Figure 1.
Isolation of XO clones from XX and XY HESCs.
A. The frequency of X or Y chromosome loss in HESCs were estimated by FISH analysis. Shown are results for X chromosome (Green) Y chromosome (Yellow) and chromosome 17 (Blue), for H9 (XX) cells, BGO1 (XY cells) and a clone of H9 that has lost one of its X chromosomes. B. Summary of the % of XO cells analyzed by FISH. The analyzed samples were either male H13 cells passage 22 and passage 44, or female H9 cells, passage 52. In each experiment 200 cells were analyzed. C. Analysis of two polymorphic markers (DXS1106 and DXS1060) on the X chromosome that are heterozygous in H9 cells. XO cells retain only one of the markers. D. PCR products of primers that distinguish between Amelogenin X and Amelogenin Y genes. XY cells (BG01) have two products whereas XX cells (H9) have only one product. XY cells that have lost the Y chromosome amplify only one band. E. Karyotype analysis of XX and XO cells. Note that in the XO cells only one X chromosome is shown.
Figure 2.
Searching for monoallelic expression in X chromosome.
A. A scheme demonstrating the differences between the haploinsufficiency hypothesis and the imprinting hypothesis to explain the phenotype in X monosomy. Green dots represent expressed alleles and red dots represent silenced alleles. Note that according to the imprinted genes hypothesis there is no expression of the remaining allele in XO cells. B. Expression levels of several genes on the X chromosome whose expression is much higher in WT than in XO HESCs. The expression levels of STS, XIST and CXorf9 are shown in 30 d EBs and of ARSE and TBL1X in undifferentiated cells. XX – average of three microarray analyses of diploid HESCs. XO – average of two XO clones that have lost the same X chromosome. C. Searching for monoallelic expression in the candidate genes by SNP analysis at the DNA and cDNA levels.
Figure 3.
Comparison of gene expression in WT and in XO cells upon in vitro and in vivo differentiation.
Gene expression levels were determined by U133A DNA microarray. Every dot represents one probe in the DNA microarray. In each plot the X axis represents fold induction in the expression levels of WT cells over XO cells (right side of the scale) and of Xo cells over WT cells (left side of the scale). The Y axis represents the P-value for each gene. For more details on the bioinformatic analysis see Materials and Methods. A. Analysis of genes specific to the placenta. B. Analysis of genes specific to the heart, fetal lung, fetal liver, fetal brain and whole blood. EBs – in vitro differentiation of HESCs. Teratoma – in vivo differentiation of HESCs. Solid vertical lines represents>2 fold higher level of expression solid horizontal line represent P-value = 0.05.
Figure 4.
Confirmation of the DNA microarray data by qRT-PCR.
Relative expression levels of WT vs. XO cells for several genes enriched in either the placenta, or in tissues that correspond to the ectoderm, endoderm or mesoderm embryonic germ layers. The genes were analyzed by qRT-PCR. The solid line represents equal expression in WT and XO cells. * represents p value<0.05 and ** represents p value<0.01.