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David C. Beebe, Jie Huang, Ying Liu; Pax6 and BMP Signaling Control The Differentiation Of The Ocular Surface Epithelia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):4737.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the function of Pax6 and BMP signaling during the development of the ocular surface epithelia.
Homozygous deletion of Pax6 or the two BMP receptors, Acvr1 (Alk2) and Bmpr1a (Alk3) was accomplished by mating the floxed mice to LeCre, which is expressed in prospective lens and all ocular surface epithelia cells. The resulting phenotypes were analyzed by standard histological methods, immunofluorescence, immunohistochemistry and in situ hybridization. Transcripts expressed in wild type or conditional knockout (CKO) tissues were isolated by laser microdissection, reverse transcribed and amplified using the NuGEN Pico kit, and quantified by Illumina BeadChip microarrays and RT-PCR analysis.
Microarray analysis of Pax6CKO lens placodes (E9.5; 9 arrays) revealed significantly decreased expression of 772 transcripts, compared to wild type littermates. Of these, 187 were also significantly decreased in BMP receptor CKO embryos (E9.5; 6 arrays). Among these co-regulated transcripts were genes known to be important for subsequent lens formation, including Maf, AP2α, Pitx2, Prox1 and Mab21l1. In contrast, several transcripts that were decreased in Pax6CKO placodes increased in BMP receptor knockouts, including the cornea-preferred gene, Lypd2 and Edaradd, which is required for eyelid closure. Microarray analysis of prospective cornea, conjunctiva and eyelid epithelia at E12.5 revealed that the transcription factor, Otx1 was expressed in the corneal and conjunctival epithelia, while Barx2 and Sox9 were selectively expressed in the prospective conjunctival epithelium. Pax6CKO surface epithelial cells showed little evidence of normal differentiation. At E12.5, Otx1 mRNA was not detected in the prospective cornea and conjunctiva and Sox9 mRNA or protein was not detected in the prospective conjunctival epithelium. Barx2 was the only region-specific marker detected in Pax6CKO eyes. At E15.5 the eyelids did not fuse, lacrimal glands did not form and cornea-specific keratin-12 (K12) expression was replaced by the epidermal keratin, K10. Skin with hair follicles formed in place of the corneal epithelium. BMP receptor CKO eyes showed normal expression of corneal and conjunctival markers, including K12, but lacrimal glands did not form and, as we reported previously, eyelids did not close.
At the lens placode stage, BMP signaling promotes lens formation, but suppresses the expression of cornea-preferred transcripts. Pax6 is required for differentiation of all ocular surface epithelia and their derivatives. At later stages, BMP signaling mediates eyelid closure and lacrimal gland formation.
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