Embryonic stem cell (ESC) pluripotency regulators have novel and critical roles in pre-blastocyst development

Abstract

OBJECTIVE: Gene regulation and reprogramming during the maternal-embryonic transition in the early embryo have remained elusive despite our understanding of the ESC gene network. Based on their transcript abundance, we hypothesized that the ESC pluripotency regulators, Sall4, Oct4, and Sox2, may be required for pre-blastocyst development, and their precise functions would help us to understand mechanisms controlling early embryo development.DESIGN: Gene-specific, antisense morpholino oligonucleotides (MOs) were injected into the cytoplasm at the 2PN stage to block translation and induce gene knockdown (KD) of each of Sall4Oct4, and Sox2, via steric hindrance. Maternal and embryonic transcripts are thus simultaneously targeted in a way that is not possible by conventional knockout mouse models.MATERIALS AND METHODS: We compared the developmental effect of each gene KD to its uninjected and mismatch controls in≥ 3 independent experiments by Student's t-test. Protein expression was tested by immunocytochemistry. Global gene expression profiles were obtained from KD and control embryos at the 2-cell stage as per protocol (PicoPure® kit, Molecular Devices; Pico-ovation®, Nugen; GeneChip® Mouse Genome 430 2.0 Array, Affymetrix).Table 1Developmental Effects of Gene KnockdownGene KnockdownStage of Arrest% Arrest MO% Arrest Mismatch% Arrest Uninjectedp-value (MO vs mismatch)Sall41- to 4-cell76.2 ± 10.94.2 ± 4.26.7 ± 6.70.013Oct41- to multicell89.6 ± 5.818.9 ± 8.100.003Sox2multicell, morula78.5 ± 11.55.6 ± 2.900.019 Open table in a new tab CONCLUSIONS: By combining MO-mediated gene KD and global gene expression profiling, we discovered that Sall4, Oct4, and Sox2, have novel and critical roles in normal embryo development. More broadly, we propose to apply our strategy to dissect the dynamic gene network that directs early embryo development. OBJECTIVE: Gene regulation and reprogramming during the maternal-embryonic transition in the early embryo have remained elusive despite our understanding of the ESC gene network. Based on their transcript abundance, we hypothesized that the ESC pluripotency regulators, Sall4, Oct4, and Sox2, may be required for pre-blastocyst development, and their precise functions would help us to understand mechanisms controlling early embryo development. DESIGN: Gene-specific, antisense morpholino oligonucleotides (MOs) were injected into the cytoplasm at the 2PN stage to block translation and induce gene knockdown (KD) of each of Sall4Oct4, and Sox2, via steric hindrance. Maternal and embryonic transcripts are thus simultaneously targeted in a way that is not possible by conventional knockout mouse models. MATERIALS AND METHODS: We compared the developmental effect of each gene KD to its uninjected and mismatch controls in≥ 3 independent experiments by Student's t-test. Protein expression was tested by immunocytochemistry. Global gene expression profiles were obtained from KD and control embryos at the 2-cell stage as per protocol (PicoPure® kit, Molecular Devices; Pico-ovation®, Nugen; GeneChip® Mouse Genome 430 2.0 Array, Affymetrix). CONCLUSIONS: By combining MO-mediated gene KD and global gene expression profiling, we discovered that Sall4, Oct4, and Sox2, have novel and critical roles in normal embryo development. More broadly, we propose to apply our strategy to dissect the dynamic gene network that directs early embryo development.