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

• Published in: Developmental biology 2012-03, Vol.363 (1), p.320-329
• Main Authors: Carbe, Christian, Hertzler-Schaefer, Kristina, Zhang, Xin
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites - genetics; Binding Sites - physiology; Blotting, Western; Embryo, Mammalian - embryology; Embryo, Mammalian - metabolism; Enhancer Elements, Genetic - genetics; Enhancer Elements, Genetic - physiology; Enhancers; Eye; Eye - embryology; Eye - metabolism; Eye lens; Eye Proteins - genetics; Eye Proteins - metabolism; Female; Gene deletion; Gene Expression Regulation, Developmental; genetically modified organisms; genomics; Glucagon; homeodomain proteins; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Hypoplasia; Immunohistochemistry; In Situ Hybridization; Insulin; lacrimal apparatus; Lacrimal Apparatus - embryology; Lacrimal Apparatus - metabolism; Lacrimal gland; Lens; Lens, Crystalline - embryology; Lens, Crystalline - metabolism; loci; Male; Meis; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Myeloid Ecotropic Viral Integration Site 1 Protein; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Paired Box Transcription Factors - genetics; Paired Box Transcription Factors - metabolism; Pancreas; Pancreas - embryology; Pancreas - metabolism; Pax6; Pax6 protein; PAX6 Transcription Factor; pknox1; Polymerase chain reaction; Prep1; Protein Binding; Regulatory sequences; Repressor Proteins - genetics; Repressor Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transcription factors
• ISSN: 0012-1606; 1095-564X
• DOI: 10.1016/j.ydbio.2011.12.038

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 3.1kb deletion (Pax6∆DP/∆DP) removed two putative pancreatic enhancers and a previously identified ectodermal enhancer, while a 450bp sub-deletion (Pax6∆PE/∆PE) eliminated only the promoter-proximal pancreatic enhancer. Immunohistochemistry and quantitative RT-PCR showed that the Pax6∆PE/∆PE pancreata had a significant decrease in Pax6, glucagon, and insulin expression, while no further reductions were observed in the Pax6∆DP/∆DP mice, indicating that only the 450bp region is required for pancreatic development. In contrast, Pax6∆DP/∆DP, but not Pax6∆PE/∆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. ► Genomic deletion analysis identifies an essential pancreatic enhancer for Pax6. ► The Pax6 ectodermal enhancer cooperates with its adjacent sequences to regulate Pax6 lens expression. ► Prep1 is dispensable for the maintenance of Pax6 lens and pancreas expression.