Transgenic Analysis of the Iridocorneal Angle-Specific Enhancer of the Zebrafish gelsolin-like 1 Gene

Abstract

Purpose:: Our long-term goal is to develop genetic tools such as gene promoters, enhancers and transcription factors to study the genetic mechanisms and to model ocular diseases in zebrafish. We previously reported that the zebrafish gelsolin-like 1 (gsnl1) gene is expressed in the eye and sub-population of the head mesenchyme during early development. Since its expression in the connective tissue at the iridocorneal angle is strong and specific, gsnl1 is a good tissue-specific regulatory tool for studying the anterior segment development. The purpose of this study is to isolate the cis-regulatory elements of gsnl1 that are responsible for this tissue-specific expression.

Methods:: A 6.4-kb genomic fragment upstream of the translational start site (ATG) of gsl1 was isolated and cloned into a Tol2-based green fluorescent protein (GFP) expression vector. This reporter plasmid was used to establish stable transgenic lines and to isolate tissue-specific regulatory elements by deletion and point mutation analyses. Embryos at 1-2 cell stages were co-injected with the expression plasmids and Tol2 transposase mRNA and analyzed for their expression of GFP in the target tissues. GFP-positive embryos were scored to examine if an expression vector contained the tissue-specific enhancer.

Results:: The stable transgenic embryos expressed GFP in the iridocorneal angle (IRA), corneal epithelium (CEP) and sub-population of the head mesenchyme at the nose (HMN). This pattern was identical to the endogenous gsnl1 gene expression. The reporter assays with multiple deletion constructs showed that each tissue-specific expression was regulated by an independent cis-regulatory element. The tissue-specific enhancers for IRA, CEP and HMN located 3871-3812, 3786-3687 and 3686-3532 base pairs (bp) upstream of the ATG, respectively. Further analysis with point mutations in the 60-bp IRA enhancer region suggested that an AT-rich region located between the positions 3836-3822 was required for the IRA-specific expression. This sequence matches the target consensus of POU class III (Brn2) and MADS type II (Mef2) transcription factors.

Conclusions:: We have successfully isolated the tissue-specific cis-regulatory elements of the zebrafish gsnl1 gene for IRA, CEP and HMN. These enhancers will be useful to generate transgenic animals for anterior eye disease models. The IRA-specific expression may be regulated by POU and/or MADS transcription factors. Further functional analyses of these genes such as knock-down experiments will enhance our understanding of genetic regulation in the anterior segment of the eye.

Keywords: anterior segment • gene/expression • transgenics/knock-outs