Functional Analysis of Six1.1 Gene Enhancer Elements During Craniofacial Development by Transgenic Zebrafish with Green Fluorescent Protein (GFP) Reporter Gene
Date Issued
2008
Date
2008
Author(s)
Lu, Chih-Kai
Abstract
The homeobox-containing Six genes, homologues to Drosophila sine oculis (so) gene, are widely expressed in many tissues during vertebrate embryogenesis. Identified in many species, Six family genes are hypothesized to play important roles in morphogenesis, organgenesis and cell differentiation.o identify the cis-regulatory elements of zebrafish six1.1, we ligated the genomic DNA promoter region from +31 to -2631 to the egfp gene and injected this construct into zebrafish embryos. The resulting reporter-EGFP expression showed spatial restrictions similar to those of six1.1 mRNA detected by in situ hybridization (i.e., mainly in somites, ventral otic vesicle and neuromasts). This transient transgenic assay suggested that the proximal 2.6 kb promoter contains some tissue-specific cis-regulatory elements. ne stable transgenic line (F1 generation) injected with six1.1 proximal 2.6 kb promoter was obtained and the EGFP reporter expression can be observed mainly in ventral otic vesicle and neuromasts.ot Matrix and UCSC-bioinformatics analyses of the Six1 genes in many species revealed five conserved sequences, designated as UCR1 (upstream conserved region 1), UCR2, UCR3, UCR4 and DCR (downstream conserved region), in the vicinity of zebrafish six1.1 and six4.1 genes, that are candidate regulatory elements. These fragments inserted upstream of the zebrafish six1 proximal promoter or the thymidine kinase (TK) basal promoter with egfp reporter gene were analyzed in zebrafish embryos. The reporter EGFP expression was observed in somites, otic vesicle, neuromasts and branchial arch. aken together, these results suggested that 2.6 kb upstream region of zebrafish six1.1 has regulatory function. And five conserved sequences harboring enhancer elements that may have enhancer redundancy and participate in the regulatory mechanism of zebrafish six1.1 and six4.1 expression patterns.
Subjects
zebrafish
conserved sequence
neuromast
somite
otic vesicle
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