Abstract
Purpose: :
PITX3 is a homeodomain transcription factor involved in a variety of human phenotypes such as cataracts, anterior segment dysgenesis and microphthalmia. PITX3 demonstrates a distinct temporal and spatial pattern of expression during development. At the same time, factors and regions that mediate this unique pattern have not yet been identified. The purpose of this study is to discover regulatory elements responsible for PITX3 expression and to identify upstream factors.
Methods: :
Human PITX3 genomic sequence was compared to sequences from other species and several conserved regions have been identified between human and mouse sequences and one with zebrafish gene. These sequences have been cloned into GFP–reporter vector and tested for transcriptional activity by injection into 1–2 cell zebrafish embryos. Then these transient transgenic fish embryos were observed for GFP fluorescence.
Results: :
The sequence element that demonstrated conservation across vertebrates (from human to zebrafish) directed expression in distinct regions during brain development that were consistent with endogenous expression of pitx3. Other constructs did not show any expression in 12–120 hours post fertilization embryos. A 3–kb promoter region of human PITX3 and 2.1 kb promoter region of zebrafish pitx3 demonstrated strong promoter activity in tissue culture but failed to direct any specific expression in transient transgenics indicating to requirement of enhancer elements. Examination of other, non–conserved, regions of PITX3 and neighboring genes is now in progress.
Conclusions: :
A complex network of specific enhancers regulates expression of PITX3 during embryonic development. A sequence element that is conserved across vertebrates was identified and found to mediate expression during brain development. Screening of other PITX3 regions is currently in progress.
Keywords: anterior segment • gene/expression • transcription factors