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J. Huang, O. Shaham, R. Ashery-Padan, D. C. Beebe; The Role of Pax6 in Lens Placode Formation. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1219.
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© ARVO (1962-2015); The Authors (2016-present)
Tissue-specific deletion of Pax6 (Pax6CKO ) in the surface ectoderm of mouse embryos at E9 causes failure of lens formation. This study sought to determine why the lens does not form in Pax6CKO ectoderm, to identify downstream targets of Pax6 and to test possible mechanisms of lens placode formation.
Wild type, heterozygous and Pax6CKO embryos were examined at the lens placode stage. The thickness of lens placode was measured and cell proliferation and cell death were characterized by BrdU and TUNEL index. Cell density was determined by morphometry. To identify genes regulated by Pax6, we collected Pax6CKO and wild type surface ectoderm by laser microdissection on E9.5. Three samples of WT and Pax6CKO RNA were reverse transcribed, the cDNA amplified using a NuGEN DNA amplification kit and the amplified material used to probe Illumina microarrays. Selected microarray data was verified by in situ hybridization. The function of selected downstream targets of Pax6 is being determined by conditional knockout in the surface ectoderm.
The lens placode did not form (thicken), and the cell number in the prospective placode was significantly decreased in Pax6CKO embryos, but this was not associated with decreased proliferation or increased cell death. Placodes were of intermediate thickness in Pax6CKO/+ ectoderm. The microarray data showed that transcripts encoding known targets of Pax6, Prox1, Mab21l1 and Maf, were decreased or absent. Of the 508 transcripts significantly decreased in Pax6CKO lens ectoderm, several encoding proteins that contribute to assembly of the extracellular matrix, such as fibronectin, versican and hyaluronan synthase, were strongly affected. Conditional knockout of fibronectin (Fn1) from the surface ectoderm resulted in small eyes, with no evidence of a lens at birth. We are collecting embryos from earlier stages and will report the effect of Fn1 deletion on lens placode formation.
It is likely that Pax6CKO surface ectoderm does not form a lens because the lens placode never forms. Failure of placode formation does not result from abnormalities in cell proliferation or death, but is associated with excessive spreading of the ectoderm. Analysis of microarray data and deletion of Fn1 raise the possibility that decreased extracellular matrix assembly might reduce adhesion between the the optic vesicle and the surface ectoderm, thus impairing placode formation, as predicted by the "restricted growth model" of Zwaan and Hendrix (Am Zool 13 1039).
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