The functional role of the Meis/Prep-binding elements in Pax6 locus during pancreas and eye development

Pax6 is an essential transcription factor for lens, lacrimal gland and pancreas development. Previous transgenic analyses have identified several Pax6 regulatory elements, but their functional significance and binding factors remain largely unknown. In this study, we generated two genomic truncations to delete three elements that were previously shown to bind to the Meis/Prep family homeoproteins. One 3.1 kb deletion (Pax6(Delta DP/Delta DP)) removed two putative pancreatic enhancers and a previously identified ectodermal enhancer, while a 450 bp sub-deletion (Pax64(Delta PE/Delta PE)) eliminated only the promoter-proximal pancreatic enhancer. Immunohistochemistry and quantitative RT-PCR showed that the Pax(Delta PE/Delta PE) pancreata had a significant decrease in Pax6, glucagon, and insulin expression, while no further reductions were observed in the Pax(Delta DP/Delta DP)mice, indicating that only the 450 bp region is required for pancreatic development. In contrast, Pax(Delta DP/Delta DP), but not Pax6(Delta PE/Delta PE) mice, developed stunted lacrimal gland and lens hypoplasia which was significantly more severe than that reported when only the ectodermal enhancer was deleted. This result suggested that the ectodermal enhancer must cooperate with its neighboring sequences to regulate the Pax6 ectodermal expression. Finally, we generated conditional knockouts of Prep1 in the lens and pancreas, but surprisingly, did not observe any developmental defects. Together, these results provide functional evidence for the independent and synergistic roles of the Pax6 upstream enhancers, and they suggest the potential redundancy of Meis/Prep protein in Pax6 regulation. (C) 2012 Elsevier Inc. All rights reserved.