain). Other upstream sequences were shown to work with the sensory vesicle enhancer to drive expression in the remainder of the CNS. An “eye enhancer” was localized to the first intron, which controls specific expression in the central portion of the sensory vesicle, including photoreceptor cells. The fourth intron was found to repress ectopic expression of the reporter gene in middle portions of the embryonic brain. Aspects of this overall regulatory organization are similar to the organization of the Pax6 homologs in mice and Drosophila, particularly the presence of intronic elements driving expression in the eye, brain and nerve cord.