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Jie Huang, Ying Liu, David Beebe; Transcriptional And Post-transcriptional Regulation Of "Fiber Cell-specific" Gene Expression In The Lens Placode. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2771.
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Microarray studies on the lens placode of wild type embryos identified transcripts encoding "lens fiber cell-specific" proteins, including β/γ-crystallins, Prox1 and MIP. The purpose of this study is to confirm that these genes are being transcribed, but not translated, at lens placode stage and to determine the extent to which their transcription is regulated by BMP signaling, Pax6 and CBP/p300.
Lens placodes of wild type or ectoderm-specific Pax6, Bmpr1a/Acvr1, or Crebbp/Ep300 knockout embryos were collected by laser microdissection on E9.5 or E10.25, total RNA was isolated, reverse transcribed and amplified using the NuGEN Pico whole transcriptome amplification kit. Microarray analysis was performed using Illumina Mouse6 whole genome microarrays. The transcription and translation of selected lens fiber cell-specific proteins were examined at lens placode or lens vesicle stages by quantitive real time PCR (qRT-PCR) after whole-transcriptome amplification using the NuGEN WT-Ovation kit, in situ hybridization, western blotting and immunostaining.
Abundant transcripts encoding beta/gamma-crystallins, Prox1 and MIP were detected in lens placodes by in situ hybridization and quantitative real time PCR. In spite of the abundance of Prox1 mRNA in the placode at E9.5, Prox1 protein was not detected until E10.5 in the prospective fiber cells at the posterior of the lens pit. Definitive antibody staining for MIP was not detected until after E10.5. Antibody staining and western blotting did not detect γ-crystallin protein in lens placodes or whole heads at E9.5. qRT-PCR showed that transcripts encoding Prox1, MIP and selected β/γ-crystallins were decreased >100-fold in the presumptive lens-forming ectoderm when Pax6 or Bmpr1a and Acvr1, the two type I BMP receptors expressed in the lens-forming ectoderm, were deleted using LeCre. Deletion of the two histone acetyltransferases that are commonly recruited by active transcription factors, Crebbp and Ep300 (CBP/p300) greatly decreased the levels of each of the "fiber cell-specific" transcripts studied.
Lens placode cells transcribe many genes that encode fiber cell-specific proteins, like β/γ-crystallins, Prox1 and MIP, but do not translate them until later in lens developmet. All of the fiber cell-specific transcripts studied were regulated by Pax6, BMP signaling and by CBP/p300. We suggest that BMP receptors may regulate Pax6 activity by promoting the phosphorylation of Pax6 to recruit CBP/p300 and transcribe genes that are later translated in fiber cells.
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