Abstract
Purpose::
The homeodomain transcription factor PITX2 is responsible for Axenfeld-Rieger syndrome in humans, an autosomal dominant disorder associated with anterior segment defects, glaucoma, dental and umbilical abnormalities. In order to identify functional elements responsible for regulation of PITX2 expression in the developing eye, we performed comparative sequence analysis and identified 14 regions of conservation between human, mice, rat, fugu and zebrafish genes. These regions were cloned into GFP-reporter plasmid containing 2.6-kb zebrafish pitx2 promoter. The created constructs were injected into 1-2 cell zebrafish embryos and the developing embryos were examined for GFP expression.
Methods::
Sequence analysis was performed using BLAST and VISTA tools. Analysis of GFP expression was performed by examination of embryos under fluorescent microscope. Transcription factor binding site analysis was performed using MatInspector software.
Results::
Out of fourteen tested regions, four were found to be associated with expression in the developing ocular tissues. One of the regions was found to target expression to the periocular mesenchyme cells in about 14% of transient transgenics (out of ~600). Three additional regions demonstrated variable ocular expression in about 5-10% of transient transgenics (out of ~400). Analysis of the identified regions for transcription factor binding sequences revealed sites for multiple proteins including members of PAX, FOX and HOX families. Expression studies involving in situ hybridization with GFP antisense riboprobe are currently ongoing and will allow detailed evaluation of ocular expression pattern controlled by the identified elements.
Conclusions::
The zebrafish pitx2 gene demonstrates expression pattern that is consistent with human and mouse genes and therefore zebrafish represents a viable system for identification of regions controlling pitx2 activation in different tissues. We have preliminary identified several ocular enhancers of pitx2 and plan to further characterize spaciotemporal expression patterns associated with these elements. Further characterization of these regions will identify upstream factors involved in PITX2 pathway.
Keywords: anterior segment • in situ hybridization • transcription factors