Effects of Ciona Intestinalis Distal-Less-B Nonexpression and Overexpression on Phenotype and Downstream Targets

Abstract

Ciona intestinalis can serve as a useful model for developmental studies in the chordate lineage due to its basal position in the chordate phylogeny. It shows a simplified chordate body plan during its development, during which important genetic pathways are conserved with vertebrates, and its developmental gene regulation can be manipulated through the insertion of transgenic DNA by electroporation. The Distalless (Dll) genes are a family of homeobox genes in chordates that are homologous to the single Dll gene of other metazoan animal groups and code for developmental factors that play a role in determining multiple developmental cell fates. These include broadly conserved roles in appendage development and sensory functions of the central nervous system, as well as more novel roles such as differentiation of the epidermis in chordates. In C. intestinalis, Dll-B transcripts are expressed throughout the prospective epidermis during gastrulation. To study the role of Ci-Dll-B in development, I have produced a transgenic dominant negative form of Ci-Dll-B by making use of the Drosophila engrailed repressor domain (EnR). I then examined its effects on development. Embryos electroporated with this construct showed defects in adhesion of cells in the epidermis. Whole mount in situ hybridization analysis of known Ci-Dll-B downstream targets showed changes in gene expression only in certain targets, suggesting a degree of redundancy in the regulation of the epidermal development program. Phenotypic analysis and immunofluorescent staining of epidermal markers suggest that Ci-Dll-B has a role in the regulation of cell adhesion or differentiation, since Ci-Dll-B knock-down alters the expression pattern of collagen and laminin. I also attempted to identify Ci-Dll-B gene targets through suppression subtractive hybridization, but was unsuccessful. These results are consistent with earlier reports that Dll genes could have a role inducing final differentiation in the epidermis. This work characterizing a key gene in epidermal development may have implications for epidermal development in other chordates, such as mammals.