The Insider Secrets With YES1 Unearthed

Immunohistochemical staining of E13.5 lenses showed a marked increase in cells that stained positive for phosphorylated histone H2AX (��-H2AX). Positivity for ��-H2AX was particularly acute in newly differentiating fiber cells at the equatorial PR 171 plane of the TKO lenses, whereas ��-H2AX positivity was high in both TKO and control lenses during late stages of fiber cell differentiation ( Fig.?4A). DNA double-strand breaks in terminally differentiating fiber cells within the lens cortex are presumably a result of normal nuclear degeneration that must occur for maturation of the translucent fibers. Apoptotic cell death, as measured by TUNEL and cleaved caspase-3 immunofluorescence, was apparent in the epithelium and fiber compartments of E13.5 TKO lenses but not in control lenses ( Fig. 4B, C, and Fig. S5C). At E16.5, apoptosis was most pronounced near the equatorial transition zone of E16.5 TKO lenses, consistent with the distribution of ��-H2AX-positive cells ( Fig.?3D). As a result, the anterior perimeter of E17.5 TKO lens sections contained 36% fewer epithelial cells than control lenses (p?=?0.0002; Fig. S5D). From these observations, YES1 we conclude that postnatal architectural collapse of TKO lenses is accompanied by massive breakdown of genetic material and initiation of an apoptotic program. In order to identify the molecular pathways impacted by loss of E2F1-3 in AZD9291 vivo, we compared global gene expression profiles in control (E2f2?/?) and TKO lenses at two developmental stages, E17.5 and P0, which precede the physical breakdown of the mutant lens. We implemented unsupervised class comparison analyses of all probesets on the array to identify genes differentially expressed between lenses. We also used an unbiased approach similar to Gene Set Enrichment Analysis ( Mootha et al., 2003) to identify a priori defined groups of genes that were significantly differentially expressed. Expression changes observed among samples were attributable to two variables: developmental time (E17.5 vs. P0) and genotype (control vs. TKO). Comparison of control lenses at the two developmental stages identified 408 genes that were downregulated and 174 that were upregulated in P0 lenses (> 1.5 fold and p?