The regulatory mechanisms and functions of Pax6 isoforms in eye development
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Pax6, a member of the paired-family transcription factors, exhibits restricted expression and essential functions to control the development and maintenance of the mammalian eye. In mammals, three different isoforms are known to be involved in diverse functions of Pax6. The goal of this study was to understand global regulatory mechanisms that control expression of different Pax6 isoforms during oculogenesis. Although previous studies have provided a wealth of information about regulatory elements located 3', within, and 5' of Pax6 transcription unit, genetic evidences from mice and human indicate that these elements act coordinately. The mechanism by which this happens to achieve tightly controlled Pax6 expression is not clear. To study Pax6 regulation and isoform expression, I developed a set of BAC transgene reporters that serve as a useful tool to study mechanism(s) by which the 3' Downstream Regulatory Region(DRR) exerts its master regulatory function to coordinate a global regulatory network. Using this transgenic mouse, I showed that the 3' region between HV break point and Elp4 exon7 is sufficient to drive reporter expression that is comparable to endogenous Pax6 in the eye. Phylogenetic foot printing and an in vitro reporter assay revealed that several evolutionarily conserved regions within DRR are capable of inducing reporter expression in a tissue specific manner. Subsequent in vivo assays using transgenic mouse showed that removal of CR2, one of the highly conserved region within the DRR, can abolish transgene expression in the entire embryo. Together, this evidence strongly argues that the Pax6 DRR is required for correct Pax6 expression in the eye. In addition to a complex regulatory mechanism, diverse functions of Pax6 isoforms add more complexity in understanding the activity of Pax6 during oculogenesis. Three isoforms including Pax6, Pax6 (5a), and Pax6 PD have been identified in mammals. Here I demonstrate that Pax6 PD is predominantly expressed in the mammalian eye and can cause microphthalmia when it is over-expressed. These results suggest that Pax6 PD has a role during eye development. Genetic analysis suggests that Pax6 PD is acting possibly as dominant negative factor against full length Pax6. Although our detailed analysis suggests that Pax6 PD is acting on signaling pathway to control lens development, what type of signaling pathway is involved in this cascade is yet to be studied.