To date, the only rigorous analysis of deletion of all macrohistones in a vertebrate animal has been conducted in zebrafish, which are not complicated by the presence of multiple isoforms during embryogenesis. Interestingly, mH2A-deficient zebrafish exhibit profound developmental defects, suggesting that a genetic double knock out of H2afy and H2afy2 in mice might exhibit a similar phenotype. If so, this will provide good evidence that the developing embryo is a far more sensitive crucible for the detection of developmental defects than cell culture-based ESC models. In addition, mH2As are found in vertebrates that do not have a female XX, male XY sex chromosome system, also suggesting that mH2A incorporation into the mammalian Xi is a relatively late and derived event in evolutionary terms. Semi-quantitative RT-PCR and quantitative real-time RTPCR assays were performed as previously described, with modifications. qRT-PCR analyses were performed using iTaq Fast SYBR Green Supermix with ROX, following the manufacturer��s recommended conditions. Data was analyzed using the comparative cycle threshold method, as described. Primers used in the study are listed in the Supplemental Information. For Western blots, 30 mg of total protein was resolved on 5-15% gradient polyacrylamide gels, transferred to Hybond PVDF membranes, and probed with an anti-mH2A1 antibody. Detection was performed using an HRP-conjugated secondary antibody and ECL reagent according to the manufacturer��s protocol. Protein band intensities were quantified using Carestream MI software. Neuroectoderm-directed differentiation of ESC lines was performed by supplementing the culture media with 100nM alltrans retionoic acid in the absence of LIF. Total RNA was isolated from cell cultures 10 days after the initiation of differentiation and subjected to RT-PCR. Analyses of Abmole Company Vorinostat growth rates were performed for 50892-23-4 undifferentiated ESCs and over the course of 10 days of atRA differentiation. Proliferation of undifferentiated ESCs was determined over two consecutive passages, using a hemocytometer counting chamber with an improved Neubauer ruling. For the atRA-differentiated samples, cells were plated at low density on gelatinized 100mm cell culture plates in duplicate cultures, and the total cell number was determined at days 5 and 10 after the initiation of differentiation.