The inner cell mass (ICM) of mammalian blastocysts consists of pluripotent epiblast and hypoblast lineages, which develop into embryonic and extraembryonic tissues, respectively. We conducted a chemical screen for regulators of epiblast identity in bovine Day 8 blastocysts. From the morula stage onward, in vitro fertilized embryos were cultured in the presence of cell-permeable small molecules targeting nine principal signaling pathway components, including TGFbeta1, BMP, EGF, VEGF, PDGF, FGF, cAMP, PI3K, and JAK signals. Using 1) blastocyst quality (by morphological grading), 2) cell numbers (by differential stain), and 3) epiblast (FGF4, NANOG) and hypoblast (PDGFRa, SOX17) marker gene expression (by quantitative PCR), we identified positive and negative regulators of ICM development and pluripotency. TGFbeta1, BMP, and cAMP and combined VEGF/PDGF/FGF signals did not affect blastocyst development while PI3K was important for ICM growth but did not alter lineage-specific gene expression. Stimulating cAMP specifically increased NANOG expression, while combined VEGF/PDGF/FGF inhibition up-regulated epiblast and hypoblast markers. The strongest effects were observed by suppressing JAK1/2 signaling with AZD1480. This treatment interfered with ICM formation, but trophectoderm cell numbers and markers (CDX2, KTR8) were not altered. JAK inhibition repressed both epiblast and hypoblast transcripts as well as naive pluripotency-related genes (KLF4, TFCP2L1) and the JAK substrate STAT3. We found that tyrosine (Y) 705-phosphorylated STAT3 (pSTAT3(Y705)) was restricted to ICM nuclei, where it colocalized with SOX2 and NANOG. JAK inhibition abolished this ICM-exclusive pSTAT3(Y705) signal and strongly reduced the number of SOX2-positive nuclei. In conclusion, JAK/STAT3 activation is required for bovine ICM formation and acquisition of naive pluripotency markers.

AIM: To identify molecular markers shared across South African esophageal squamous cell carcinoma (ESCC) cell lines using cytogenetics, fluorescence in situ hybridization (FISH) and single nucleotide polymorphism (SNP) array copy number analysis. METHODS: We used conventional cytogenetics, FISH, and multicolor FISH to characterize the chromosomal rearrangements of five ESCC cell lines established in South Africa. The whole genome copy number profile was established from 250K SNP arrays, and data was analyzed with the CNAT 4.0 and GISTIC software. RESULTS: We detected common translocation breakpoints involving chromosomes 1p11-12 and 3p11.2, the latter correlated with the deletion, or interruption of the EPHA3 gene. The most significant amplifications involved the following chromosomal regions and genes: 11q13.3 (CCND1, FGF3, FGF4, FGF19, MYEOV), 8q24.21(C-MYC, FAM84B), 11q22.1-q22.3 (BIRC2, BIRC3), 5p15.2 (CTNND2), 3q11.2-q12.2 (MINA) and 18p11.32 (TYMS, YES1). The significant deletions included 1p31.2-p31.1 (CTH, GADD45 alpha, DIRAS3), 2q22.1 (LRP1B), 3p12.1-p14.2 (FHIT), 4q22.1-q32.1 (CASP6, SMAD1), 8p23.2-q11.1 (BNIP3L) and 18q21.1-q21.2 (SMAD4, DCC). The 3p11.2 translocation breakpoint was shared across four cell lines, supporting a role for genes involved at this site, in particular, the EPHA3 gene which has previously been reported to be deleted in ESCC. CONCLUSION: The finding that a significant number of genes that were amplified (FGF3, FGF4, FGF19, CCND1 and C-MYC) or deleted (SFRP2 gene) are involved in the Wnt and fibroblast growth factor signaling pathways, suggests that these pathways may be activated in these cell lines. (C) 2011 Baishideng. All rights reserved.