Background Compared to the emerging embryonic stem cell (ESC) gene network, little is known about the dynamic gene network that directs reprogramming in the early embryo. We hypothesized that Oct4, an ESC pluripotency regulator that is also highly expressed at the 1- to 2-cell stages in embryos, may be a critical regulator of the earliest gene network in the embryo. Methodology/Principal Findings Using antisense morpholino oligonucleotide (MO)-mediated gene knockdown, we show that Oct4 is required for development prior to the blastocyst stage. Specifically, Oct4 has a novel and critical role in regulating genes that encode transcriptional and post-transcriptional regulators as early as the 2-cell stage. Our data suggest that the key function of Oct4 may be to switch the developmental program from one that is predominantly regulated by post-transcriptional control to one that depends on the transcriptional network. Further, we propose to rank candidate genes quantitatively based on the inter-embryo variation in their differential expression in response to Oct4 knockdown. Of over 30 genes analyzed according to this proposed paradigm, Rest and Mta2, both of which have established pluripotency functions in ESCs, were found to be the most tightly regulated by Oct4 at the 2-cell stage. Conclusions/Significance We show that the Oct4-regulated gene set at the 1- to 2-cell stages of early embryo development is large and distinct from its established network in ESCs. Further, our experimental approach can be applied to dissect the gene regulatory network of Oct4 and other pluripotency regulators to deconstruct the dynamic developmental program in the early embryo.
References
[1]
Ralston A, Rossant J (2008) Cdx2 acts downstream of cell polarization to cell-autonomously promote trophectoderm fate in the early mouse embryo. Dev Biol 313: 614–629.
[2]
Barratt CL, St John JC, Afnan M (2004) Clinical challenges in providing embryos for stem-cell initiatives. Lancet 364: 115–118.
[3]
Hochedlinger K, Jaenisch R (2006) Nuclear reprogramming and pluripotency. Nature 441: 1061–1067.
[4]
Park IH, Zhao R, West JA, Yabuuchi A, Huo H, et al. (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451: 141–146.
[5]
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131: 861–872.
[6]
Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, et al. (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448: 318–324.
[7]
Hamatani T, Carter MG, Sharov AA, Ko MS (2004) Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell 6: 117–131.
[8]
Wang QT, Piotrowska K, Ciemerych MA, Milenkovic L, Scott MP, et al. (2004) A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo. Dev Cell 6: 133–144.
[9]
Zeng F, Schultz RM (2005) RNA transcript profiling during zygotic gene activation in the preimplantation mouse embryo. Dev Biol 283: 40–57.
[10]
Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, et al. (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95: 379–391.
[11]
Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24: 372–376.
[12]
Palmieri SL, Peter W, Hess H, Scholer HR (1994) Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev Biol 166: 259–267.
[13]
Scholer HR, Ruppert S, Suzuki N, Chowdhury K, Gruss P (1990) New type of POU domain in germ line-specific protein Oct-4. Nature 344: 435–439.
[14]
Haraguchi S, Saga Y, Naito K, Inoue H, Seto A (2004) Specific gene silencing in the pre-implantation stage mouse embryo by an siRNA expression vector system. Mol Reprod Dev 68: 17–24.
[15]
Kim MH, Yuan X, Okumura S, Ishikawa F (2002) Successful inactivation of endogenous Oct-3/4 and c-mos genes in mouse preimplantation embryos and oocytes using short interfering RNAs. Biochem Biophys Res Commun 296: 1372–1377.
[16]
Hara KT, Oda S, Naito K, Nagata M, Schultz RM, et al. (2005) Cyclin A2-CDK2 regulates embryonic gene activation in 1-cell mouse embryos. Dev Biol 286: 102–113.
[17]
Gore AV, Maegawa S, Cheong A, Gilligan PC, Weinberg ES, et al. (2005) The zebrafish dorsal axis is apparent at the four-cell stage. Nature 438: 1030–1035.
[18]
Imai KS, Levine M, Satoh N, Satou Y (2006) Regulatory blueprint for a chordate embryo. Science 312: 1183–1187.
[19]
Sumanas S, Larson JD (2002) Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics? Brief Funct Genomic Proteomic 1: 239–256.
[20]
Yamada L, Shoguchi E, Wada S, Kobayashi K, Mochizuki Y, et al. (2003) Morpholino-based gene knockdown screen of novel genes with developmental function in Ciona intestinalis. Development 130: 6485–6495.
[21]
Stefanovic S, Puceat M (2007) Oct-3/4: not just a gatekeeper of pluripotency for embryonic stem cell, a cell fate instructor through a gene dosage effect. Cell Cycle 6: 8–10.
[22]
Zhou Q, Chipperfield H, Melton DA, Wong WH (2007) A gene regulatory network in mouse embryonic stem cells. Proc Natl Acad Sci U S A 104: 16438–16443.
[23]
Masutani M, Sonenberg N, Yokoyama S, Imataka H (2007) Reconstitution reveals the functional core of mammalian eIF3. Embo J 26: 3373–3383.
[24]
Amano H, Itakura K, Maruyama M, Ichisaka T, Nakagawa M, et al. (2006) Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5). BMC Dev Biol 6: 11.
[25]
Kim SK, Suh MR, Yoon HS, Lee JB, Oh SK, et al. (2005) Identification of developmental pluripotency associated 5 expression in human pluripotent stem cells. Stem Cells 23: 458–462.
[26]
Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, et al. (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453: 539–543.
[27]
Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, et al. (2007) Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 25: 500–510.
[28]
Chew JL, Loh YH, Zhang W, Chen X, Tam WL, et al. (2005) Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells. Mol Cell Biol 25: 6031–6046.
[29]
Loh YH, Wu Q, Chew JL, Vega VB, Zhang W, et al. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat Genet 38: 431–440.
[30]
Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, et al. (2005) Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280: 24731–24737.
[31]
Bengtsson M, Stahlberg A, Rorsman P, Kubista M (2005) Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. Genome Res 15: 1388–1392.
[32]
Chang HH, Hemberg M, Barahona M, Ingber DE, Huang S (2008) Transcriptome-wide noise controls lineage choice in mammalian progenitor cells. Nature 453: 544–547.
[33]
Warren L, Bryder D, Weissman IL, Quake SR (2006) Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc Natl Acad Sci U S A 103: 17807–17812.
[34]
Singh SK, Kagalwala MN, Parker-Thornburg J, Adams H, Majumder S (2008) REST maintains self-renewal and pluripotency of embryonic stem cells. Nature 453: 223–227.
[35]
Liang J, Wan M, Zhang Y, Gu P, Xin H, et al. (2008) Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells. Nat Cell Biol 10: 731–739.
[36]
Yuan H, Corbi N, Basilico C, Dailey L (1995) Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3. Genes Dev 9: 2635–2645.
[37]
Jiang J, Chan YS, Loh YH, Cai J, Tong GQ, et al. (2008) A core Klf circuitry regulates self-renewal of embryonic stem cells. Nat Cell Biol 10: 353–360.
