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Sheldon Rowan, Trevor Siggers, Yingzi Yue, Martha L. Bulyk, Richard L. Maas; Signaling Downstream of the Notch Pathway and Prep1 Transcription Factor Controls Pax6 Spatiotemporal Dynamics. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5883.
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© ARVO (1962-2015); The Authors (2016-present)
Pax6 is essential for the development of the vertebrate lens and regulates genes essential for lens fiber cell differentiation. The gene regulatory network that controls the precise temporal changes in Pax6 expression levels and its eventual restriction to the lens epithelium is just beginning to be defined. We undertook a bifurcated approach to identify lens regulatory molecules via two different genetic models that perturb lens development.
Notch signaling was activated in the lens using Pax6 regulatory sequences that direct GFPCre expression. Lens placodes from control and activated-Notch embryos were isolated using laser-capture microscopy and RNA was extracted and hybridized to a DNA microarray to identify Notch regulated genes. Prep1 putative target genes were computationally identified on the basis of phylogenetic conservation of pairs of lower-affinity binding sites, as is the mode for activation of Pax6. Target genes were further evaluated for their expression in activated-Notch and Prep1 mutant embryos.
Gene sets from both analyses were highly enriched for transcription regulators and lens regulatory molecules. Filtering of the data revealed regulators that were affected by both genetic perturbations. Two such factors, Bach2 and Sox1, exhibited dynamic expression patterns, eventually becoming highly expressed in primary fiber cells, concomitant with the down-regulation of Pax6. Biochemical and reporter data suggest that both factors bind to and repress Pax6 regulatory sequences.
We identify here a set of genes that coordinate lens development and differentiation via convergence on Pax6 regulatory elements. In particular, we identify two different transcriptional regulators that are initially activated by Prep1 in an affinity-dependent fashion in the lens, but become restricted to fiber cells via Notch signaling, where they then repress Pax6 to allow fiber cell differentiation. Our work defines the composition and function of a novel Type II incoherent feedback loop, which appears to be a hallmark of Pax6 regulation in the lens.
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