REVIEW ARTICLE Signaling pathways and preimplantation development of mammalian embryos Yong Zhang1, Zhaojuan Yang1 and Ji Wu1,2 School of Life Science and Biotechnology, Shanghai Jiao Tong University, China Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education of China, Shanghai Jiao Tong University, China Keywords development; preimplantation embryo; signaling pathways; signaling transduction network; stage-specific expression pattern Correspondence J Wu, School of Life Science and Biotechnology, Shanghai Jiao Tong University, no 800, Dongchuan Road, Minhang District, Shanghai, 200240, China Fax: 86 21 3420 4051 Tel: 86 21 3420 4933 E-mail: jiwu@sjtu.edu.cn The mammalian preimplantation embryo is a critical and unique stage in embryonic development This stage includes a series of crucial events: the transition from oocyte to embryo, the first cell divisions, and the establishment of cellular contacts These events are regulated by multiple signaltransduction pathways In this article we describe patterns of stage-specific expression in several signal-transduction pathways and try to give a profile of the signaling transduction network in preimplantation development of mammalian embryo (Received 17 April 2007, revised 12 June 2007, accepted July 2007) doi:10.1111/j.1742-4658.2007.05980.x An embryo is a stage in the development of plants, invertebrate and vertebrate animals Embryonic development is a key event in the organism and is under rigorous control Preimplantation growth is one of the early embryonic development processes, from a singlecell zygote, to a morula, to a blastocyst Furthermore, preimplantation development is critical in establishing a viable mammalian pregnancy During this period, the zygote initiates its first cell division and the first lineage cell begins to differentiate into the inner cell mass and the trophectoderm These processes are complex and are regulated by various cell-signaling pathways Each signal-transduction pathway is primarily responsible for one or several related biological processes, such as cell division, growth, differentiation, migration, apoptosis, transformation, immune response and polarity By combining several functions, such as cross-linking and other interactions, these pathways form a complicated signaling network Successful embryo development requires functional signaling networks, and any disruption to these networks may lead to abnormal development or fatal disease Although there is a reasonably sound understanding of the specific events associated with mammalian preimplantation embryo development, including activation of the zygotic genome, development of the anterior– posterior axis, compaction, and blastocyst formation, little is known about the intracellular signaling pathways that regulate these events [1–6] Several signaltransduction pathways have been shown to be involved Abbreviations BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; ERK, extracellular signal-regulated protein kinase; JAK, Janus-activated kinase; JNK, Jun N-terminal kinase; LRP, lipoprotein receptor-related protein; MAPK, mitogen-activated protein kinase; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns-3,4,5-P3, phosphatidylinositol-3,4,5-triphosphate; PtdIns-4,5-P2, phosphatidylinositol4,5-diphosphate; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; Wnt, Wingless FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS 4349 Signaling pathways in preimplantation development Y Zhang et al in this process, including mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PtdIns3K) ⁄ Akt, Wingless (Wnt) ⁄ b-catenin, Notch, bone morphogenetic protein (BMP)–Smad, transforming growth factor (TGF)-b, Hedgehog, and Janus-activated kinase (JAK) ⁄ signal transducer and activator of transcription (STAT) signaling pathways Moreover, these signaling pathways play a central role in the embryonic development processes of other vertebrate and invertebrate animals [7–13] Detailed mechanisms of these signaling pathways are now better understood, and most have been reviewed previously [14,15] This article describes the patterns of stage-specific expression of several signal-transduction pathways and the signaling transduction network in the preimplantation development of the mammalian embryo Stage-specific expression pattern of several signal-transduction pathways in the preimplantation embryo We review the existing evidence for the presence of each signaling pathway during preimplantation embryo development, and summarize the stage-specific expression pattern of each signaling pathway (Fig 1) Oocyte Zygote 2-Cell MAPK pathways MAPK pathways transmit signals from ligand–receptor interactions and convert them into a variety of cellular responses, ranging from apoptosis to immune responses, as well as proliferation, differentiation, growth and embryonic development The MAPK superfamily of proteins can be subdivided into four separate signaling cascades: extracellular signal-regulated protein kinase (ERK), Jun N-terminal kinase (JNK), p38 and ERK5 or big MAP kinase pathway [16–19] All are highly conserved throughout eukaryotic systems Preimplantation embryos utilize MAPK pathways to relay signals from the external environment in order to prepare appropriate responses and adaptations to a changing milieu It is therefore important to figure out the roles of MAPK pathways during preimplantation embryo development Using RT-PCR and immunostaining, 10 transcripts of MAPK signaling pathway members have been detected in unfertilized eggs and ⁄ or zygotes These genes include SOS1 (Son of sevenless 1), RSK1 (ribosomal S6 kinase 1) and MAPK ⁄ ERK2, the expression of which is lowest in unfertilized eggs; RSK3 and MAPK ⁄ ERK5 are expressed at extremely low levels in blastocysts; and GAB1 (Grb2-associated binder 1) 4-Cell 8-Cell Marula Stage 16-Cell 32-Cell Blastocyst Wnt-4 Wnt Wnt-3a Notch Notch-1, Notch-2, Jag-1, Jag-2, DII-3, Rbpshu, Dtx-2 Notch-4, DII-4 Notch-3, DII-1, Dtx-1 BMPR-II BMP ActR-1 BMRP-1B BMRP-1A Akt PtdIns3K 80Kda and 110Kda subunit of PtdIns3K Raf1 MEK-1, MEK-2, MEK-5, MAPK/ERK1 MAPK SOS1, GAB1 MAPK/ERK2 JAK-STAT MAPK/ERK5, RSK3 STAT5 Fig Stage-specific expression of several signal-transduction pathways in the preimplantation development of the mammalian embryo Red, Wnt signaling pathway; blue, Notch signaling pathway; green, BMP signaling pathway; yellow, PtdIns3K signaling pathway; gray, JAK-STAT signaling pathway 4350 FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Zhang et al Raf1, Rafb, MEK (MAPK or ERK kinase)-1, -2, -5, and MAPK ⁄ ERK1 are detected in unfertilized eggs and blastocysts Transcripts and the protein localization of p38-regulated and -activated kinase, p38 MAPK, MK2 and hsp25 have also been observed throughout murine preimplantation embryo development These proteins have been detected in trophoblasts on embryonic day (E)3.5, when they mediate mitogenic fibroblast growth factor signals from the embryo or colony-stimulating factor-1 signals from the uterus [8,20] The phosphorylation state and position of the phosphoproteins in the cells suggest that they might function in mediating mitogenic signals Raf1 is expressed abundantly in unfertilized eggs and throughout preimplantation embryo development Expression of MEK-1, -2, -5, and MAPK ⁄ ERK1 is lowest in unfertilized eggs, and gradually increases throughout the blastocyst stage SOS1 and GAB1 are also expressed at a low level in unfertilized eggs, but at the beginning of the two-cell stage expression abruptly increases and continues throughout preimplantation embryo development MAPK ⁄ ERK2 could not be detected in unfertilized eggs but was detected at the two-cell stage; it also increased throughout preimplantation embryo development This is in accordance with activation of the zygotic genome MAPK ⁄ ERK5 and RSK3 mRNA was abundantly and increasingly detected in unfertilized eggs up to the eight-cell ⁄ compaction stage, but was not detectable at the blastocyst stage [21,22] According to some experimental results, the JNK or p38 MAPK pathway is required for development from the 8–16-cell stage to the blastocyst stage, and p38 MAPK is a regulator of filamentous actin during preimplantation embryo development [22] Active JNK and p38 MAPK pathways are required for cavity formation during mouse preimplantation embryo development, because inhibition of such signaling pathways, excluding the ERK pathway, inhibits cavity formation [23] Maternal RNA of fibroblast growth factor receptor substrate (FRS2alpha), GAB1, growth factor receptor-bound protein 2(GRB2), SOS1, Raf-B and Raf1 genes may delay the presence of the lethal phenotype of null mutations These genes are considered to be postimplantation lethal knockouts of the genes for lipophilic MAPK pathway proteins They are all expressed at the protein level in the cytoplasm or in the cell membrane of E3.5 embryos, at a time when the first known mitogenic intercellular communication takes place It is still not clear why the lethality of these null mutants arises after implantation [24] Signaling pathways in preimplantation development Wnt signaling pathway The Wnt signaling pathway consists of 19 Wnt genes encoding secreted proteins [25], 10 Wnt receptors composed of Frizzled genes, and low-density lipoprotein receptor-related protein (LRP) 5–6 as coreceptors participating in signal transmission [26] Antagonists of Wnt signals include two categories [27] Fzb (frizzled-b) with its four homologs forms the secreted frizzle-related protein (Sfrp) family, which can block activation of the receptor through binding to Wnt proteins directly [28] Dickkopf-1 (Dkk1) and its three homologs can bind to and inactivate the LRP coreceptors [29–31] There are several intracellular components of the Wnt signal-transduction pathway The canonical Wnt pathway (b-catenin pathway) is the best characterized, and includes a series of phosphorylation reactions that eventually activate target genes in the nucleus Signal pathways triggered by Wnts (Wnt1, -2, -2b, -3, -3a, -6, -7b, -8a and -8b) belong to this phosphorylation mechanism The signal-transduction pathway activated by other Wnts (Wnt4, -5a, and -11) is regulated by noncanonical pathways involving the intracellular signaling cascade of Ca2+ or JNK b-Catenin is present in the eggs and early embryos of some vertebrate species; it is the first essential component of the signal-transduction pathway that leads to formation of the endogenous dorsal–ventral axis Studies of immunoreactivity of total b-catenin in preimplantation embryos, from the two-cell stage to the blastocyst stage, have shown that b-catenin accumulates on the cell surface rather than in the nucleus [32– 34] It has been shown that endogenous b-catenin accumulates in the prospective dorsal side of the embryo as early as the first division, and continues to accumulate in the cytoplasm of all animal and vegetal blastomeres, to a greater extent on the prospective dorsal side than on the ventral side, during the early cleavage stages By the 16- and 32-cell stages, b-catenin accumulates in the dorsal but not the ventral nuclei when zygotic transcription begins The pattern of b-catenin accumulation after cortical rotation thus reflects the distribution of the transplantable dorsaldetermining activity The nonphosphorylated isoform of b-catenin accumulates in response to Wnt signaling [35] Recent studies have shown that b-catenin is necessary and sufficient for formation of the dorsal axis, and that it accumulates in cells that give rise to the dorsal side of the embryo These results indicate that the Wnt ⁄ b-catenin signaling pathway is not active in embryos until the blastocyst stage They also show that activation of the Wnt signaling pathway is sufficient to FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS 4351 Signaling pathways in preimplantation development Y Zhang et al maintain the pluripotency of embryonic stem cells, and that b-catenin is localized in the nuclei of the inner cell mass, but not trophoblast cells in the blastocyst [8,26,36] This suggests that Wnts may participate in cell determination in preimplantation embryos Recently, the expression patterns of several Wnts during preimplantation stages have been reported, and mRNAs encoding for Wnt1, -2b, -3, -3a, -4, -5a, -5b, -6, -7a, -7b, -10b and -11 have been described [7,8,34] Transcripts of Wnt3a, -6, -7b, -9a and -10b have been detected in blastocysts, and Wnt1 and -4, Sfrp1 and Dkk1 are highly expressed at this stage [37] Receptors (Fz2, frizzled-2 and Fz4, frizzled-4), intracellular signal transducers and modifiers [Dishevelled (Dsh), adenomatous polyposis coli (APC), axin], as well as nuclear effectors (e.g homologs of Drosophila arm, Tcf and groucho) are also present in blastocysts [8] Transcripts of Wnt3a are found at the 2-cell stage, decreased at the 4- ⁄ 8-cell stages, and are strongly expressed in compact 8- and 16-cell and early blastocysts The source of the Wnt3a transcripts in 2-cell embryos, i.e whether of maternal or embryonic origin, is not clear, because the major gene expression transition from the maternal to zygotic stage occurs in the late two-cell embryo [38] The onset of expression of Wnt4 is observed in the 4- ⁄ 8cell stages, and is more strongly expressed at the 8- and 16-cell and blastocyst stages Both Wnt3a and -4 transcripts have been detected in some precompact 4- ⁄ 8-cell stages, with consistent expression detected in all compact 8- and 16-cell and blastocyst stages [8] Primers specific for Wnt11 amplified the expected size product at the blastocyst stage, as well as in 10-week whole fetus libraries during human preimplantation embryo development [39] These data suggest that Wnts play a role in cell development and in cellular interactions occurring in preimplantation embryo development By analyzing the expression levels of all 19 Wnt genes and their 11 antagonists in mouse blastocysts, pregastrula, gastrula and neurula stages, new expression domains for Wnt2b and Sfrp1 have been found in the future primitive streak at the posterior side and in the anterior visceral endoderm before the initiation of gastrulation Moreover, the anterior visceral endoderm expresses three secreted Wnt antagonists (Sfrp1, Sfrp5 and Dkk1) in partially overlapping domains Notably, the predominant expression of Wnt1 and Sfrp1 in the inner cell mass, and of Wnt9a in the mural trophoblast and inner cell mass surrounding the blastocele, suggests that the Wnt signal-transduction pathway plays a novel role in preimplantation embryo development 4352 The PtdIns3K/Akt signal transduction pathway PtdIns3Ks consist of three types of enzymes, but they can produce lipid secondary messengers by phosphorylation of plasma-membrane phosphoinositides at the 3¢OH group of the inositol ring [40] Class PtdIns3Ks include a catalytic subunit (110 kDa, p110) and an adaptor ⁄ regulatory subunit They can be subgrouped into class 1A and 1B PtdIns3Ks according to their different catalytic subunits Class 1B PtdIns3Ks encompass a p110r catalytic subunit, associated with a 101 kDa (p101) adaptor subunit [40–43] Class 1A PtdIns3Ks are activated through binding of the Src homology (SH2) domain in the adaptor subunit to autophosphorylated tyrosine kinase receptors, or to nonreceptor tyrosine kinases in the cytoplasm, such as the Src family kinases or JAK kinases Activation of class 1B kinases occurs in the binding of the catalytic subunit to heterotrimeric GTP-binding proteins or G proteins Activated PtdIns3Ks preferentially phosphorylate phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2) in vivo, to produce phosphatidylinositol 3,4,5 triphosphate (PtdIns-3,4,5P3) [42] In turn, the production of PtdIns-3,4,5-P3 is regulated by the phosphates phosphatase and tensin homolog deleted on chromosome 10 which catalyzes the dephosphorylation of PtdIns-3,4,5-P3 to PtdIns4,5-P2 [44,45] A wide variety of signal-transduction proteins, including Akt, interact with PtdIns3K-generated phosphorylated phosphoinositides via lipid-binding pleckstrin homology domains [46] This facilitates recruitment of these proteins to the plasma membrane and their subsequent activation Akt, a well-known serine–threonine kinase mediator of survival signals is the best characterized downstream target of PtdIns3K It is a central player in multiple signaling pathways, and acts as a transducer of many functions initiated by growth factor receptors that activate PtdIns3K [47] The PtdIns3K ⁄ Akt signaling pathway is a major pathway that has been found to regulate cell survival downstream of activated growth-factor receptors The expression and function of this pathway have been documented during early and late stages of the reproductive process, including in murine preimplantation embryos PtdIns3K signaling is required to suppress apoptosis in preimplantation embryos, because programmed cell death is rapidly induced by inhibition of PtdIns3K with LY294002 [48] Riley et al [13] found, using confocal immunofluorescence microscopy and western blot analysis, that the p85 and p110 subunits of PtdIns3K and Akt are expressed from the one-cell stage through to the blastocyst stage of murine preimplantation embryo development These proteins are FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Zhang et al localized predominantly at the cell surface at the onecell stage through to the morula stage Both PtdIns3K and Akt exhibit an apical staining pattern in trophectoderm cells at the blastocyst stage Phosphorylated Akt was determined throughout murine preimplantation embryo development, and its presence at the plasma membrane is a reflection of its activation status Inhibition of Akt activity has significant effects on the normal physiology of the blastocyst Specifically, inhibition of this pathway results in a reduction in insulin-stimulated glucose uptake Moreover, inhibiting Akt activity can cause a significant delay in blastocyst hatching, a developmental step facilitating implantation Taken together, these data demonstrate the presence and function of the PtdIns3K ⁄ Akt pathway in mammalian preimplantation embryos These results further our knowledge of the PtdIns3K ⁄ Akt signaling pathway [13] The Notch signaling pathway The Notch signaling pathway is evolutionarily conserved, and it is essential for cell fate decisions in many different tissues in multicellular organisms The data show that the Notch signaling pathway blocks differentiation towards a primary differentiation fate in a cell, rather than directing the cell to a second, alternative differentiation program, or forcing the cell to remain in an undifferentiated state [14,49–53] Relatively few signal proteins are involved in the function of the Notch signaling pathway, in which signals from the cell surface are conveyed to the nuclear transcription machinery The Notch receptor is synthesized in the endoplasmic reticulum, undergoes maturation in the trans-Golgi network, and is transferred to the cell surface, where it interacts with ligands from neighboring cells This interaction occurs only when cells are in physical contact with each other The Notch receptor is activated by this interaction and is prototypically cleaved, releasing the Notch intracellular domain which translocates from the membrane to the nucleus, where it interacts with the CSL DNA-binding protein (CBF1 or Rbpsuh in vertebrates, suppressor of hairless in Drosophila, Lag-1 in Caenorhabditis elegans) to regulate selected target gene expression [53,54] The Notch signaling pathway is modulated by numerous accessory proteins, such as members of the Deltex family [50] Cormier et al [9] systematically examined the expression profiles of genes that directly or indirectly participate in the Notch signaling pathway in preimplantation embryo development These include Notch1–4, Jagged1–2 (Jag1–2), Delta-like1 (Dll-1), Rbpsuh and Deltex1 (Dtx1) Notch1, -2, Jag1–2, Dll-3, Signaling pathways in preimplantation development Rbpsuh and Dtx2 transcripts are synthesized in unfertilized oocytes and at later blastocyst stages; Notch4 and Dll-4 mRNAs can be detected from the two-cell stage to the hatched blastocyst stage; and Notch3, Dll-1 and Dtx1 mRNAs are found in two-cell embryos and in hatched blastocysts, but are absent or present at a low levels at the morula stage These results suggest that the Notch signaling pathway may be active during these stages [9] Using cDNA microarray technology, researchers have also found that other genes of the Notch pathway are expressed in the mouse embryo, such as homologs of Drosophila N, Delta, deltex, fringe, serrate and presenilin [8] The JAK–STAT signaling pathway The JAK–STAT5 signaling pathway plays a crucial role in the growth and differentiation of mammalian cells RT-PCR analysis shows the expression of STAT5 throughout preimplantation embryo development; inhibiting the activation of JAK might interfere with the localization of STAT5 to the nucleus, and reduce the embryo development rate, suggesting that the JAK–STAT5 signaling pathway has a key function in preimplantation embryo development [55] The BMP signaling pathway BMPs are members of the TGF-b superfamily of growth factors, which plays a critical role in developmental and regenerative processes BMPs were originally identified as regulators of bone formation in rodents [56] More than 30 BMPs have been identified to date BMPs are broadly conserved across the animal kingdom, including vertebrates, arthropods and nematodes The BMPs fulfill their signaling function by binding to a heterodimeric complex of two transmembrane receptors, type and type 2, which have serine–threonine kinase activity [57–59] When ligand binding is required for type receptor activation, the kinase activity of the type receptors is constitutive Although BMPs can bind to each of these weakly, and subsequently recruit the second subunit, optimal ligand binding is achieved when both type and type receptors are present The type receptor transphosphorylates the type receptor by ligand binding The type receptor then phosphorylates members of the Smad family of transcription factors which are subsequently translocated to the nucleus, activating the expression of target genes [60–62] At the very beginning of the preimplantation stage, embryonic polarity and spatial patterns start to FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS 4353 Signaling pathways in preimplantation development Y Zhang et al develop [10,11] BMP receptors (BMPRs) are essential for this process, and BMPs exert their function by binding to BMPRs In the preimplantation mouse embryo, large-scale cDNA analysis has been performed and has provided some insight into the phased gene expression patterns [12] Activation of the Xenopus BMP signaling pathway is coincident with the onset of zygotic transcription, but requires maternal signaling proteins Analysis of the expression profiles of several BMPRs has shown that BMPR-II mRNAs are present in the zygote, two-cell and blastocyst stages However, no BMPR-II mRNA can be detected at the four-cell and morula stages Expression of ActR-I one of the BMPR-Is, similar to BMPR-II, can be observed at the zygote, two- and four-cell, and late blastocyst stages, but not at the uncompacted or compacted morula stages BMPR-IA mRNA is detected only in blastocysts; BMPR-IB transcripts are found at all stages from the zygote to the uncompacted morula, but are absent from the compacted morula and blastocysts Because maternal gene products are degraded rapidly after the start of zygotic transcription [63], transcripts of BMPR-IB at the one- and two-cell stages are probably maternal derivations However, at the four-cell and uncompacted morula stages, the transcripts may be from the embryonic genome Therefore, one- and two-cell stage embryos may have the ability to respond to BMPs, either via an ActR-I ⁄ BMPR-II receptor complex, or by forming a BMPR-IB ⁄ BMPRII receptor complex Transcripts of BMPs have been found in preimplantation embryos However, some researchers have detected several BMP proteins, mitotic arrest-deficient proteins, and other components of the BMP signaling pathway, as well as homologs of the receptors, in blastocysts, using cDNA microarray analysis BMP6, a member of the 60A subgroup of BMPs, is expressed in diverse sites in the developing mouse embryo from preimplantation onwards [8,64] A profile of the signal transduction network in the preimplantation development of the mammalian embryo All of the above-mentioned signaling pathways in preimplantation development are essential for various cell events, such as cell proliferation, differentiation and growth, as well as apoptosis, and interactions between them have been established in many other types of cells and biological processes, suggesting that there exists a complex signal network controlling mammalian preimplantation development SOX7 protein, one of the SRY box-containing transcription factors (SOX proteins), can repress Wnt 4354 signaling by inhibiting the ability of TGF ⁄ lymphoid enhancer factor–b-catenin to transactivate a T-cell factor ⁄ lymphoid enhancer factor-dependent reporter construct [65] Dickkopf-1 (Dkk1) is another potent antagonist of Wnt signaling [29] It specifically blocks Wnt ⁄ b-catenin signaling by interacting with low-density lipoprotein receptor-related protein [66] Its expression is regulated by the Ap-1 family member c-Jun and it is activated by BMP-4 to induce apoptosis [67] Both Dkk1 and SOX7 have been identified during mouse preimplantation embryo development as direct targets of the p38 and JNK pathways [23] Inhibition of the p38 or JNK pathway leads to decreased expression of Dkk1 and SOX7 [23] Dishevelled (Dsh ⁄ Dvl) proteins are important transducers for divergent Wnt pathways that lead to different cell events: cell proliferation, apoptosis, or differentiation [68,69] Recently, this type of protein has been identified in mouse oocytes and during preimplantation embryo development, and has an important function in the regulation of cell adhesion in mouse blastocysts [70] The changes in expression of Dvl proteins are coincident with those of b-catenin and p120 catenin transcription during preimplantation embryo development, implying upregulation of Wnt signaling activity before implantation [70] Furthermore, Dvls can induce JNK MAPK signaling [71–73] The reason might be that Dvl can form Wnt-induced complexes with Rac and Rho, and Rac stimulates JNK activity independent of Rho [72,73] Rac-1 protein has been demonstrated throughout murine preimplantation embryo development and is a potential regulator of E-cadherin ⁄ catenin interactions during this development progress [74] The expression profiles of some genes that directly or indirectly participate in the Notch signaling pathway, including Notch1–4, Jag1–2, Dll-1, Rbpsuh and Dtx1, have been observed in mammalian preimplantation embryos [9] In other biological progresses, Notch signaling has been suggested to repress the activity of p38 MAPK by specifically inducing the expression of MKP-1, a member of the dual-specificity MAPK phosphatase [75] Notch negatively regulates the JNK pathway by interfering with the interaction between JNK and JNK-interacting protein (JIP1) [76] During C elegans vulval development, LIN-12 ⁄ NOTCH inactivates MAPK to specify the secondary fate through up-regulation of isoenzymes from Candida rugosa lipase (LIP1) transcription [77] Ras activation interferes with endocytic routing of LIN-12, resulting in downregulation of LIN-12 ⁄ Notch [78] Whether the interaction between the Notch and MAPK signaling pathways similarly exists during preimplantation FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Zhang et al embryo development needs to be clarified by further research Some proto-oncogenes, including c-fos, c-jun, c-haras and c-myc, have been studied in bovine preimplantation blastocysts [79,80] The c-fos, c-jun and c-ha-ras transcripts, as well as c-Fos, c-Jun and c-Myc proteins have been detected in 13–14-day-old preimplantation blastocysts [80] Wang et al investigated whether Ras mRNA is expressed in trophoblasts in E3.5 embryos [61] The PtdIns3K ⁄ Akt signaling pathway, which is a major pathway for regulating cell survival downstream of activated growth factor receptors [81], has also been demonstrated throughout murine preimplantation embryo development [13] The proto-oncogene Ras may suppress c-Myc-induced apoptosis, via activation of the PtdIns3K ⁄ Akt pathway [82] Sears et al [83] found that Ras can control Myc protein (a regulator of the cell cycle and essential for cell growth) accumulation via the PtdIns3K ⁄ Akt pathway by downregulating the kinase GSK-3 that promotes Myc degradation However, Ras regulates the accumulation of Myc activity by stabilizing the Myc protein [84], depending on the action of the Raf ⁄ ERK pathway [85] Furthermore, STAT5 has been identified throughout preimplantation embryo development using RT-PCR [55] It has been shown that STAT5 may induce cell proliferation by activating PtdIns3K, by interacting with p85 and Grb2-associated binder-2 (Gab2) [86,87] Nyga et al have found that Gab2 seems to activate ERK1 ⁄ via PtdIns3K in Ba ⁄ F3 cells that express constitutively activated STAT5 [87] TEL-JAK2 can constitutively activate the PtdIns3K signaling pathway independent of the STAT5 pathway [88] This illustrates the complex interactions among these signaling pathways during mammalian preimplantation embryo development (Fig 2), even if it is just a profile of the signaling network In different preimplantation events and different species, there should be a different regulative pattern of this transduction network For example, Wnt ⁄ b-catenin and BMP signal pathways play a key role in the polarization of frog eggs [89,90] In Xenopus, stabilization of b-catenin leads to the activation of dorsal-specific genes, while a member of the Nodal family of TGF-b signals, Squint, serves as a dorsal determinant in zebrafish embryos [91,92] However, which part of this network is essential for triggering the first cell division and which is essential for the first cell differentiation in preimplantation development? These questions need further study Moreover, much more research is required on the whole signaltransduction network in preimplantation embryo development Signaling pathways in preimplantation development Ras JAKs Notch Rac MKP-1 Raf Wnt STAT5 DvI Bmp-4 PI3K ERK p38 JNK Dkk1 Myc c-Jun AKT Cell Growth division, apoptosis differentiation SOX7 active repress directly indirectly Fig Signal network predicted during mammalian preimplantation embryo development.STAT5 may regulate preimplantation development by activating PtdIns3K Ras may suppress c-Myc-induced apoptosis by activation of the PtdIns3K ⁄ Akt pathway Ras can therefore stabilize the Myc protein, depending on the action of the Raf ⁄ ERK pathway Notch signaling represses the activity of p38 MAPK by specifically inducing the expression of MKP-1 Dvl proteins are important transducers for the Wnt ⁄ b-catenin pathway Dvls can forms Wnt-induced complexes with Rac, and Rac can induce JNK ⁄ MAPK signaling Dkk1 and SOX7 are antagonists of Wnt signaling Both are identified as direct targets of the p38 and JNK pathways Perspectives Preimplantation development is a unique and critical stage during embryo development It is not only the very beginning of a new life, but also the beginning of many biological reactions Understanding the role of signaling pathways in embryos is important for knowledge about life processes However, due to the difficulties in manipulating preimplantation embryos, progression in this field has been delayed, especially in discovering how these signaling cascades function during each particular event To date, little of our knowledge about these cascades has come from direct experimental evidence, some has come from indirect experimental observation, and much is speculation based on the cascades functions in somatic cells The fact is that we still know little about this process, and dozens of questions remain unanswered, such as which signaling pathway triggers the activation of the zygote genome? Is it exogenous or endogenous? What is the exact means of maternal signaling proteins passing on new life? Which signaling cascade participates in cavity formation during development of the blastocyst? Which cascades participate in preimplantation embryo FEBS Journal 274 (2007) 4349–4359 ª 2007 The Authors Journal compilation ª 2007 FEBS 4355 Signaling pathways in preimplantation development Y Zhang et al apoptosis, and explain why 15–50% of embryos die during preimplantation development? 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[8,64] A profile of the signal transduction network in the preimplantation development of the mammalian embryo All of the above-mentioned signaling pathways in preimplantation development are... Rac, and Rac can induce JNK ⁄ MAPK signaling Dkk1 and SOX7 are antagonists of Wnt signaling Both are identified as direct targets of the p38 and JNK pathways Perspectives Preimplantation development. .. describes the patterns of stage-specific expression of several signal-transduction pathways and the signaling transduction network in the preimplantation development of the mammalian embryo Stage-specific