REVIEW ARTICLE
Signaling pathways and preimplantation development
of mammalian embryos
Yong Zhang
1
, Zhaojuan Yang
1
and Ji Wu
1,2
1 School of Life Science and Biotechnology, Shanghai Jiao Tong University, China
2 Key Laboratory of Cell Differentiation and Apoptosis of Ministry of Education of China, Shanghai Jiao Tong University, China
An embryo is a stage in the development of plants,
invertebrate and vertebrate animals. Embryonic devel-
opment is a key event in the organism and is under
rigorous control. Preimplantation growth is one of the
early embryonic development processes, from a single-
cell 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 com-
plex and are regulated by various cell-signaling path-
ways. Each signal-transduction pathway is primarily
responsible for one or several related biological pro-
cesses, 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 net-
works, 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 pre-
implantation 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 path-
ways that regulate these events [1–6]. Several signal-
transduction pathways have been shown to be involved
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
(Received 17 April 2007, revised 12 June
2007, accepted 5 July 2007)
doi:10.1111/j.1742-4658.2007.05980.x
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 establish-
ment of cellular contacts. These events are regulated by multiple signal-
transduction 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.
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-P
3
, phosphatidylinositol-3,4,5-triphosphate; PtdIns-4,5-P
2
, phosphatidylinositol-
4,5-diphosphate; STAT, signal transducer and activator of transcription; TGF, transforming growth factor; Wnt, Wingless.
FEBS Journal 274 (2007) 4349–4359 ª2007 The Authors Journal compilation ª2007 FEBS 4349
in this process, including mitogen-activated pro-
tein kinase (MAPK), phosphatidylinositol 3-kinase
(PtdIns3K) ⁄Akt, Wingless (Wnt) ⁄b-catenin, Notch,
bone morphogenetic protein (BMP)–Smad, transform-
ing growth factor (TGF)-b, Hedgehog, and Janus-acti-
vated 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 expres-
sion pattern of each signaling pathway (Fig. 1).
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 1 pathway [16–19]. All are highly
conserved throughout eukaryotic systems. Preimplanta-
tion 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 (ribo-
somal 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)
Zygote 2-Cell 4-Cell 8-Cell
Oocyte 16-Cell 32-Cell Blastocyst
Marula Stage
Wnt Wnt-4
Wnt-3a
Notch Notch-1, Notch-2, Jag-1, Jag-2, DII-3, Rbpshu, Dtx-2
BMPR-II
Notch-3, DII-1, Dtx-1
BMP ActR-1
BMRP-1B
PtdIns3K Akt
80Kda and 110Kda subunit of PtdIns3K
BMRP-1A
Notch-4, DII-4
MAPK
Raf1
MEK-1, MEK-2, MEK-5, MAPK/ERK1
SOS1, GAB1
MAPK/ERK2
MAPK/ERK5, RSK3
STAT5
JAK-STAT
Fig. 1. 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.
Signaling pathways in preimplantation development Y. Zhang et al.
4350 FEBS Journal 274 (2007) 4349–4359 ª2007 The Authors Journal compilation ª2007 FEBS
Raf1,Rafb,MEK (MAPK or ERK kinase)-1,-2,-5,
and MAPK ⁄ERK1 are detected in unfertilized eggs
and blastocysts. Transcripts and the protein localiza-
tion of p38-regulated and -activated kinase, p38
MAPK, MK2 and hsp25 have also been observed
throughout murine preimplantation embryo develop-
ment. These proteins have been detected in tropho-
blasts 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 detec-
ted 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 ⁄com-
paction 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 2 (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 cyto-
plasm 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].
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 lipopro-
tein 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 core-
ceptors [29–31]. There are several intracellular compo-
nents of the Wnt signal-transduction pathway. The
canonical Wnt pathway (b-catenin pathway) is the
best characterized, and includes a series of phospho-
rylation 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-trans-
duction pathway activated by other Wnts (Wnt4, -5a,
and -11) is regulated by noncanonical pathways
involving the intracellular signaling cascade of Ca
2+
or JNK.
b-Catenin is present in the eggs and early embryos
of some vertebrate species; it is the first essential com-
ponent of the signal-transduction pathway that leads
to formation of the endogenous dorsal–ventral axis.
Studies of immunoreactivity of total b-catenin in pre-
implantation embryos, from the two-cell stage to the
blastocyst stage, have shown that b-catenin accumu-
lates 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 dor-
sal 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 dorsal-
determining activity. The nonphosphorylated isoform
of b-catenin accumulates in response to Wnt signaling
[35]. Recent studies have shown that b-catenin is neces-
sary 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
Y. Zhang et al. Signaling pathways in preimplantation development
FEBS Journal 274 (2007) 4349–4359 ª2007 The Authors Journal compilation ª2007 FEBS 4351
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 [Dishev-
elled (Dsh), adenomatous polyposis coli (APC), axin],
as well as nuclear effectors (e.g. homologs of Dro-
sophila 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 embry-
onic 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- ⁄8-
cell 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 cellu-
lar 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 expres-
sion 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 endo-
derm 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.
The PtdIns3K/Akt signal transduction pathway
PtdIns3Ks consist of three types of enzymes, but they
can produce lipid secondary messengers by phosphory-
lation of plasma-membrane phosphoinositides at the
3¢OH group of the inositol ring [40]. Class 1
PtdIns3Ks include a catalytic subunit (110 kDa, p110)
and an adaptor ⁄regulatory subunit. They can be sub-
grouped 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 sub-
unit to autophosphorylated tyrosine kinase receptors,
or to nonreceptor tyrosine kinases in the cytoplasm,
such as the Src family kinases or JAK kinases. Activa-
tion of class 1B kinases occurs in the binding of
the catalytic subunit to heterotrimeric GTP-binding
proteins or G proteins. Activated PtdIns3Ks pre-
ferentially phosphorylate phosphatidylinositol-4,5-di-
phosphate (PtdIns-4,5-P
2
)in vivo, to produce
phosphatidylinositol 3,4,5 triphosphate (PtdIns-3,4,5-
P
3
) [42]. In turn, the production of PtdIns-3,4,5-P
3
is
regulated by the phosphates phosphatase and tensin
homolog deleted on chromosome 10 which catalyzes
the dephosphorylation of PtdIns-3,4,5-P
3
to PtdIns-
4,5-P
2
[44,45]. A wide variety of signal-transduction
proteins, including Akt, interact with PtdIns3K-gener-
ated phosphorylated phosphoinositides via lipid-bind-
ing 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 repro-
ductive process, including in murine preimplantation
embryos. PtdIns3K signaling is required to suppress
apoptosis in preimplantation embryos, because pro-
grammed 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 pre-
implantation embryo development. These proteins are
Signaling pathways in preimplantation development Y. Zhang et al.
4352 FEBS Journal 274 (2007) 4349–4359 ª2007 The Authors Journal compilation ª2007 FEBS
localized predominantly at the cell surface at the one-
cell stage through to the morula stage. Both PtdIns3K
and Akt exhibit an apical staining pattern in trophec-
toderm cells at the blastocyst stage. Phosphorylated
Akt was determined throughout murine preimplanta-
tion embryo development, and its presence at the
plasma membrane is a reflection of its activation sta-
tus. 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 implanta-
tion. Taken together, these data demonstrate the pres-
ence 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 con-
served, and it is essential for cell fate decisions in many
different tissues in multicellular organisms. The data
show that the Notch signaling pathway blocks differ-
entiation towards a primary differentiation fate in a
cell, rather than directing the cell to a second, alterna-
tive 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 sig-
nals from the cell surface are conveyed to the nuclear
transcription machinery. The Notch receptor is synthe-
sized in the endoplasmic reticulum, undergoes matura-
tion 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 prototyp-
ically 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 regu-
late 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 pre-
implantation embryo development. These include
Notch1–4, Jagged1–2 (Jag1–2), Delta-like1 (Dll-1),
Rbpsuh and Deltex1 (Dtx1). Notch1,-2,Jag1–2,Dll-3,
Rbpsuh and Dtx2 transcripts are synthesized in unfer-
tilized 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-bsuperfamily of
growth factors, which plays a critical role in develop-
mental and regenerative processes. BMPs were origi-
nally 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 nema-
todes.
The BMPs fulfill their signaling function by binding
to a heterodimeric complex of two transmembrane
receptors, type 1 and type 2, which have serine–threo-
nine kinase activity [57–59]. When ligand binding is
required for type 1 receptor activation, the kinase
activity of the type 2 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 1 and type 2 recep-
tors are present. The type 2 receptor transphosphory-
lates the type 1 receptor by ligand binding. The type 1
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
Y. Zhang et al. Signaling pathways in preimplantation development
FEBS Journal 274 (2007) 4349–4359 ª2007 The Authors Journal compilation ª2007 FEBS 4353
