Abstract
Purpose: :
The similar lens phenotypes observed in mice with mutations in either Pax6 or AP–2α transcription factors implies that these genes may work together to regulate specific signalling cascades during lens development. In this study we examine the overlapping expression patterns of AP–2α and Pax6 in the developing lens and further examine their potential co–operative roles through the creation of double heterozygote mice.
Methods: :
Co–localization of Pax6 and AP–2α expression patterns were performed on sections of mouse embryos at embryonic days 13.5, 16.5 and 18.5 using immunofluorescence. To test potential co–operativity between these two proteins, mice heterozygous for Pax6 were bred with those heterozygous for AP–2α to produce double heterozygous AP–2α+/K.0/Pax6+/lacZ mice. Hematoxylin and Eosin (H&E) stained sections of post–natal stages, from the double heterozygotes, were then compared to that of single heterozygote and wild–type mice.
Results: :
Examination of all three developmental stages demonstrated distinct co–localization of AP–2α and Pax6 protein in the anterior lens epithelium. However, Pax6 expression continued further into the transitional zone of the lens, than that of AP–2α, in the region where epithelial cells differentiate into fiber cells. Investigation of H&E stained sections of post–natal mutant mice showed that while the single Pax6 heterozygote mice (n=8) exhibited remnants of a corneal–lenticular adhesion, the lens and cornea were physically separated. In contrast, the double heterozygotes (n=19) displayed distinct lens stalks that joined the lens and cornea, protruding towards the surface of the cornea.
Conclusions: :
Co–localization of AP–2α and Pax6 is mainly observed in the proliferating central lens epithelium. The more severe phenotype observed in the double heterozygote mice as compared to the Pax6 single heterozygotes suggests that Pax6 and AP–2α may work synergistically to control lens development. Further investigation into the co–operative role that these regulators may have in controlling downstream expression will be carried out using proteomic and genomic approaches.
Keywords: development • transcription factors • immunohistochemistry