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David C. Beebe, Jie Huang, Ying Liu, Alina Oltean, Benjamin A. Filas, Larry A. Taber; The Mechanism Of "Retinal Placode" Formation And Its Importance For Optic Cup Invagination. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1187.
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Anophthalmia and microphthalmia result from failure of interactions between the optic vesicle and lens-forming head ectoderm. We noted that the distal optic vesicle, the future optic cup, thickens in mirror symmetry with the the lens placode. Our studies of lens placode formation suggested a model of "retinal placode" and optic cup formation.
Wild type, Pax6 ectoderm-specific conditional knockout (Pax6ECKO), and fibronectin (Fn1) ERTam-Cre knockout mouse embryos were analyzed. Cell morphology, number, volume and proliferation (BrdU or EdU labeling ) were assessed during lens and retinal placode formation. Extracellular matrix (ECM) was stained with the PAS reaction and Alcian Blue. Genes regulated by Pax6 were identified by laser dissection of wild type and Pax6ECKO ectoderm, amplification with the NuGEN Pico kit and Illumina Mouse6 microarrays.
Pax6 is required in the ectoderm for lens placode formation. Removal of the early lens placode or deletion of Pax6 in the ectoderm prevented the invagination of the optic vesicle to form the optic cup. We noted that, during the formation of the lens placode, the distal region of the optic vesicle thickened in mirror-symmetry with the lens placode, forming what we term the "retinal placode." After the optic vesicle contacts the ectoderm, an abundant ECM accumulates and the area of contact between the tissues becomes fixed. Deletion of Pax6 in the ectoderm decreased the expression of several genes encoding components of the ECM and markedly decreased the ECM between the optic vesicle and ectoderm. The contact area between these tissues increased, rather than being constrained, and the lens and retinal placodes failed to form. In Pax6ECKO eyes, cell proliferation in the (wild type) distal optic vesicle cells decreased. When the ectoderm was heterozygous for Pax6, the lens and retinal placodes were of intermediate thickness. Fibronectin is required for proper assembly of and adhesion to the ECM. Deletion of Fn1 at E8.5 increased the contact area between the optic vesicle and the ectoderm, prevented formation of the lens and retinal placodes and prevented invagination of the optic vesicle to form the optic cup.
Adhesion to the ECM between optic vesicle and ectoderm and continued cell proliferation in these tissues results in the formation of the lens and retinal placodes. Pax6 is important in the ectoderm for production of this ECM and for promoting proliferation in the retinal placode. In our model, cell proliferation in the retinal placode drives the invagination of the optic cup.
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