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W. Liu, O. V. Lagutin, M. Mende, A. Streit, G. Oliver; Functional Analysis of Six3 in Mammalian Eye Development. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1307. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous results in our laboratory and other laboratories show that Six3 is involved in mammalian forebrain and eye development. Recent analysis of Six3-null mutants in our laboratory demonstrated that Six3 repression of Wnt1 is essential for mammalian forebrain regionalization. In Six3-null mutants the eye development is never initiated. This study is aimed to investigate the roles of Six3 in eye development.
LoxP/Cre conditional knockout approach in mouse was used. For this purpose, a floxed Six3 allele was generated by ES cell targeting. For lens, Pax6 Le-Cre (lens lineage Cre line), CAGG-Cre-ER (tamoxifen inducible ubiquitous Cre line) were used. Expression analysis of the key lens markers either by immunohistochemistry or in situ hybridization was performed. Further biochemical analysis including electrophoretic mobility shift assay (EMSA), luciferase reporter assay and ChIP assay were carried out. Electroporation of chick embryos in ovo was also executed.For neural retina progenitor specification, Six3-Cre line (active in neural retina lineage from around E8.5) was used.
Conditional deletion of mouse Six3 in the presumptive lens ectoderm (PLE) disrupted lens formation. In the most severe cases, lens induction and specification were defective, and the lens placode and lens were absent. In Six3-mutant embryos, Pax6 was strongly downregulated, and Sox2 was absent in the lens preplacodal ectoderm. The lens phenotypes of the mutants were highly dependent on the temporal Six3 deletion: early Six3 deletion caused the failure of lens specification, but late Six3 deletion did not. Using ChIP, EMSA, and luciferase reporter assays, we determined that Six3 activates Pax6 and Sox2 expression. Misexpression of mouse Six3 into chick embryos promoted the ectopic expansion of the ectodermal Pax6 expression domain.Conditional deletion of mouse Six3 in neural retina lineage by using Six3-Cre line resulted in severe defects in neural retina progenitor specification. In the most severe cases, the specification of neural retina progenitors totally failed and the cells in whole optic vesicle adopted the fate as retina pigment epithelial cells.
Six3 directly activates Pax6 and probably also Sox2 in the PLE and regulates cell autonomously the earliest stages of mammalian lens induction. Six3 is positioned at the top of the regulatory pathway leading to lens formation. At meantime, Six3 is required for neural retina progenitor specification.
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