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P.H. Mathers, V.A. Voronina, C. Wilson, S.V. Kozlov, M. Lewandoski; The Rx Homeobox Gene Is Required For Both Neural Retina And Lens Placode Induction . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2249.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the functional role of the mammalian Rx homeobox gene during retinal development and in doing so, help to understand the molecular program that directs retinal and lens formation. Methods: A conditional deletion allele of the mouse Rx gene was generated by ES cell targeting and shown to convert to a Rx null allele upon exposure to Cre recombinase. We crossed this allele to a mouse line expressing Cre under the control of the Foxg1 promoter, effectively eliminating Rx protein activity only in the developing optic vesicle from embryonic day (E) 9 onward. Rx–deleted embryos were analyzed morphologically, immunocytologically, and molecularly to evaluate the defects caused by Rx inactivation during retinal development. Co–transfection experiments were performed with human RX protein and the BMP–4 promoter in Cos7 cells. Results: Deletion of Rx after optic vesicle formation, but before the formation of the optic cup, leads to an absence of neural retinal cell specification, as evidenced by the lack of Chx10 and CyclinD1 expression in mutant optic vesicles. In addition, the lens placode fails to form, with concomitant lack of Sox2 and Pax6 expression in the overlying surface ectoderm. Growth factors involved in the induction of ocular tissue, such as BMP–4, FGF–8, and FGF–15, are also reduced in the mutant optic vesicle. In co–transfection experiments, Rx causes a 6–fold increase in BMP–4 promoter activity, suggesting a mechanism by which Rx may promote lens placode formation. The mutant optic vesicles continue to develop into RPE, with activation of Mitf, Otx2, and Trp–2 at E9.5 and pigment deposition by E11.5, suggesting terminal differentiation. Conclusions: We previously showed that Rx activity is necessary for the formation of the optic vesicle. Here we demonstrate that Rx is also crucial at later developmental stages for the specification of neural retinal cell fate and for the induction of the lens placode. The ability of Rx to stimulate BMP–4 expression provides a mechanism for lens placode induction that is dependent on neural retinal formation. In the absence of Rx, the distal optic vesicle takes on an RPE–specific fate, suggesting that Rx may actively inhibit RPE fate during normal neural retinal development, and therefore, inactivation of Rx may be required for normal RPE formation. These findings highlight the importance of the Rx gene in early ocular developmental patterning and retinal cell specification.
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