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Instructions for Assembling the Early Mammalian Embryo

Article  in  Developmental Cell · June 2018

DOI: 10.1016/j.devcel.2018.05.013

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Developmental Cell

Perspective

Instructions for Assembling

the Early Mammalian Embryo
Melanie D. White,1 Jennifer Zenker,1 Stephanie Bissiere,1 and Nicolas Plachta1,2,*
1Instituteof Molecular and Cell Biology, A*STAR, 61 Biopolis Drive, Singapore 138673
2Department of Biochemistry, NUS, Singapore
*Correspondence: plachtan@imcb.a-star.edu.sg
https://doi.org/10.1016/j.devcel.2018.05.013

The preimplantation mouse embryo is a simple self-contained system, making it an excellent model to
discover how mammalian cells function in real time and in vivo. Work over the last decade has revealed
some key morphogenetic mechanisms that drive early development, yielding rudimentary instructions for
the generation of a mammalian embryo. Here, we review the instructions revealed thus far, and then discuss
remaining challenges to discover upstream factors controlling cell fate determination, test the role of mech-
anisms based on biological noise, and take advantage of recent technological developments to advance the
spatial and temporal resolution of our current understanding.

The Preimplantation Embryo Is a Simple, Self-Contained
System
A central question in developmental biology is how complex
forms and functions arise from the simple starting point of a sin-
gle cell. During morphogenesis, spatiotemporal order and
patterning spontaneously emerge through local interactions at
cellular and subcellular scales (Davies, 2017; Sasai, 2013). This
process of self-organization occurs in the absence of external
control and results in the emergence of higher order properties
that cannot be accounted for by the properties of the individual
components. In this way, large numbers of cells can organize
into complex tissues by obeying simple rules of local interaction
without reference to a global pattern. Such self-organization has
the advantage that it is relatively robust to error, highly scalable,
and retains the capacity for self-repair.
The preimplantation mouse embryo is an excellent system for
studying how simple cells organize dynamically into increasingly
complex structures. The mouse embryo executes a develop-
mental program from zygote to hatched blastocyst without the
requirement for external input and both cellular and subcellular
elements of the embryo interact dynamically to generate emer-
gent patterned structures. These elements consist of physical
properties such as cell shape, adhesion and polarity, biochem-
ical signaling and feedback loops, genetically encoded informa-
tion, and stochastically generated variation (Nissen et al., 2017;
White et al., 2016c; Wennekamp et al., 2013). Adaptive self-orga-
nization in response to the changing interactions between these
elements enables robust regulative development of the embryo
without the external imposition of order. In this way, the embryo
progresses from a single cell into a complex multi-layered struc-
ture consisting of multiple cell lineages prior to implantation.
In practical terms, the preimplantation mouse embryo repre-
sents an excellent midway point between complex tissues and
organs, and simplistic cell culture systems (Figure 1). Progres-
sion through the entire preimplantation stage can proceed
in vitro, in the absence of maternal tissues, without any discern-
ible consequences on subsequent development (Whitten and
Biggers, 1968). Even before fertilization, the mouse egg can be
isolated and studied directly under the microscope, making it
possible to dissect mechanisms of mammalian meiosis. When
isolated after fertilization, the one-cell embryo undergoes
cleavage divisions and develops as a self-contained group of
blastomeres enclosed by the zona pellucida (ZP) and indepen-
dent of interactions with extracellular matrices or tissues. This
developing embryo can be imaged in such ex utero conditions
until the blastocyst stage when it is ready for implantation into
the uterus (Behringer, 2014; Brinster, 1963). The accessibility
of the cultured mouse embryo to imaging and genetic manipula-
tions while retaining the advantage of a physiological in vivo
system has placed this system at the center of research in
mammalian genetics, physiology, and development.
Although there is some variability in timing, the major events of
preimplantation development are well conserved across
mammalian species (Reijo Pera and Prezzoto, 2016). From
mouse to human, most mammals follow a general sequence of
events consisting of fertilization followed by early development
without embryonic transcription; cleavage divisions in the
absence of growth; compaction to form a morula; and, finally,
generation of a blastocyst. Early studies manipulating cells of
the mouse embryo demonstrated that this developmental pro-
gram is not predetermined, as in the case of lower organisms
such as Drosophila, Caenorhabditis elegans, and Xenopus, but
consists of varying division patterns and flexibility of cell fate
that can compensate for cell loss (Hoppe and Whitten, 1972;
Tarkowski and Wroblewska, 1967; Tarkowski, 1959) and even
artificial rearrangements (Ziomek and Johnson, 1982; Ziomek
et al., 1982). These initial findings hinted at what is now thought
to be a complex interaction of regulatory mechanisms working in
concert to gradually drive cells toward separate fates. Intrinsic to
this regulative process are not only subcellular processes but
also intercellular interactions that lead to a progressive refine-
ment of cellular identity. This plasticity, and the consequent abil-
ity to compensate in the face of internal or external disturbances,
may be especially important in the context of mammalian devel-
opment where significant energy and resources are invested into
each embryo.
Recent advances in live-cell imaging, computational cell
tracking, and optogenetics are beginning to yield the details

Developmental Cell 45, June 18, 2018 ª 2018 Elsevier Inc. 667

underlying these regulatory mechanisms and reveal how cells
interact with their neighbors to resolve their fates within the intact
developing embryo. Although some of the elements driving the
self-organization of the preimplantation embryo have been iden-
tified, extensive work is still required to understand how multiple
mechanisms operating at the level of the cell nucleus, cytoskel-
eton, and cell-cell interactions are integrated to fully assemble
the embryo.
Preimplantation Development from Oocyte to
Blastocyst
Cytoskeletal Changes Mediate the Transition from
Oocyte to Fertilized Embryo
The transformation from an egg to an embryo requires precise
coordination of a series of intricate processes that unite the
haploid genomes of each parent into a single diploid genome
of a new living organism. However, preparation for these events
begins long before fertilization with the maturation of the egg
precursor cell, or oocyte (Figure 2). Within the ovary, oocytes
are stored in a quiescent state, arrested in prophase of the first
meiotic division. Surrounding support cells provide the oocyte
668 Developmental Cell 45, June 18, 2018
Developmental Cell
Perspective
Figure 1. Comparison of Model Systems for
Studying Mammalian Development
(A) A mouse embryonic stem cell culture system.
This system has the advantage of being simple
and highly accessible for manipulations but is not
in a physiological context. Cells are labeled for the
Oct4 transcription factor (green) and a nuclear
marker (red).
(B) A 16-cell stage preimplantation mouse embryo
labeled for actin (green), microtubules (red), and
DNA (blue). The preimplantation embryo com-
bines the advantages of simplicity and accessi-
bility with the physiological relevance of an in vivo
system.
(C) A post-implantation gastrulation stage embryo
labeled for the cell membrane (orange) and DNA
(blue). By this stage the embryo has complex
architecture and is relatively inaccessible for im-
aging and manipulation. Graphs in right panels are
schematic representations of the relative advan-
tages and disadvantages of each model system.
Scale bars represent 10 mm in (A) and (B) and
30 mm in (C).
with maternal mRNAs and proteins that
will be required to sustain development
through the first stages of life (Li and
Albertini, 2013). In response to a surge
of luteinizing hormone released from the
pituitary gland, the oocyte resumes
meiosis, breaks down its nucleus, and
assembles a microtubule spindle around
its chromosomes (Bury et al., 2017). In
a critical first step of symmetry breaking,
the cortex of the oocyte softens and
thickens, promoting migration of the
spindle toward the nearest cortex
(Chaigne et al., 2013). This off-center
positioning of the spindle ensures the
asymmetry of the first meiotic division.
Unlike spindle positioning in somatic
cells, which typically depends on astral
microtubules, oocytes lack centrosomes, and therefore migra-
tion of the meiotic spindle is driven by actin polymerization (Yi
et al., 2013; Dumont et al., 2007). After spindle migration, the
close proximity of the chromosomes to the cortex induces the
formation of a cortical actomyosin cap (Deng et al., 2007). An
actin flow is established that drives cytoplasmic streaming to
maintain spindle positioning and the first polarization events
(Yi et al., 2011). The oocyte then undergoes a division that is
extremely asymmetric in size and discards half of its chromo-
somes into a small cell, called the polar body, which will later
degenerate. Recent high-resolution imaging studies have re-
vealed that actin filaments also infiltrate the meiotic spindle
and are crucial for correct alignment and segregation of the
chromosomes (Mogessie and Schuh, 2017). Immediately after
this division, the meiosis II spindle assembles around the chro-
mosomes just under the cortex and the oocyte remains arrested
at this stage, awaiting fertilization. Completion of the maturation
process can occur spontaneously in isolated oocytes in vitro
(Edwards, 1965), indicating that it proceeds by self-organiza-
tion. Fusion of a sperm with the mature oocyte triggers the
completion of meiosis II with another asymmetric division that
Developmental Cell

Perspective

Figure 2. Instructions for Assembling a Mouse Embryo from Oocyte to Compacted Morula
An overview of the main mechanisms currently known to direct preimplantation development from an oocyte to a compacted morula. Scale bars represent 10 mm.

generates a second polar body, translation of maternal mRNAs,
and the progression from meiosis to mitosis (Clift and Schuh,
2013). The first mitotic division in the newly formed zygote is
also dependent on actin networks (Chaigne et al., 2016).
However unlike in the oocyte, the zygotic spindle is positioned
centrally and the subsequent division is symmetric, thereby pro-
ducing two daughter cells, or blastomeres, of similar sizes.
Thus, the transition from meiosis to mitosis is complete
(Figure 2).
Early Heterogeneities among Cells of the Embryo
Upon fertilization, the maternal chromosomes and the sperm
chromatin decondense and form separate pronuclei. Within
each pronucleus there is a distinct program of epigenetic remod-
eling of the parental DNA to remove gamete-specific modifica-
tions and transform the zygote to a totipotent state. Changes
in DNA methylation, histone modifications, and activation of
retrotransposons all contribute to nuclear remodeling (Habibi
and Stunnenberg, 2017; Jachowicz et al., 2017; Burton and

Developmental Cell 45, June 18, 2018 669


Torres-Padilla, 2014). It has been proposed that differences
between cells of the embryo do not arise until the 16-cell stage
when the cells first adopt inner and outer positions (Alarcon
and Marikawa, 2005; Motosugi et al., 2005; Hiiragi and Solter,
2004). Indeed, initial studies revealed no differences between
early blastomeres in gene expression; however, these studies
were restricted to the analysis of selected candidate genes
(Wicklow et al., 2014; Guo et al., 2010; Ralston and Rossant,
2008; Dietrich and Hiiragi, 2007). Furthermore, although blasto-
meres can retain the capacity to differentiate into either inner
cell mass (ICM) or trophectoderm until at least the 16-cell stage
(Suwinska et al., 2008; Ziomek et al., 1982; Rossant and Vijh,
1980; Tarkowski and Wroblewska, 1967), mounting evidence
suggests that heterogeneities that bias cells toward one fate or
the other may arise earlier. Lineage tracing studies revealed a
cell fate bias in some blastomeres at the four-cell stage suggest-
ing that early blastomeres may not contribute equally to all line-
ages (Tabansky et al., 2013; Piotrowska-Nitsche et al., 2005;
Fujimori et al., 2003), although this effect may not be conserved
among all mouse strains (Alarcon and Marikawa, 2005). Analysis
of epigenetic modifications at the four-cell stage has revealed an
asymmetry between blastomeres in the levels of dimethylation of
histone H3 at arginines 17 and 26 (H3R17 and H3R26) and in the
levels of the chromatin modifiers Carm1 (Torres-Padilla et al.,
2007) and PRDM14 (Burton et al., 2013). The levels of H3 methyl-
ation are proposed to influence cell fate determination, as those
four-cell blastomeres displaying higher levels of methylation at
H3R17 and H3R26 tend to contribute more progeny to the ICM
of the embryo (Figure 2) (Burton et al., 2013; Torres-Padilla
et al., 2007). Overexpression of CARM1 leads to increased
H3R26 methylation and was associated with elevated expres-
sion of the ICM fate markers Nanog and Sox2 and positioning
of progeny within the ICM (Torres-Padilla et al., 2007).
Recently, the development of photoactivatable fluorescence
correlation spectroscopy (paFCS) techniques has made it
possible to probe transcription factor dynamics in vivo (Zhao
et al., 2017; Kaur et al., 2013; Plachta et al., 2011). Application
of paFCS within the embryo revealed that the increases in
Carm1-mediated histone H3 methylation promote DNA binding
of Sox2, a transcription factor important for maintaining pluripo-
tency, likely by increasing the accessibility of Sox2 binding sites
(White et al., 2016a). This increased Sox2-DNA binding is
thought to result in upregulation of Sox2-dependent genes
linked to pluripotency (Goolam et al., 2016; White et al., 2016a)
biasing cells toward an embryonic fate. How the variability in his-
tone H3 methylation arises de novo at the four-cell stage remains
unclear, but it may originate from interplay between cell cleavage
orientations (Zernicka-Goetz et al., 2009), differences in gene
expression (Shi et al., 2015; Biase et al., 2014; Piras et al.,
2014) resulting from noise-excitable mechanisms (Eldar and
Elowitz, 2010), or the possible asymmetric distribution of uniden-
tified factors.
The transition from oocyte to embryo occurs prior to zygotic
genome activation and proceeds without RNA transcription. In
fact, early development relies almost entirely on maternally pro-
vided factors deposited in the egg, many of which execute func-
tions involved in protein transport, protein localization, and cell
cycle (Gao et al., 2017; Zhou and Dean, 2015). These factors
may be unevenly distributed into each daughter cell during divi-
670 Developmental Cell 45, June 18, 2018
Developmental Cell
Perspective
sion in a process known as a partitioning error (Shi et al., 2015;
Huh and Paulsson, 2011). In the absence of transcription, parti-
tioning errors likely account for the initial introduction of
variability between cells. One such variably distributed factor
may relate to mitochondrial rRNAs, which have recently been
shown to become progressively heterogeneous by the end of
the two-cell stage (Zheng et al., 2016). Whether and how this
might influence or translate into asymmetries arising at the
four-cell stage remains to be determined.
Minor activation of zygotic transcription is first detected at the
late one-cell stage with higher activity in the male pronucleus
than the female pronucleus (Zhou and Dean, 2015). However,
the majority of zygotic genome activation first occurs at the
two-cell stage and contributes to the preparation of basic cellular
machinery involved in functions such as protein translation, cell
metabolism, and RNA processing (Gao et al., 2017). From this
point on, the newly synthesized embryonic products gradually
replace the maternally supplied factors and become the main
regulators of development.
Polarization and Compaction
The first major morphological changes in the embryo begin
with the onset of polarization and compaction at the eight-
cell stage. Until the early eight-cell stage, each cell within the
embryo is morphologically similar. Within 1–3 hr of reaching
the eight-cell stage, blastomeres begin to display the first out-
ward signs of radial polarity with the establishment of distinct
apical and basolateral domains (Figure 2) (Ziomek and John-
son, 1980). During this time, the actin cytoskeleton is reorgan-
ized to form an apical pole rich in microvilli and there is a polar-
ized redistribution of cytoplasmic organelles and cytoskeletal
elements (Ducibella et al., 1977). The contact-free surface of
each blastomere forms the apical domain, characterized by
clustering of the microvilli and the progressive localization of
the actin-binding protein Ezrin (Louvet et al., 1996), and the
Par3-Par6-aPKC complex (Vinot et al., 2005). The adhesion
molecule E-cadherin (Vestweber et al., 1987) accompanied
by Par-1 (Vinot et al., 2005), Jam-1 (Thomas et al., 2004),
and Na+/K+ ATPase (Watson and Kidder, 1988) become
restricted to the basolateral cell-cell contacts and the first
epithelial-like tissue architecture of the embryo is established
(Johnson, 2009). Although E-cadherin-mediated intercellular
adhesion is not required for the initiation of polarization in iso-
lated blastomeres (Ziomek and Johnson, 1980), it is necessary
for correct segregation of the apical and basolateral domains
and therefore controls the timing and axis of polarization (White
et al., 2016b; Stephenson et al., 2010). The process of polari-
zation does not require transcription or translation at the
eight-cell stage (Levy et al., 1986), suggesting it is instead
regulated through post-translational mechanisms (Johnson,
2009). The exact molecular trigger for polarization of the mouse
embryo remains to be revealed, but recent work has elucidated
some of the signaling pathways involved (Zhu et al., 2017).
Accumulation of actomyosin at the apical cortex is initiated
by phospholipase C-mediated PIP2 hydrolysis, which activates
protein kinase C (PKC). This in turn activates RhoA, leading to
polarization of the actin network. The Par3-Par6-aPKC com-
plex is then recruited to the apical surface in a manner that
likely requires additional unknown factors. Polarization of the
Par complex excludes actomyosin, forming a mature apical
Developmental Cell
Perspective
domain (Zhu et al., 2017), which is proposed to exhibit reduced
cortical contractility (Maitre et al., 2016).
Concomitant with the establishment of basolateral domains in
the embryo is the process of compaction. This results in maximi-
zation of cell-cell contact area and flattening of the outer surface
of the blastomeres to form a tightly packed mass of cells with
indistinct cell boundaries (White et al., 2016b; Ducibella and
Anderson, 1975). Embryo compaction is critical for blastocyst
formation and ongoing development (Ducibella and Anderson,
1975). Due to the requirement for E-cadherin in compaction, it
was widely believed that increases in intercellular adhesion
were responsible for this morphological change. However, it
has recently been demonstrated that there are no differences
in the expression or mobility of E-cadherin during compaction
(Samarage et al., 2015), and it is unclear whether E-cadherin
trans-ligation could even generate sufficient forces to achieve
this cellular deformation (Maitre et al., 2012). Live imaging of
E-cadherin during compaction revealed long membrane protru-
sions, classified as filopodia, that extend from the apical border
of cells in the eight-cell embryo and adhere via E-cadherin to the
top of their neighboring cells (Fierro-Gonzalez et al., 2013). In
addition to E-cadherin, the filopodia contain proteins that link it
to the cytoskeleton and the unconventional myosin motor
protein, myosin-10. Molecular manipulation of any of these com-
ponents disrupts compaction and acute femtosecond laser
ablation of filopodia results in rapid rounding and separation
of neighboring cells. These findings indicate that filopodia
contribute to the tensile force required to change cell shape dur-
ing compaction (Figure 2). Additional support for a role of tensile
force in cell shape change during compaction was provided us-
ing micropipette aspiration and measurement of blastomere
deformation (Maitre et al., 2015). This demonstrated a progres-
sive increase in cortical tension throughout compaction driven
by pulsed actomyosin contractions, and indicated that junctional
E-cadherin acts to redistribute actomyosin contractility away
from cell-cell contacts. How E-cadherin-dependent filopodia
and pulsatile actomyosin contractility are each initiated and
regulated remains to be determined. However, the accessibility
of the preimplantation embryo makes this an ideal system to
study these basic biological processes, which are commonly
employed throughout morphogenesis in many systems (Lecuit
et al., 2011).
Formation of the Pluripotent ICM
Compaction and embryo polarization are followed by the first
spatial segregation of cells into two separate populations that
will establish the first distinct cell lineages (Mihajlovic and Bruce,
2017) (Figure 2). During the eight- to 16-cell stage, the first cells
will be positioned in the interior of the embryo. These inner cells
are completely surrounded by basolateral membrane and cell-
cell contacts, and are therefore apolar. They will subsequently
differentiate to form the primitive endoderm, which gives rise
to extra-embryonic membranes, and the pluripotent epiblast,
from which the entire embryo proper is derived. The cells on
the outside of the embryo retain an apical surface, which is
devoid of cell-cell contacts and exposed to the exterior. These
cells are therefore polarized and most of them will differentiate
to form the trophectoderm, which gives rise to the placenta.
This spatial segregation of cells creates the first discrete niche
inside the embryo and generates structural asymmetries that
direct lineage divergence. Consequently, regulation of cell repo-
sitioning during this stage is critical for the correct development
of the embryo.
Traditionally, it was assumed that ‘‘asymmetric’’ cell divisions
allocated inner cells by cleaving the parental cell perpendicular
to the embryo’s surface and pushing one daughter cell into the
ICM as a direct result of the scission (Cockburn and Rossant,
2010; Johnson and Ziomek, 1981). However, division-indepen-
dent cell internalization events were also suggested to contribute
varying numbers of inner cells (Anani et al., 2014; Watanabe
et al., 2014; Yamanaka et al., 2010; Plusa et al., 2005).
Combining live imaging with computational membrane segmen-
tation enabled quantitative tracking of changes in cell shape and
position, and revealed that asymmetric divisions are relatively
rare (Samarage et al., 2015). Instead, most cells divide symmet-
rically, or at an oblique angle, on the surface of the embryo and
then one of the daughter cells may internalize through a process
of apical constriction. Laser ablations of the cortical actomyosin
network revealed the development of anisotropies in the magni-
tude and directionality of tensile forces acting at cell junctions
and the apical cell cortex. Cells that will internalize to form the
ICM display increased levels of myosin II relative to their neigh-
bors (Anani et al., 2014), resulting in higher tensile forces that
drive constriction of their apical membrane (Samarage et al.,
2015). Although many of these internalization events occur
following cell rearrangements due to divisions in the embryo,
the exact molecular mechanisms initiating the asymmetries in
cortical tension between cells is still an open question. One
classic model suggests that the asymmetric inheritance of the
apical domain during cell division determines cell fate. Recent
work suggests that the apical domain recruits a mitotic spindle
pole to ensure its asymmetric inheritance (Korotkevich et al.,
2017). Presence of the apical domain is thought to minimize
cortical tension to prevent cell internalization and trigger tro-
phectoderm fate specification (Maitre et al., 2016). However,
the inheritance of the apical domain during cell division has not
been demonstrated at sufficiently high temporal resolution by
live imaging in whole embryos, and some have proposed
that apical polarity may be reestablished following cytokinesis
(Watanabe et al., 2014; Zenker et al., 2018).
In addition to remodeling of actin networks, dynamic changes
in microtubule organization are essential for the formation of the
ICM (Zenker et al., 2017). Whereas in most animal cells the
centrosome is the main microtubule organizing center (MTOC)
(Conduit et al., 2015), the cells of the mouse embryo do not
form centrosomes until the blastocyst stage (Clift and Schuh,
2015). This suggested a random microtubule organization during
preimplantation development. However, recent work revealed a
different form of non-centrosomal microtubule organization
within the early embryo (Zenker et al., 2017). Following cell divi-
sion, the cytokinetic bridge is not abscised but instead it is
retained throughout interphase and connects each pair of sister
cells within the embryo. This microtubule bridge accumulates
Nezha/CAMSAP3, a protein that stabilizes microtubule minus
ends at non-centrosomal sites (Akhmanova and Hoogenraad,
2015) and enables the bridge to direct the growth of microtu-
bules into the cell.
One function of this non-centrosomal MTOC is to direct the
transport of endosomes carrying E-cadherin to the cell
Developmental Cell 45, June 18, 2018 671

membrane. Moreover, as cells internalize to form the ICM, their
bridge displays increased CAMSAP3 levels, higher microtubule
density, and more E-cadherin transport, which is proposed to
facilitate the expansion of the basolateral cell membrane of the
inner cells (Figure 2). Beyond E-cadherin, the microtubules of
the interphase bridge may also transport other proteins essential
for cell polarity or cell fate determination. Moreover, the mainte-
nance of a shared cytoskeleton between sister cells likely en-
ables mechanical coupling between cells.
The establishment of populations of inner and outer cells with
different patterns of cell-cell contacts creates structural asym-
metries that are critical for the expression of fate-specifying
markers. Each cell relies on the balance between cell-cell adhe-
sion and polarity to regulate the activity of the Hippo signaling
pathway and downstream lineage-specific genes. The transcrip-
tion factors Gata2, Gata3, and Cdx2 promote and maintain tro-
phectoderm differentiation (Home et al., 2017; Ralston et al.,
2010). Tead4 and its coactivator, Yap1, maintain the expression
of Gata3 and Cdx2 in outer cells in a Hippo-dependent manner
(Nishioka et al., 2009). Yap1 shuttles between the nucleus and
the cytoplasm and its phosphorylation state determines its sub-
cellular localization (Sasaki, 2017).
In the outer cells, Yap1 translocates into the nucleus to activate
expression of trophectoderm-specific genes, whereas, in the
inner cells, Yap1 is phosphorylated and therefore excluded from
the nucleus (Nishioka et al., 2009). Both cell-cell adhesion and
polarity act to regulate the junctional localization of Amot, a key
member of the Hippo pathway required for the phosphorylation
of Yap1 (Hirate et al., 2013). In apolar inner cells, Amot indirectly
associates with E-cadherin and Nf2 at the junctions
encompassing the cell, where it is activated through phosphoryla-
tion by Lats kinase (Cockburn et al., 2013; Hirate et al., 2013; Nish-
ioka et al., 2009). Phosphorylated Amot then acts together with
Lats to enhance the phosphorylation and nuclear exclusion of
Yap1, allowing the expression of pluripotency-associated genes.
By contrast, in the polarized outer cells, the presence of an
apical domain sequesters Amot from the junctions and localizes
it apically where it binds to cortical F-actin and remains unphos-
phorylated (Hirate et al., 2013). Recent work demonstrated that
Rho kinase also acts to stabilize Amot binding to F-actin and
inhibit the interaction between Amot and Nf2 (Shi et al., 2017).
As Amot is absent from the junctional complexes, the Hippo
signaling pathway remains inactive and unphosphorylated
Yap1 is retained in the nucleus to drive a trophectoderm-specific
gene expression program (Sasaki, 2017; Ralston et al., 2010).
Blastocyst Formation and Hatching
Once the embryo has established separate populations of inner
and outer cells, it is ready to transition to a fluid-filled blastocyst.
The blastocyst cavity begins as microlumens that emerge in the
intercellular spaces within the embryo (Marikawa and Alarcon,
2012). These microlumens are formed by exocytosis of vesicles
or vacuoles from the basal membrane of the outer cells (Figure 3)
(Aziz and Alexandre, 1991). Sodium ions are actively transported
across the outer cell layer through transmembrane pumps (Wat-
son and Barcroft, 2001; Benos et al., 1985). The differential
sodium ion concentration generates an osmotic gradient that en-
ables fluid to be pumped into the embryo (Barcroft et al., 2003),
enlarging and coalescing the microlumens into a single cavity.
Expansion of this cavity requires the formation of a paracellular
672 Developmental Cell 45, June 18, 2018
Developmental Cell
Perspective
permeability barrier that seals the embryo from the exterior and
resists the increasing hydrostatic pressure. This barrier is medi-
ated by the establishment of tight junctions between the outer
cells (Wang et al., 2008), forming the trophectoderm: the first
epithelium of the embryo. Recent work demonstrated that assem-
bly of the permeability barrier begins with the formation of cortical
actin rings on the apical surface of outer cells at the 16-cell stage
(Zenker et al., 2018). Unlike stereotypical actin rings, which are
contractile, these actin rings expand to the cell-cell junctions
where they recruit and stabilize components of adherens and tight
junctions. Coupling of the actin rings to the junctions triggers a
tension-dependent zippering mechanism that extends along the
junctions and seals the embryo, enabling blastocyst expansion.
As the blastocyst cavity expands, it breaks the radial symme-
try of the embryo and causes clustering of the inner cells at the
embryonic pole. This allows for a separation of cell layers that
may be critical for the further differentiation of cells of the ICM.
Until the early blastocyst stage, the ICM is a homogeneous pop-
ulation of cells co-expressing lineage markers including Nanog,
Gata6, and Oct-4 (Ohnishi et al., 2014). However, as the blasto-
cyst expands, a reciprocal expression of Nanog and Gata6 leads
to the formation of two distinct populations, the primitive endo-
derm and the epiblast, in a heterogeneous ‘‘salt-and-pepper’’
distribution (Figure 3) (Chazaud et al., 2006). The early variations
in gene expression are known to be propagated by FGF4/FGFR2
signaling (Krawchuk et al., 2013; Guo et al., 2010); however, their
temporal regulation is not yet fully understood (Bessonnard
et al., 2017). Epiblast precursors expressing Nanog secrete
FGF4 to drive Gata6 expression and primitive endoderm fate
specification in their neighbor cells (Nissen et al., 2017; Lanner
and Rossant, 2010). Recent evidence suggests that the hetero-
geneities in FGF4/FGFR2 signaling may be initiated by variations
in Sox2/Oct4-dependent Fgf4 transcription that arise from fluc-
tuating Sox2 expression levels (Mistri et al., 2018). In addition
to FGFR2, FGFR1 has recently been shown to be required for
the differential transduction of the FGF4 signal within the ICM
(Kang et al., 2017; Molotkov et al., 2017).
Following cell fate specification, the precise spatial arrange-
ment of the two-cell populations occurs through cell migration,
apoptosis, polarization, and adhesion. By the time of blastocyst
implantation, the primitive endoderm is a morphologically
distinct epithelial sheet of polarized cells facing the blastocyst
cavity, while the cells of the pluripotent epiblast are enclosed
by the primitive endoderm and trophectoderm (Saiz and Plusa,
2013; Lanner and Rossant, 2010).
To complete preimplantation development and implant into
the uterus, the embryo must finally hatch out of the protective
shell formed by the ZP (Figure 2). Blastocyst hatching has
been suggested to occur as a combined result of localized pro-
teolytic lysis of the ZP and mechanical pressure exerted by the
expanding blastocyst (Seshagiri et al., 2009; Perona and Was-
sarman, 1986). The embryo then gradually emerges from the
ZP in a poorly defined process that is regulated by dynamic
cellular trophectodermal projections, molecular signaling fac-
tors, and proteases released by both the blastocyst and the
maternal endometrium. From this point on, the preimplantation
stage is over and the embryo must establish interactions with
the uterus and undergo implantation to support the increasingly
complex demands of its continuing development.
Developmental Cell

Perspective

Figure 3. Instructions for Assembling a Mouse Embryo from Compacted Morula to Hatched Blastocyst
An overview of the main mechanisms currently known to direct preimplantation development from a compacted morula to a hatched blastocyst ready for
implantation. Scale bars represent 10 mm.

Future Challenges standing of how the embryo is formed. There are undoubtedly
Although research to date has yielded many insights into the additional mechanisms to discover, especially regarding the
myriad processes occurring during preimplantation develop- establishment and amplification of initial heterogeneities among
ment, substantial challenges remain to complete our under- blastomeres and the role of forces and changes in mechanical

Developmental Cell 45, June 18, 2018 673


Figure 4. Progressive Refinement of Cell Fate
Biases in cell fate may arise at the four-cell stage due to a combination of biological noise and early heterogeneities between blastomeres in epigenetic
modifications (red triangles, green hexagons) and transcription factor binding to DNA. These early differences may be progressively amplified and refined until the
first two lineages diverge at the blastocyst stage.
properties in fate determination. A significant challenge ahead is
to identify crosstalk between the diverse processes identified in
the early embryo and to understand how these mechanisms are
integrated to progressively induce cells to transform into all the
requisite components of the embryo. Technological advance-
ments and the implementation of approaches used in other sys-
tems are likely to be critical to addressing these questions.
How to Discover New Morphogenetic Processes
Despite recent progress in revealing morphogenetic processes
controlling the assembly of the early mammalian embryo
(Figure 2), numerous additional cellular events will likely be
revealed as we broaden our technical spectrum of observation.
Advances in live-cell imaging, super-resolution and electron
microscopy (EM) techniques, and computational analysis will
allow us to image faster events, occurring in the order of minutes
or seconds, and the more microscopic organization of molecules
and organelles within cells of intact embryos.
Imaging methods providing faster time resolution could enable
the visualization of the movement of signaling vesicles, or the
reorganization of cytoskeletal elements, as cells change their
fate, shape, and positions in vivo. For instance, imaging experi-
ments in Drosophila embryos showed multiple endosomes car-
rying signaling molecules shuttling rapidly between cells during
asymmetric division (Derivery et al., 2015; Coumailleau et al.,
2009). Advances in light-sheet-based imaging technologies
may reveal if similar mechanisms of rapid endosome transport
enable cell-cell communication in the mouse embryo (Whitehead
et al., 2017). In particular, as recent studies demonstrated the
presence of cellular protrusions (Fierro-Gonzalez et al., 2013)
674 Developmental Cell 45, June 18, 2018
Developmental Cell
Perspective
and microtubule bridges (Zenker et al., 2017) between the cells
of the embryo, it would be interesting to determine if these
cellular structures or organelles enable the transport of vesicles
or signaling molecules as a mechanism of cell-cell communi-
cation.
It will also be important to explore how rapid changes in cell
shape in the mouse embryo correlate with redistributions of
signaling molecules, particularly those controlling the cytoskel-
eton (Munjal and Lecuit, 2014). Pushing the application of fast-
acquisition imaging techniques could enable discoveries of
changes in the distribution of second messengers, or the meta-
bolic or mechanical state of cells at the whole-embryo level.
At the subcellular level, super-resolution microscopy has not
yet been extensively applied to mouse embryos, yet these tech-
niques will prove tremendously useful to reconstruct the spatial
arrangement of most organelles within early blastomeres. More-
over, the combination of EM with powerful computer segmenta-
tion techniques has provided a previously inconceivable level of
detail, uncovering the organization of brain synapses at the
nanometer scale (Hildebrand et al., 2017). This type of approach
is being applied in early mouse embryos to reveal the organiza-
tion of vesicular structures transported along microtubule tracks
in the intact embryo (Zenker et al., 2017). Together with ad-
vances in histology methods to visualize fluorescently labeled
proteins in EM sections, these methods will significantly extend
our understanding of subcellular organization within the embryo.
Finally, it will also be valuable to explore the more macro-
scopic mechanisms regulating the assembly of the early mouse
embryo. The timing of cell divisions may be tightly controlled
Developmental Cell
Perspective
by structures providing intercellular communication within the
embryo. For example, cells extending filopodia on top of neigh-
boring cells appear to inhibit the division of their neighbors
(Fierro-Gonzalez et al., 2013). Moreover, cells connected via
the microtubule interphase bridge undergo division in a tightly
coordinated manner (Zenker et al., 2017). When a cell enters
mitosis, the depolymerization of its microtubule network is prop-
agated along the interphase bridge toward the connected sister
cell, which only then enters mitosis. This suggests a mechanism
whereby signals triggering mitosis could travel between sister
cells via the interphase bridge.
These open problems can greatly benefit from the use of
mathematical models predicting changes in embryo architecture
(Yu and Fernandez-Gonzalez, 2017). However, we should
remain cautious about reductionist approaches that inadver-
tently simplify the morphology and structural asymmetries that
exist between cells in vivo.
What Is Upstream of Changes in Epigenetics,
Transcription Factor-DNA Interactions, and Gene
Expression?
Despite the identification of early epigenetic and transcriptional
asymmetries, it remains an open question how these asymme-
tries initially develop and exactly how they are amplified to
direct lineage divergence (Figure 4). The emergence of
Figure 5. Regulation of Mechanical Forces
Driving Embryo Formation
Signaling pathways and mechanical properties
within the embryo regulate actomyosin contrac-
tility and force production. Anisotropies in tensile
forces constrict the apical surface of cells, causing
them to internalize to form the inner cell mass.
patterning from homogeneous cell popu-
lations requires symmetry breaking,
which may be initiated by stochastic
processes. Recently, the application of
single-cell RNA sequencing technology
to the preimplantation embryo revealed
that differences in the transcriptome
initially arise between blastomeres as
early as the first cleavage division (Shi
et al., 2015; Biase et al., 2014; Piras
et al., 2014). This first variability is intro-
duced by partitioning errors unevenly
distributing substances into each
daughter cell after division. Although
some of these errors may be subse-
quently corrected, genes with a low
expression level are disproportionately
affected by partitioning errors (Shi
et al., 2015). This results in the establish-
ment of differential gene expression
between blastomeres. Early heterogene-
ities between blastomeres may be
further amplified by transcriptional noise
following zygotic gene activation during
the two-cell stage (Piras et al., 2014).
Random stochastic fluctuations, or
noise, in the levels and activities of interacting genes and pro-
teins underlies the divergence of genetically identical cells in
many biological systems (Eldar and Elowitz, 2010). Gene
expression noise can be generated intrinsically by transcrip-
tional bursts, or extrinsically by the propagation of fluctuations
in the expression of one gene to cause fluctuations in expres-
sion of downstream genes. The introduction of biological noise
into a system enables the probabilistic differentiation of genet-
ically identical cells while maintaining the flexibility to respond
to external factors (Balazsi et al., 2011). Within the embryo,
positive and negative feedback loops fine-tune the initial differ-
ences and affect the ratio of opposing lineage specifiers to bias
cells toward different fates (Shi et al., 2015). In this way, cells
can slowly advance toward a specific fate but still maintain a
time window during which noise can act to correct any organi-
zational errors.
How inter-blastomere differences introduced at the two-cell
stage by partitioning error and transcriptional noise relate to
the observed differences in epigenetic modifications (Torres-Pa-
dilla et al., 2007), transcription factor dynamics (White et al.,
2016a), and gene expression (Goolam et al., 2016; Shi et al.,
2015; Biase et al., 2014) at the four-cell stage and beyond
remains to be determined. Resolving this requires improvements
in the precise quantification of levels and dynamics of molecules
Developmental Cell 45, June 18, 2018 675

at single-cell resolution without amplification and the capacity to
track these molecules over time in living embryos (Zhao
et al., 2016).
Tilting the Balance of Mechanical Forces
Most morphogenetic processes are driven by changes in the
balance of mechanical forces within and between cells (Munjal
and Lecuit, 2014). Forces driving changes in cell shape are
generated within the cell by actomyosin networks and trans-
mitted to neighboring cells and the extracellular environment
via adhesion-mediated connections. In the preimplantation em-
bryo, the main forces at play are cortical tension and cell-cell
adhesion mediated by adhesion molecules like E-cadherin.
Consistent with many other developmental systems, changes
in adhesion and cortical tension also drive morphological
changes required to assemble the mouse embryo. For example,
changes in both of these forces are important for embryo
compaction, and anisotropies in tensile forces drive the internal-
ization of the cells that form the ICM (Maitre et al., 2015; Samar-
age et al., 2015).
However, the key open challenge is to understand what mech-
anisms tilt the balance between these mechanical forces to
direct morphogenesis. Two main mechanisms are likely in place:
signaling pathways controlling the function or localization of key
molecules mediating cell-cell adhesion and cortical tension, and
changes in mechanical properties within the embryo (Figure 5).
For example, in cells and other systems, myosin-based contrac-
tility can be activated by signaling through the myosin light-chain
kinase pathway, which commonly responds to intracellular cal-
cium, or via signaling through the Rho kinase pathway (Priya
and Yap, 2015). E-cadherin is recruited to junctions in response
to myosin activation and Rho signaling stabilizes junctional
integrity (Hoffman and Yap, 2015). Actin regulators such as
WAVE and Arp2/3 can be recruited to junctions to differentially
regulate actin organization and connect adhesion to contractility
(Brieher and Yap, 2013). While many of these pathways have
been described in detail in other systems, their regulation is rela-
tively unexplored in the preimplantation embryo. The application
of optogenetic techniques for controlling protein localization has
recently demonstrated how emerging technologies may enable
further dissection of these pathways in vivo (Zhu et al., 2017).
Less is known, however, about changes in mechanical proper-
ties in vivo, since few techniques enable their measurement in
complex systems like embryos and tissues. For instance, it is
possible that, as blastomeres undergo cleavage divisions, they
may be subject to changes in intracellular pressure, cytoplasmic
viscosity, or stiffness. Dynamic changes in these mechanical
properties would affect the embryo’s response to internal forces.
It will therefore be important to apply new methods to measure
mechanical properties in the embryo, which could include new
tools for measuring intracellular pressure (Gomez-Martinez
et al., 2013); optical tweezers, microindenters, or cantilevers to
apply forces and measure responses; and magnetically respon-
sive ferrofluid microdroplets that enable highly precise measure-
ments of mechanical properties such as viscosity in tissues and
embryos (Serwane et al., 2017; Campas, 2016). It should be
emphasized that mechanical forces remain the main executers
of embryo patterning, yet future experiments would enable us
to examine how signaling pathways and changes in mechanical
properties are intertwined to affect these forces in vivo.
676 Developmental Cell 45, June 18, 2018
Developmental Cell
Perspective
Conclusion
Decades of research have revealed basic mechanisms underly-
ing key events during preimplantation development and yielded
some preliminary instructions on how to assemble a mammalian
embryo. These instructions should be viewed as an initial guide
and are likely to be refined and replaced over time as emerging
technologies facilitate new discoveries.
The high plasticity and regulative nature of the mammalian
embryo ensures that the identification of deterministic processes
is more complicated than in non-mammalian systems. Although
blastomeres isolated from the embryo can reproduce key
morphogenetic milestones such as polarization and compac-
tion, they may do so via regulative processes. Thus results
from such experiments should be interpreted with caution due
to the disruption of the normal geometry of cell-cell contacts
and mechanical forces within the embryo (Humiecka
et al., 2017).
Recent work also advises restraint in translating findings in
mouse embryos directly to humans. Although mouse and human
embryos share many similarities, there are important differences
in the timing of key events (Jukam et al., 2017) and the
functioning of key lineages specifiers (Fogarty et al., 2017).
Future work adapting murine stem cell models of embryogenesis
(Harrison et al., 2017) to human cells, and utilizing human
embryos where possible (Shahbazi et al., 2016), will help to
define such species-specific permutations of common develop-
mental processes.
ACKNOWLEDGMENTS
This work was supported by funds from the DFG (ZE99711), SNF
(PBLAP3145877), and HFSP (LT000164/2015) to J.Z. and from A*STAR,
EMBO, and HHMI-Wellcome Trust to N.P. We apologize to those colleagues
whose work we are unable to review here due to space limitations.
REFERENCES
Akhmanova, A., and Hoogenraad, C.C. (2015). Microtubule minus-end-target-
ing proteins. Curr. Biol. 25, R162–R171.
Alarcon, V.B., and Marikawa, Y. (2005). Unbiased contribution of the first two
blastomeres to mouse blastocyst development. Mol. Reprod. Dev. 72,
354–361.
Anani, S., Bhat, S., Honma-Yamanaka, N., Krawchuk, D., and Yamanaka, Y.
(2014). Initiation of Hippo signaling is linked to polarity rather than to cell posi-
tion in the pre-implantation mouse embryo. Development 141, 2813–2824.
Aziz, M., and Alexandre, H. (1991). The origin of the nascent blastocoele in
preimplantation mouse embryos ultrastructural cytochemistry and effect of
chloroquine. Roux Arch. Dev. Biol. 200, 77–85.
Balazsi, G., van Oudenaarden, A., and Collins, J.J. (2011). Cellular decision
making and biological noise: from microbes to mammals. Cell 144, 910–925.
Barcroft, L.C., Offenberg, H., Thomsen, P., and Watson, A.J. (2003). Aquaporin
proteins in murine trophectoderm mediate transepithelial water movements
during cavitation. Dev. Biol. 256, 342–354.
Behringer, R. (2014). Manipulating the Mouse Embryo: A Laboratory Manual
(Cold Spring Harbor Laboratory Press).
Benos, D.J., Biggers, J.D., Balaban, R.S., Mills, J.W., and Overstrom, E.W.
(1985). Developmental aspects of sodium-dependent transport processes of
preimplantation rabbit embryos. Soc. Gen. Physiol. Ser. 39, 211–235.
Bessonnard, S., Coqueran, S., Vandormael-Pournin, S., Dufour, A., Artus, J.,
and Cohen-Tannoudji, M. (2017). ICM conversion to epiblast by FGF/ERK
Developmental Cell
Perspective
inhibition is limited in time and requires transcription and protein degradation.
Sci. Rep. 7, 12285.
Biase, F.H., Cao, X., and Zhong, S. (2014). Cell fate inclination within 2-cell and
4-cell mouse embryos revealed by single-cell RNA sequencing. Genome Res.
24, 1787–1796.
Brieher, W.M., and Yap, A.S. (2013). Cadherin junctions and their cytoskele-
ton(s). Curr. Opin. Cell Biol. 25, 39–46.
Brinster, R.L. (1963). A method for in vitro cultivation of mouse ova from two-
cell to blastocyst. Exp. Cell Res. 32, 205–208.
Burton, A., Muller, J., Tu, S., Padilla-Longoria, P., Guccione, E., and Torres-Pa-
dilla, M.E. (2013). Single-cell profiling of epigenetic modifiers identifies
PRDM14 as an inducer of cell fate in the mammalian embryo. Cell Rep. 5,
687–701.
Burton, A., and Torres-Padilla, M.E. (2014). Chromatin dynamics in the regula-
tion of cell fate allocation during early embryogenesis. Nat. Rev. Mol. Cell Biol.
15, 723–734.
Bury, L., Coelho, P.A., Simeone, A., Ferries, S., Eyers, C.E., Eyers, P.A., Zer-
nicka-Goetz, M., and Glover, D.M. (2017). Plk4 and Aurora A cooperate in
the initiation of acentriolar spindle assembly in mammalian oocytes. J. Cell
Biol. 216, 3571–3590.
Campas, O. (2016). A toolbox to explore the mechanics of living embryonic
tissues. Semin. Cell Dev. Biol. 55, 119–130.
Chaigne, A., Campillo, C., Gov, N.S., Voituriez, R., Azoury, J., Umana-Diaz, C.,
Almonacid, M., Queguiner, I., Nassoy, P., Sykes, C., et al. (2013). A soft cortex
is essential for asymmetric spindle positioning in mouse oocytes. Nat. Cell
Biol. 15, 958–966.
Chaigne, A., Campillo, C., Voituriez, R., Gov, N.S., Sykes, C., Verlhac, M.H.,
and Terret, M.E. (2016). F-actin mechanics control spindle centring in the
mouse zygote. Nat. Commun. 7, 10253.
Chazaud, C., Yamanaka, Y., Pawson, T., and Rossant, J. (2006). Early lineage
segregation between epiblast and primitive endoderm in mouse blastocysts
through the Grb2-MAPK pathway. Dev. Cell 10, 615–624.
Clift, D., and Schuh, M. (2013). Restarting life: fertilization and the transition
from meiosis to mitosis. Nat. Rev. Mol. Cell Biol. 14, 549–562.
Clift, D., and Schuh, M. (2015). A three-step MTOC fragmentation mechanism
facilitates bipolar spindle assembly in mouse oocytes. Nat. Commun. 6, 7217.
Cockburn, K., Biechele, S., Garner, J., and Rossant, J. (2013). The Hippo
pathway member Nf2 is required for inner cell mass specification. Curr. Biol.
23, 1195–1201.
Cockburn, K., and Rossant, J. (2010). Making the blastocyst: lessons from the
mouse. J. Clin. Invest. 120, 995–1003.
Conduit, P.T., Wainman, A., and Raff, J.W. (2015). Centrosome function and
assembly in animal cells. Nat. Rev. Mol. Cell Biol. 16, 611–624.
Coumailleau, F., Furthauer, M., Knoblich, J.A., and Gonzalez-Gaitan, M.
(2009). Directional Delta and Notch trafficking in Sara endosomes during
asymmetric cell division. Nature 458, 1051–1055.
Davies, J.A. (2017). Adaptive self-organization in the embryo: its importance to
adult anatomy and to tissue engineering. J. Anat. 232, 524–533.
Deng, M., Suraneni, P., Schultz, R.M., and Li, R. (2007). The Ran GTPase me-
diates chromatin signaling to control cortical polarity during polar body extru-
sion in mouse oocytes. Dev. Cell 12, 301–308.
Derivery, E., Seum, C., Daeden, A., Loubery, S., Holtzer, L., Julicher, F., and
Gonzalez-Gaitan, M. (2015). Polarized endosome dynamics by spindle asym-
metry during asymmetric cell division. Nature 528, 280–285.
Dietrich, J.E., and Hiiragi, T. (2007). Stochastic patterning in the mouse pre-im-
plantation embryo. Development 134, 4219–4231.
Ducibella, T., and Anderson, E. (1975). Cell shape and membrane changes in
the eight-cell mouse embryo: prerequisites for morphogenesis of the blasto-
cyst. Dev. Biol. 47, 45–58.
Ducibella, T., Ukena, T., Karnovsky, M., and Anderson, E. (1977). Changes in
cell surface and cortical cytoplasmic organization during early embryogenesis
in the preimplantation mouse embryo. J. Cell Biol. 74, 153–167.
Dumont, J., Million, K., Sunderland, K., Rassinier, P., Lim, H., Leader, B., and
Verlhac, M.H. (2007). Formin-2 is required for spindle migration and for the late
steps of cytokinesis in mouse oocytes. Dev. Biol. 301, 254–265.
Edwards, R.G. (1965). Maturation in vitro of mouse, sheep, cow, pig, rhesus
monkey and human ovarian oocytes. Nature 208, 349–351.
Eldar, A., and Elowitz, M.B. (2010). Functional roles for noise in genetic circuits.
Nature 467, 167–173.
Fierro-Gonzalez, J.C., White, M.D., Silva, J.C., and Plachta, N. (2013). Cad-
herin-dependent filopodia control preimplantation embryo compaction. Nat.
Cell Biol. 15, 1424–1433.
Fogarty, N.M.E., McCarthy, A., Snijders, K.E., Powell, B.E., Kubikova, N., Bla-
keley, P., Lea, R., Elder, K., Wamaitha, S.E., Kim, D., et al. (2017). Genome
editing reveals a role for OCT4 in human embryogenesis. Nature 550, 67–73.
Fujimori, T., Kurotaki, Y., Miyazaki, J., and Nabeshima, Y. (2003). Analysis of
cell lineage in two- and four-cell mouse embryos. Development 130,
5113–5122.
Gao, Y., Liu, X., Tang, B., Li, C., Kou, Z., Li, L., Liu, W., Wu, Y., Kou, X., Li, J.,
et al. (2017). Protein expression landscape of mouse embryos during pre-im-
plantation development. Cell Rep. 21, 3957–3969.
Gomez-Martinez, R., Hernandez-Pinto, A.M., Duch, M., Vazquez, P., Zinoviev,
K., de la Rosa, E.J., Esteve, J., Suarez, T., and Plaza, J.A. (2013). Silicon chips
detect intracellular pressure changes in living cells. Nat. Nanotechnol. 8,
517–521.
Goolam, M., Scialdone, A., Graham, S.J., Macaulay, I.C., Jedrusik, A., Hupa-
lowska, A., Voet, T., Marioni, J.C., and Zernicka-Goetz, M. (2016). Heterogene-
ity in Oct4 and Sox2 targets biases cell fate in 4-cell mouse embryos. Cell
165, 61–74.
Guo, G., Huss, M., Tong, G.Q., Wang, C., Li Sun, L., Clarke, N.D., and Robson,
P. (2010). Resolution of cell fate decisions revealed by single-cell gene expres-
sion analysis from zygote to blastocyst. Dev. Cell 18, 675–685.
Habibi, E., and Stunnenberg, H.G. (2017). Transcriptional and epigenetic con-
trol in mouse pluripotency: lessons from in vivo and in vitro studies. Curr. Opin.
Genet. Dev. 46, 114–122.
Harrison, S.E., Sozen, B., Christodoulou, N., Kyprianou, C., and Zernicka-
Goetz, M. (2017). Assembly of embryonic and extraembryonic stem cells to
mimic embryogenesis in vitro. Science 356, https://doi.org/10.1126/science.
aal1810.
Hiiragi, T., and Solter, D. (2004). First cleavage plane of the mouse egg is not
predetermined but defined by the topology of the two apposing pronuclei. Na-
ture 430, 360–364.
Hildebrand, D.G.C., Cicconet, M., Torres, R.M., Choi, W., Quan, T.M., Moon,
J., Wetzel, A.W., Scott Champion, A., Graham, B.J., Randlett, O., et al.
(2017). Whole-brain serial-section electron microscopy in larval zebrafish. Na-
ture 545, 345–349.
Hirate, Y., Hirahara, S., Inoue, K., Suzuki, A., Alarcon, V.B., Akimoto, K., Hirai,
T., Hara, T., Adachi, M., Chida, K., et al. (2013). Polarity-dependent distribution
of angiomotin localizes Hippo signaling in preimplantation embryos. Curr. Biol.
23, 1181–1194.
Hoffman, B.D., and Yap, A.S. (2015). Towards a dynamic understanding of
cadherin-based mechanobiology. Trends Cell Biol. 25, 803–814.
Home, P., Kumar, R.P., Ganguly, A., Saha, B., Milano-Foster, J., Bhattacharya,
B., Ray, S., Gunewardena, S., Paul, A., Camper, S.A., et al. (2017). Genetic
redundancy of GATA factors in the extraembryonic trophoblast lineage
ensures the progression of preimplantation and postimplantation mammalian
development. Development 144, 876–888.
Hoppe, P.C., and Whitten, W.K. (1972). Does X chromosome inactivation
occur during mitosis of first cleavage? Nature 239, 520.
Huh, D., and Paulsson, J. (2011). Random partitioning of molecules at cell
division. Proc. Natl. Acad. Sci. USA 108, 15004–15009.
Developmental Cell 45, June 18, 2018 677

Humiecka, M., Szpila, M., Klos, P., Maleszewski, M., and Szczepanska, K.
(2017). Mouse blastomeres acquire ability to divide asymmetrically before
compaction. PLoS One 12, e0175032.
Jachowicz, J.W., Bing, X., Pontabry, J., Boskovic, A., Rando, O.J., and Torres-
Padilla, M.E. (2017). LINE-1 activation after fertilization regulates global chro-
matin accessibility in the early mouse embryo. Nat. Genet. 49, 1502–1510.
Johnson, M.H. (2009). From mouse egg to mouse embryo: polarities, axes,
and tissues. Annu. Rev. Cell Dev. Biol. 25, 483–512.
Johnson, M.H., and Ziomek, C.A. (1981). The foundation of two distinct cell
lineages within the mouse morula. Cell 24, 71–80.
Jukam, D., Shariati, S.A.M., and Skotheim, J.M. (2017). Zygotic genome acti-
vation in vertebrates. Dev. Cell 42, 316–332.
Kang, M., Garg, V., and Hadjantonakis, A.K. (2017). Lineage establishment and
progression within the inner cell mass of the mouse blastocyst requires FGFR1
and FGFR2. Dev. Cell 41, 496–510.e5.
Kaur, G., Costa, M.W., Nefzger, C.M., Silva, J., Fierro-Gonzalez, J.C., Polo,
J.M., Bell, T.D., and Plachta, N. (2013). Probing transcription factor diffusion
dynamics in the living mammalian embryo with photoactivatable fluorescence
correlation spectroscopy. Nat. Commun. 4, 1637.
Korotkevich, E., Niwayama, R., Courtois, A., Friese, S., Berger, N., Buchholz,
F., and Hiiragi, T. (2017). The apical domain is required and sufficient for the
first lineage segregation in the mouse embryo. Dev. Cell 40, 235–247.e7.
Krawchuk, D., Honma-Yamanaka, N., Anani, S., and Yamanaka, Y. (2013).
FGF4 is a limiting factor controlling the proportions of primitive endoderm
and epiblast in the ICM of the mouse blastocyst. Dev. Biol. 384, 65–71.
Lanner, F., and Rossant, J. (2010). The role of FGF/Erk signaling in pluripotent
cells. Development 137, 3351–3360.
Lecuit, T., Lenne, P.F., and Munro, E. (2011). Force generation, transmission,
and integration during cell and tissue morphogenesis. Annu. Rev. Cell Dev.
Biol. 27, 157–184.
Levy, J.B., Johnson, M.H., Goodall, H., and Maro, B. (1986). The timing of
compaction: control of a major developmental transition in mouse early
embryogenesis. J. Embryol. Exp. Morphol. 95, 213–237.
Li, R., and Albertini, D.F. (2013). The road to maturation: somatic cell interac-
tion and self-organization of the mammalian oocyte. Nat. Rev. Mol. Cell Biol.
14, 141–152.
Louvet, S., Aghion, J., Santa-Maria, A., Mangeat, P., and Maro, B. (1996). Ezrin
becomes restricted to outer cells following asymmetrical division in the preim-
plantation mouse embryo. Dev. Biol. 177, 568–579.
Maitre, J.L., Berthoumieux, H., Krens, S.F., Salbreux, G., Julicher, F., Paluch,
E., and Heisenberg, C.P. (2012). Adhesion functions in cell sorting by mechan-
ically coupling the cortices of adhering cells. Science 338, 253–256.
Maitre, J.L., Niwayama, R., Turlier, H., Nedelec, F., and Hiiragi, T. (2015). Pul-
satile cell-autonomous contractility drives compaction in the mouse embryo.
Nat. Cell Biol. 17, 849–855.
Maitre, J.L., Turlier, H., Illukkumbura, R., Eismann, B., Niwayama, R., Nedelec,
F., and Hiiragi, T. (2016). Asymmetric division of contractile domains couples
cell positioning and fate specification. Nature 536, 344–348.
Marikawa, Y., and Alarcon, V.B. (2012). Creation of trophectoderm, the first
epithelium, in mouse preimplantation development. Results Probl. Cell Differ.
55, 165–184.
Mihajlovic, A.I., and Bruce, A.W. (2017). The first cell-fate decision of mouse
preimplantation embryo development: integrating cell position and polarity.
Open Biol. 7, https://doi.org/10.1098/rsob.170210.
Mistri, T.K., Arindrarto, W., Ng, W.P., Wang, C., Lim, L.H., Sun, L., Chambers,
I., Wohland, T., and Robson, P. (2018). Dynamic changes in Sox2 spatio-tem-
poral expression promote the second cell fate decision through Fgf4/Fgfr2 sig-
nalling in preimplantation mouse embryos. Biochem. J. 475, 1075–1089.
Mogessie, B., and Schuh, M. (2017). Actin protects mammalian eggs against
chromosome segregation errors. Science 357, https://doi.org/10.1126/sci-
ence.aal1647.
678 Developmental Cell 45, June 18, 2018
Developmental Cell
Perspective
Molotkov, A., Mazot, P., Brewer, J.R., Cinalli, R.M., and Soriano, P. (2017).
Distinct requirements for FGFR1 and FGFR2 in primitive endoderm develop-
ment and exit from pluripotency. Dev. Cell 41, 511–526.e4.
Motosugi, N., Bauer, T., Polanski, Z., Solter, D., and Hiiragi, T. (2005). Polarity
of the mouse embryo is established at blastocyst and is not prepatterned.
Genes Dev. 19, 1081–1092.
Munjal, A., and Lecuit, T. (2014). Actomyosin networks and tissue morphogen-
esis. Development 141, 1789–1793.
Nishioka, N., Inoue, K., Adachi, K., Kiyonari, H., Ota, M., Ralston, A., Yabuta,
N., Hirahara, S., Stephenson, R.O., Ogonuki, N., et al. (2009). The Hippo
signaling pathway components Lats and Yap pattern Tead4 activity to distin-
guish mouse trophectoderm from inner cell mass. Dev. Cell 16, 398–410.
Nissen, S.B., Perera, M., Gonzalez, J.M., Morgani, S.M., Jensen, M.H., Snep-
pen, K., Brickman, J.M., and Trusina, A. (2017). Four simple rules that are suf-
ficient to generate the mammalian blastocyst. PLoS Biol. 15, e2000737.
Ohnishi, Y., Huber, W., Tsumura, A., Kang, M., Xenopoulos, P., Kurimoto, K.,
Oles, A.K., Arauzo-Bravo, M.J., Saitou, M., Hadjantonakis, A.K., et al. (2014).
Cell-to-cell expression variability followed by signal reinforcement progres-
sively segregates early mouse lineages. Nat. Cell Biol. 16, 27–37.
Perona, R.M., and Wassarman, P.M. (1986). Mouse blastocysts hatch in vitro
by using a trypsin-like proteinase associated with cells of mural trophecto-
derm. Dev. Biol. 114, 42–52.
Piotrowska-Nitsche, K., Perea-Gomez, A., Haraguchi, S., and Zernicka-Goetz,
M. (2005). Four-cell stage mouse blastomeres have different developmental
properties. Development 132, 479–490.
Piras, V., Tomita, M., and Selvarajoo, K. (2014). Transcriptome-wide variability
in single embryonic development cells. Sci. Rep. 4, 7137.
Plachta, N., Bollenbach, T., Pease, S., Fraser, S.E., and Pantazis, P. (2011).
Oct4 kinetics predict cell lineage patterning in the early mammalian embryo.
Nat. Cell Biol. 13, 117–123.
Plusa, B., Frankenberg, S., Chalmers, A., Hadjantonakis, A.K., Moore, C.A.,
Papalopulu, N., Papaioannou, V.E., Glover, D.M., and Zernicka-Goetz, M.
(2005). Downregulation of Par3 and aPKC function directs cells towards the
ICM in the preimplantation mouse embryo. J. Cell Sci. 118, 505–515.
Priya, R., and Yap, A.S. (2015). Active tension: the role of cadherin adhesion
and signaling in generating junctional contractility. Curr. Top. Dev. Biol.
112, 65–102.
Ralston, A., Cox, B.J., Nishioka, N., Sasaki, H., Chea, E., Rugg-Gunn, P., Guo,
G., Robson, P., Draper, J.S., and Rossant, J. (2010). Gata3 regulates tropho-
blast development downstream of Tead4 and in parallel to Cdx2. Development
137, 395–403.
Ralston, A., and 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.
Reijo Pera, R.A., and Prezzoto, L. (2016). Species-specific variation among
mammals. Curr. Top. Dev. Biol. 120, 401–420.
Rossant, J., and Vijh, K.M. (1980). Ability of outside cells from preimplantation
mouse embryos to form inner cell mass derivatives. Dev. Biol. 76, 475–482.
Saiz, N., and Plusa, B. (2013). Early cell fate decisions in the mouse embryo.
Reproduction 145, R65–R80.
Samarage, C.R., White, M.D., Alvarez, Y.D., Fierro-Gonzalez, J.C., Henon, Y.,
Jesudason, E.C., Bissiere, S., Fouras, A., and Plachta, N. (2015). Cortical ten-
sion allocates the first inner cells of the mammalian embryo. Dev. Cell 34,
435–447.
Sasai, Y. (2013). Cytosystems dynamics in self-organization of tissue architec-
ture. Nature 493, 318–326.
Sasaki, H. (2017). Roles and regulations of Hippo signaling during preimplan-
tation mouse development. Dev. Growth Differ. 59, 12–20.
Serwane, F., Mongera, A., Rowghanian, P., Kealhofer, D.A., Lucio, A.A., Hock-
enbery, Z.M., and Campas, O. (2017). In vivo quantification of spatially varying
mechanical properties in developing tissues. Nat. Methods 14, 181–186.
 Developmental Cell
Perspective
Seshagiri, P.B., Sen Roy, S., Sireesha, G., and Rao, R.P. (2009). Cellular and
molecular regulation of mammalian blastocyst hatching. J. Reprod. Immunol.
83, 79–84.
Shahbazi, M.N., Jedrusik, A., Vuoristo, S., Recher, G., Hupalowska, A., Bolton,
V., Fogarty, N.M., Campbell, A., Devito, L.G., Ilic, D., et al. (2016). Self-organi-
zation of the human embryo in the absence of maternal tissues. Nat. Cell Biol.
18, 700–708.
Shi, J., Chen, Q., Li, X., Zheng, X., Zhang, Y., Qiao, J., Tang, F., Tao, Y., Zhou,
Q., and Duan, E. (2015). Dynamic transcriptional symmetry-breaking in pre-im-
plantation mammalian embryo development revealed by single-cell RNA-seq.
Development 142, 3468–3477.
Shi, X., Yin, Z., Ling, B., Wang, L., Liu, C., Ruan, X., Zhang, W., and Chen, L.
(2017). Rho differentially regulates the Hippo pathway by modulating the inter-
action between Amot and Nf2 in the blastocyst. Development 144, 3957–3967.
Stephenson, R.O., Yamanaka, Y., and Rossant, J. (2010). Disorganized epithe-
lial polarity and excess trophectoderm cell fate in preimplantation embryos
lacking E-cadherin. Development 137, 3383–3391.
Suwinska, A., Czolowska, R., Ozdzenski, W., and Tarkowski, A.K. (2008). Blas-
tomeres of the mouse embryo lose totipotency after the fifth cleavage division:
expression of Cdx2 and Oct4 and developmental potential of inner and outer
blastomeres of 16- and 32-cell embryos. Dev. Biol. 322, 133–144.
Tabansky, I., Lenarcic, A., Draft, R.W., Loulier, K., Keskin, D.B., Rosains, J.,
Rivera-Feliciano, J., Lichtman, J.W., Livet, J., Stern, J.N., et al. (2013). Devel-
opmental bias in cleavage-stage mouse blastomeres. Curr. Biol. 23, 21–31.
Tarkowski, A.K. (1959). Experiments on the development of isolated blasto-
meres of mouse eggs. Nature 184, 1286–1287.
Tarkowski, A.K., and Wroblewska, J. (1967). Development of blastomeres of
mouse eggs isolated at the 4- and 8-cell stage. J. Embryol. Exp. Morphol.
18, 155–180.
Thomas, F.C., Sheth, B., Eckert, J.J., Bazzoni, G., Dejana, E., and Fleming,
T.P. (2004). Contribution of JAM-1 to epithelial differentiation and tight-junc-
tion biogenesis in the mouse preimplantation embryo. J. Cell Sci. 117,
5599–5608.
Torres-Padilla, M.E., Parfitt, D.E., Kouzarides, T., and Zernicka-Goetz, M.
(2007). Histone arginine methylation regulates pluripotency in the early mouse
embryo. Nature 445, 214–218.
Vestweber, D., Gossler, A., Boller, K., and Kemler, R. (1987). Expression and
distribution of cell adhesion molecule uvomorulin in mouse preimplantation
embryos. Dev. Biol. 124, 451–456.
Vinot, S., Le, T., Ohno, S., Pawson, T., Maro, B., and Louvet-Vallee, S. (2005).
Asymmetric distribution of PAR proteins in the mouse embryo begins at the
8-cell stage during compaction. Dev. Biol. 282, 307–319.
Wang, H., Ding, T., Brown, N., Yamamoto, Y., Prince, L.S., Reese, J., and Pa-
ria, B.C. (2008). Zonula occludens-1 (ZO-1) is involved in morula to blastocyst
transformation in the mouse. Dev. Biol. 318, 112–125.
Watanabe, T., Biggins, J.S., Tannan, N.B., and Srinivas, S. (2014). Limited pre-
dictive value of blastomere angle of division in trophectoderm and inner cell
mass specification. Development 141, 2279–2288.
Watson, A.J., and Barcroft, L.C. (2001). Regulation of blastocyst formation.
Front. Biosci. 6, D708–D730.
Watson, A.J., and Kidder, G.M. (1988). Immunofluorescence assessment of
the timing of appearance and cellular distribution of Na/K-ATPase during
mouse embryogenesis. Dev. Biol. 126, 80–90.
Wennekamp, S., Mesecke, S., Nedelec, F., and Hiiragi, T. (2013). A self-orga-
nization framework for symmetry breaking in the mammalian embryo. Nat.
Rev. Mol. Cell Biol. 14, 454–461.
White, M.D., Angiolini, J.F., Alvarez, Y.D., Kaur, G., Zhao, Z.W., Mocskos, E.,
Bruno, L., Bissiere, S., Levi, V., and Plachta, N. (2016a). Long-lived binding of
Sox2 to DNA predicts cell fate in the four-cell mouse embryo. Cell 165, 75–87.
View publication stats
White, M.D., Bissiere, S., Alvarez, Y.D., and Plachta, N. (2016b). Mouse
embryo compaction. Curr. Top. Dev. Biol. 120, 235–258.
White, M.D., Zenker, J., Bissiere, S., and Plachta, N. (2016c). How cells change
shape and position in the early mammalian embryo. Curr. Opin. Cell Biol.
44, 7–13.
Whitehead, L.W., McArthur, K., Geoghegan, N.D., and Rogers, K.L. (2017).
The reinvention of twentieth century microscopy for three-dimensional imag-
ing. Immunol. Cell Biol. 95, 520–524.
Whitten, W.K., and Biggers, J.D. (1968). Complete development in vitro of the
pre-implantation stages of the mouse in a simple chemically defined medium.
J. Reprod. Fertil. 17, 399–401.
Wicklow, E., Blij, S., Frum, T., Hirate, Y., Lang, R.A., Sasaki, H., and Ralston, A.
(2014). HIPPO pathway members restrict SOX2 to the inner cell mass where it
promotes ICM fates in the mouse blastocyst. PLoS Genet. 10, e1004618.
Yamanaka, Y., Lanner, F., and Rossant, J. (2010). FGF signal-dependent
segregation of primitive endoderm and epiblast in the mouse blastocyst.
Development 137, 715–724.
Yi, K., Rubinstein, B., Unruh, J.R., Guo, F., Slaughter, B.D., and Li, R. (2013).
Sequential actin-based pushing forces drive meiosis I chromosome migration
and symmetry breaking in oocytes. J. Cell Biol. 200, 567–576.
Yi, K., Unruh, J.R., Deng, M., Slaughter, B.D., Rubinstein, B., and Li, R. (2011).
Dynamic maintenance of asymmetric meiotic spindle position through Arp2/3-
complex-driven cytoplasmic streaming in mouse oocytes. Nat. Cell Biol. 13,
1252–1258.
Yu, J.C., and Fernandez-Gonzalez, R. (2017). Quantitative modelling of epithe-
lial morphogenesis: integrating cell mechanics and molecular dynamics.
Semin. Cell Dev. Biol. 67, 153–160.
Zenker, J., White, M.D., Templin, R.M., Parton, R.G., Thorn-Seshold, O., Bis-
siere, S., and Plachta, N. (2017). A microtubule-organizing center directing
intracellular transport in the early mouse embryo. Science 357, 925–928.
Zenker, J., White, M.D., Gasnier, M., Alvarez, Y.D., Lim, H.Y.G., Bissiere, S.,
Biro, M., and Plachta, N. (2018). Expanding actin rings zipper the mouse
embryo for blastocyst formation. Cell 173, 776–791.
Zernicka-Goetz, M., Morris, S.A., and Bruce, A.W. (2009). Making a firm deci-
sion: multifaceted regulation of cell fate in the early mouse embryo. Nat. Rev.
Genet. 10, 467–477.
Zhao, Z.W., White, M.D., Alvarez, Y.D., Zenker, J., Bissiere, S., and Plachta, N.
(2017). Quantifying transcription factor-DNA binding in single cells in vivo with
photoactivatable fluorescence correlation spectroscopy. Nat. Protoc. 12,
1458–1471.
Zhao, Z.W., White, M.D., Bissiere, S., Levi, V., and Plachta, N. (2016). Quanti-
tative imaging of mammalian transcriptional dynamics: from single cells to
whole embryos. BMC Biol. 14, 115.
Zheng, Z., Li, H., Zhang, Q., Yang, L., and Qi, H. (2016). Unequal distribution of
16S mtrRNA at the 2-cell stage regulates cell lineage allocations in mouse em-
bryos. Reproduction 151, 351–367.
Zhou, L.Q., and Dean, J. (2015). Reprogramming the genome to totipotency in
mouse embryos. Trends Cell Biol. 25, 82–91.
Zhu, M., Leung, C.Y., Shahbazi, M.N., and Zernicka-Goetz, M. (2017). Actomy-
osin polarisation through PLC-PKC triggers symmetry breaking of the mouse
embryo. Nat. Commun. 8, 921.
Ziomek, C.A., and Johnson, M.H. (1980). Cell surface interaction induces
polarization of mouse 8-cell blastomeres at compaction. Cell 21, 935–942.
Ziomek, C.A., and Johnson, M.H. (1982). The roles of phenotype and position
in guiding the fate of 16-cell mouse blastomeres. Dev. Biol. 91, 440–447.
Ziomek, C.A., Johnson, M.H., and Handyside, A.H. (1982). The developmental
potential of mouse 16-cell blastomeres. J. Exp. Zool 221, 345–355.
Developmental Cell 45, June 18, 2018 679