Functional Analysis of Six1 Gene Enhancer Elements During Craniofacial Development by Transgenic Zebrafish with Green Fluorescent Protein (GFP) Reporter Gene and Tol2 transposon system

he Six1 homeobox gene plays a critical roles in vertebrate organogenesis. Mice deficient for Six1 show severe defects in organs such as skeletal muscle, kidney, thymus, sensory organs and ganglia derived from cranial placodes. Mutations in human SIX1 cause Branchio-Oto-Renal syndrome(BOR), characterized by hearing loss and branchial defects. Six1 genes are hypothesized to play important roles in organgenesis and cell differentiation.
Depending on the previous study, there are a lot of conserved sequences around the six1 gene downstream and upstream,including 5 conserved sequences, namly UCR1 (upstream conserved region 1), UCR2, UCR3, UCR4 and DCR (downstream conserved region), The previous study used six1 proximal 130bp and 280bp promoter fragments, which may contain enhancer elements, to delineate the UCRs and DCR activity. These fragments were inserted upstream of the zebrafish six1 proximal promoter(111bp) in Tol-2 transposon elements. The UCR1 and UCR2 are located within the six1 2.6 kb proximal region.
I analyzed the highly conserved sequences UCRs and DCR in conjugation with six1 111bp proximal fragment by transgenic zebrafish assay. 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, otic vesicle and neuromasts).These
results revealed enhancers which can influence six1 gene expression.
Taken together, these results suggested that these conserved sequences contain enhancers which can influence six1 gene expression.