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.

Expression of the Distalless-B gene in Ciona is regulated by a pan-ectodermal enhancer module

“…1) was subcloned in pBlueSTAR-1 by Cre -mediated excision. The reporter gene cassette from TV13 (Irvine et al, 2008), was inserted into a BglII site in the fifth exon of Ci-Dll-B using the InFusion® Dry-Down PCR Cloning Kit (Clontech) to complete the CiDB-A construct.…”

“…Transgenes were delivered to single-celled embryos by electroporation, and lacZ reporter signal detected, as previously described (Irvine et al, 2008). Embryos were reared to mid to late gastrula stage and the number of animal hemisphere quadrants with detectable ß-galactosidase staining counted for each normally developing embryo.…”

Study of Cis-regulatory Elements in the Ascidian Ciona intestinalis

“…No more conservation was seen in alignments of amphioxus with human, zebrafish, or lamprey DNA. (CREs: [44-47, 29]).…”

Optimized conditions for transgenesis of the ascidian Ciona using square wave electroporation

“…The construct CiP6-2.5UI4 was made by inserting a 2.5-kb DNA fragment upstream of the C. intestinalis Pax6 gene, including the basal promoter, into the AscI and NotI sites of the lacZ expression vector pTV13, and a 3.3-kb fragment from C. intestinalis Pax6 intron 4 into the RsrII and PacI sites of TV13 (Irvine et al 2008). …”