Current topics in developmental biology, volume 112

CURRENT TOPICS IN
DEVELOPMENTAL BIOLOGY
“A meeting-ground for critical review and discussion of developmental processes”
A.A. Moscona and Alberto Monroy (Volume 1, 1966)

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Paul M. Wassarman
Department of Developmental and Regenerative Biology
Icahn School of Medicine at Mount Sinai
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FOUNDING ADVISORY BOARD
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Seymour S. Cohen
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S. Spiegelman
Hewson W. Swift
E.N. Willmer
Etienne Wolff


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CONTRIBUTORS
Elias H. Barriga
Cell and Developmental Biology Department, University College London, London,
United Kingdom
Deanna L. Benson
Fishberg Department of Neuroscience, Friedman Brain Institute and the Graduate School of
Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA
Nicholas H. Brown
Department of Physiology, Development and Neuroscience, The Gurdon Institute,
University of Cambridge, Cambridge, United Kingdom
Alexander N. Combes
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Brisbane,
Queensland, Australia
Jamie A. Davies
Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom
Andrew J. Ewald
Department of Cell Biology, Center for Cell Dynamics, and Department of Oncology, Johns

Hopkins University School of Medicine, Baltimore, Maryland, USA
Franc¸ois Fagotto
Department of Biology, McGill University, Montre´al, Que´bec, Canada
Lauren G. Friedman
Fishberg Department of Neuroscience, Friedman Brain Institute and the Graduate
School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai,
New York, USA
Cara J. Gottardi
Cellular and Molecular Biology, and Medicine, Northwestern University Feinberg School of
Medicine, Chicago, Illinois, USA
Benjamin M. Hogan
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland,
Australia
George W. Huntley
Fishberg Department of Neuroscience, Friedman Brain Institute and the Graduate School of
Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, USA
Anne Karine Lagendijk
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland,
Australia

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Contributors

Terry Lechler
Department of Dermatology, and Department of Cell Biology, Duke University Medical
Center, Durham, North Carolina, USA

Melissa H. Little
Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, Victoria,
Australia
Aidan P. Maartens
Department of Physiology, Development and Neuroscience, The Gurdon Institute,
University of Cambridge, Cambridge, United Kingdom
Meghan T. Maher
Department of Biology, Washington University in St. Louis, St. Louis, Missouri, USA
Kenji Mandai
Division of Pathogenetic Signaling, Kobe University Graduate School of Medicine, and
CREST, Japan Science and Technology Agency, Kobe, Japan
Roberto Mayor
Cell and Developmental Biology Department, University College London, London, United
Kingdom
Pierre D. McCrea
Department of Genetics, University of Texas MD Anderson Cancer Center; Program in
Genes & Development, Graduate School in Biomedical Sciences, Houston, Texas, USA
Masahiro Mori
CREST, Japan Science and Technology Agency; Division of Neurophysiology, Department
of Physiology and Cell Biology, Kobe University Graduate School of Medicine, and Faculty
of Health Sciences, Kobe University Graduate School of Health Sciences, Kobe, Japan
Nicolas Plachta
European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative
Medicine Institute, Monash University, Clayton, Victoria, Australia
Rashmi Priya
Division of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience,
The University of Queensland, Brisbane, Queensland, Australia
Yoshiyuki Rikitake
CREST, Japan Science and Technology Agency; Division of Signal Transduction,
Department of Biochemistry and Molecular Biology, and Division of Cardiovascular

Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine,
Kobe, Japan
Katja R€
oper
MRC-Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge,
United Kingdom
Pierre Savagner
IRCM, Institut de Recherche en Cance´rologie de Montpellier, INSERM U896, Institut
re´gional du Cancer Universite´ Montpellier1, Montpellier, France


Contributors

xiii

Eliah R. Shamir
Department of Cell Biology, Center for Cell Dynamics, and Department of Oncology, Johns
Hopkins University School of Medicine, Baltimore, Maryland, USA
Kaelyn D. Sumigray
Department of Dermatology, and Department of Cell Biology, Duke University Medical
Center, Durham, North Carolina, USA
Yoshimi Takai
Division of Pathogenetic Signaling, Kobe University Graduate School of Medicine, and
CREST, Japan Science and Technology Agency, Kobe, Japan
Melanie D. White
European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative
Medicine Institute, Monash University, Clayton, Victoria, Australia
Alpha S. Yap
Division of Cell Biology and Molecular Medicine, Institute for Molecular Bioscience,
The University of Queensland, Brisbane, Queensland, Australia

PREFACE
Cell adhesion is a fundamental determinant of development in metazoan
organisms. For over a century—from the early observations of HV Wilson,
through the seminal studies of Townes and Holtfreter, and since—we have
endeavored to understand how adhesion helps make multicellular organisms
more than just the sum of their parts. We now know that physical interactions between cells and their environment (other cells and components of
the extracellular matrix) influence critical parameters of development,
including tissue cohesion, cellular patterning, differentiation, and population
control. These diverse functional effects reflect the complex ways in which
distinct adhesion systems interact with cellular processes such as signaling,
the cytoskeleton, and membrane trafficking. In this volume, we aim to survey recent developments in understanding how the cellular and molecular
mechanisms of adhesion determine the development of organisms and their
constituent organs.
The early chapters in this volume endeavor to define some of the key
processes that allow adhesion to influence development. Melanie White
and Nicolas Plachta review how adhesion cooperates with the cytoskeleton
to drive the earliest cellular events in the preimplantation mouse embryo:
compaction, change in cell shape, polarity, and cell fate. Franc¸ois Fagotto
then addresses one of the long-standing problems in developmental biology:
understanding how boundaries are formed in the embryo. Building on the
long-standing realization that boundaries reflect physical differences
between populations of cells, Fagotto outlines how different cell–cell adhesion systems may cooperate with the cytoskeleton to segregate cell
populations at boundaries.
We then have a series of chapters that focus on the mechanisms by which
cadherin cell adhesion molecules influence animal development. Here, a
major advance has come from the realization that cadherins cooperate with
the contractile apparatus, that is, the actomyosin cytoskeleton. Accordingly,
Rashmi Priya and Alpha Yap discuss the molecular and cellular mechanisms

that allow cadherin adhesion systems to physically interact with, and also
regulate, the actomyosin cytoskeleton. Katja R€
oper then addresses how
cooperation between cell–cell adhesion and contractility determines morphogenesis in the early Drosophila embryo. In their chapter, Pierre McCrea,
Meghan Maher, and Cara Gottardi broaden the discussion to review how
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xvi

Preface

cadherins and their associated proteins signal to the nucleus, a paradigm that
underlies canonical Wnt signaling and also impinges on other fundamental
developmental pathways, such as the Hippo signaling pathway.
Of course, cadherins are not the only adhesion systems that influence
development. Another large family of cell–cell adhesion molecules are
the nectins and nectin-like proteins. Kenji Mandai, Yoshimi Takai, and their
colleagues discuss the fundamental cell biology of nectins and review how
these molecules affect the development of many organs in the body. Aidan
Maartens and Nicholas Brown then outline developments in understanding
how integrin cell–matrix adhesion molecules contribute to Drosophila development, including notable developments in how integrins influence cell
fate, cell migration, and cell polarity.
The two subsequent chapters focus on developmental processes that
integrate adhesion, signaling, and the cytoskeleton. Pierre Savagner discusses
the concept of epithelial-to-mesenchymal transition, providing a historical
and conceptual framework for this complex phenomenon, with its often
controversial mechanistic underpinnings. Elias Barriga and Roberto Mayor
then take the example of neural crest migration to consider how adhesive
events generate collective patterns of cell migration.

Finally, we examine how cell adhesion influences the development of
individual organs. Anne Lagendijk and Benjamin Hogan review how cell
signaling and cell–cell adhesion cooperate during vascular development.
Eliah Shamir and Andrew Ewald focus on how individual and collective cell
migration are regulated by cell–cell adhesion to drive epithelial morphogenesis of the mammary gland. Kaelyn Sumigray and Terry Lechler review how
multiple junctions (adherens, tight, and desmosomes) contribute to development of the epidermis as a fundamental biological barrier in the body.
Lauren Friedman, Deanna Benson, and George Huntley consider the role
that cadherins play in the nervous system, with a particular focus on understanding their role in synapse formation and the generation of synaptic
networks, the bases of neural activity. And in the final chapter of this volume, Alexander Combes, Jamie Davies, and Melissa Little discuss how cell
adhesion drives self-organization in the embryonic kidney, providing
insights relevant to tissue engineering and regenerative medicine.
We hope that the contributions in this volume illustrate some of the different perspectives that are now being used to understand how cell adhesion
contributes to development. A final perspective lies in the relationship
between development and disease. Many of the cellular mechanisms and
biological processes that we consider are also implicated in disease. Thus,


Preface

xvii

we also sought, where possible, to highlight how basic biology illuminates
our understanding of disease and vice versa. We hope that these reviews will
then be a useful guide to students of fundamental biology and pathology.
And we will be well pleased if they prompt further research at the interface
between these disciplines.
ALPHA S. YAP


CHAPTER ONE

How Adhesion Forms the Early
Mammalian Embryo
Melanie D. White, Nicolas Plachta1
European Molecular Biology Laboratory (EMBL) Australia, Australian Regenerative Medicine Institute,
Monash University, Clayton, Victoria, Australia
1
Corresponding author: e-mail address:

Contents
1. The Mouse Preimplantation Embryo as a Model of Adhesion in Mammalian
Development
1.1 Adhesion molecules in the preimplantation mouse embryo
2. Adhesion Regulates Cell Shape
3. Adhesion Controls Cell Polarity
4. Adhesion Determines Cell Fate
5. Emerging Technologies to Study Adhesion
6. Questions for the Future
References

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5
7
7
9
11
13

Abstract

The early mouse embryo is an excellent system to study how a small group of initially
rounded cells start to change shape and establish the first forms of adhesion-based
cell–cell interactions in mammals in vivo. In addition to its critical role in the structural
integrity of the embryo, we discuss here how adhesion is important to regulate cell polarity and cell fate. Recent evidence suggests that adherens junctions participate in signaling
pathways by localizing key proteins to subcellular microdomains. E-cadherin has been
identified as the main player required for the establishment of adhesion but other mechanisms involving additional proteins or physical forces acting in the embryo may also contribute. Application of new technologies that enable high-resolution quantitative
imaging of adhesion protein dynamics and measurements of biomechanical forces will
provide a greater understanding of how adhesion patterns the early mammalian embryo.

1. THE MOUSE PREIMPLANTATION EMBRYO
AS A MODEL OF ADHESION IN MAMMALIAN
DEVELOPMENT
Most research on adhesion has been performed on cells in tissue culture due to their availability and ease of manipulation. However, it is only
Current Topics in Developmental Biology, Volume 112
ISSN 0070-2153
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2015 Elsevier Inc.
All rights reserved.

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Melanie D. White and Nicolas Plachta

during true cellular differentiation within an embryo that the contribution of
adhesion to development can be examined directly. The mouse preimplantation embryo provides an ideal system to study adhesion mechanisms that

are based exclusively on cell–cell interactions. A glycoprotein membrane,
the zona pellucida, encloses the preimplantation embryo so cell–cell adhesion
can be studied in the complete absence of extracellular matrix interactions.
Preimplantation development naturally occurs within the oviduct, but it
can be recapitulated in vitro without adversely affecting the developmental
potential of embryos (Summers & Biggers, 2003). Mouse embryos can be
easily removed from the maternal oviducts and cultured in simple media
conditions. Under these ex utero conditions, the embryos develop almost
as rapidly as they do in utero and if transferred back to the uterus they can
implant and continue developing to produce viable offspring.
During the first 2 days of development, the fertilized mouse egg
undergoes three cleavage divisions to produce an 8-cell embryo
(Fig. 1A). At this stage, the cells are round and visibly indistinguishable.
The first major cell morphological changes begin as the 8-cell embryo
undergoes compaction. Concomitant with a rise in intercellular adhesion,
the cells flatten their membranes against each other, maximizing contact
and forming a highly packed mass. This process of increased adhesion and
embryo compaction occurs ubiquitously during preimplantation

Figure 1 Imaging preimplantation development in the mouse embryo. (A) DIC
images showing development of mouse embryo from 1-cell to blastocyst stage.
(B) Microinjection of mRNA or DNA into the pronucleus allows visualization of proteins
of interest throughout preimplantation development. In the example shown, the membrane is labeled with mCherry and the nucleus is labeled with H2B-GFP. ICM, inner cell
mass; TE, trophectoderm.


Adhesion in the Early Mouse Embryo

3

development in different mammalian species and is an absolute requirement
for embryo viability. This process is very similar in mouse and humans, making the preimplantation mouse embryo an ideal model to study the role of
adhesion in cell shape, position, and fate in early mammalian development.
In addition, the cells of the mouse embryo are relatively large, facilitating
imaging of subcellular processes. Furthermore, there are many available
genetic tools that are applicable in the mouse for manipulation of proteins
of interest. Pronuclear microinjection of mRNA or DNA is a wellestablished technique for expression of exogenous proteins and mouse
embryos are resilient enough to withstand this process with high efficiency
(Fig. 1B). Moreover, thousands of genetically modified animals are now
available carrying targeted endogenous genes or expressing various transgenic constructs.

1.1. Adhesion molecules in the preimplantation
mouse embryo
Early studies identified a critical role for calcium-dependent adhesion in
embryo compaction, subsequent spatial segregation of the inner cell mass
(ICM) and formation of the first differentiated tissue, the trophectoderm
(Fleming, Sheth, & Fesenko, 2001). Interfering with adhesion by chelating
calcium ions or using antibodies targeting a cell surface glycoprotein
decompacted embryos and prevented blastocyst formation (Ducibella &
Anderson, 1975; Wales, 1970; Whitten, 1971). In 1981, this cell surface glycoprotein was identified as uvomorulin, now more commonly known as
E-cadherin (Hyafil, Babinet, & Jacob, 1981).
Although usually expressed in epithelial cell layers, E-cadherin is also
expressed from the very early stages of development. It is initially maternally
derived in the oocyte and at the 2-cell stage, de novo E-cadherin zygotic synthesis starts (Vestweber, Gossler, Boller, & Kemler, 1987). Embryos lacking
zygotic E-cadherin are preimplantation lethal. They do undergo compaction due to residual maternal E-cadherin but fail to form a blastocyst
(Larue, Ohsugi, Hirchenhain, & Kemler, 1994). Using siRNAs to knockdown E-cadherin expression in just half of the embryo prevents those cells
from integrating into the compacting embryo (Fig. 2) (Fierro-Gonzalez,
White, Silva, & Plachta, 2013). Embryos lacking both maternal and zygotic
E-cadherin are unable to compact or form a blastocyst and they appear as
loose aggregates of cells (Stephenson, Yamanaka, & Rossant, 2010). Deleting maternal E-cadherin alone delays compaction until the late morula stage

but embryos then develop normally due to zygotic E-cadherin expression


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Melanie D. White and Nicolas Plachta

Figure 2 E-cadherin is required for cell–cell adhesion and embryo compaction.
(A) Microinjection of one cell at the 2-cell stage results in an embryo expressing a control
siRNA and a membrane-Cherry marker in half of its cells. The transgenic cells have normal morphology and integrate into the compacting embryo mass. (B) An siRNA
targeting E-cadherin reduces cell–cell adhesion in the transgenic half of the embryo.
The nontransgenic cells compact normally but the E-cadherin knockdown cells are very
spherical and do not integrate into the embryo mass. (C) Treating the embryo with the
DECMA-1 E-cadherin function-blocking antibody reduces adhesion and causes all cells
to become very spherical. The embryo does not compact. Scale bar ¼ 10 μm.

Figure 3 E-cadherin localization changes during preimplantation development.
E-cadherin is distributed throughout the membrane until the late 8-cell stage. Then,
it begins to accumulate in cell–cell junctions and is predominantly localized to basolateral regions by the 16-cell stage.

(De Vries et al., 2004). Adhesion does not develop until the late morula stage
in these embryos indicating that although E-cadherin and its binding partners are expressed (Ohsugi et al., 1996; Sefton, Johnson, & Clayton, 1992;
Vestweber et al., 1987), they are not required to form functional adhesion
complexes at very early stages.
E-cadherin is uniformly distributed in the cell membrane until the 8-cell
stage when PKC-α-mediated phosphorylation of β-catenin, a key protein
regulating E-cadherin intracellular signaling, is thought to activate the compaction process (Fig. 3; Pauken & Capco, 1999). Compaction can be
blocked by inhibition of PKC-α and induced early at the 2- and 4-cell stages

Adhesion in the Early Mouse Embryo

5

by PKC-α activation (Ohsugi, Ohsawa, & Semba, 1993; Winkel, Ferguson,
Takeichi, & Nuccitelli, 1990). E-cadherin accumulates basolaterally, forming adherens junctions between cells and connecting to the actin cytoskeleton via catenin proteins (Ozawa, Ringwald, & Kemler, 1990). As adhesion
initiates, the actin cytoskeleton is reorganized to define the orientation of the
first cellular polarity in the embryo (Stephenson et al., 2010).
After compaction has occurred, tight junctions begin to assemble at
apicolateral cell–cell junctions in a process that requires prior activation of
E-cadherin-mediated adhesion (Fleming, McConnell, Johnson, &
Stevenson, 1989; Ohsugi, Larue, Schwarz, & Kemler, 1997). The timing
of tight junction formation is tightly regulated by staggered expression of
the constituent proteins from the 8- to 32-cell stage (Sheth et al., 1997).
E-cadherin-mediated adhesion may also stabilize tight junction proteins,
preventing their turnover once they are assembled at the membrane
( Javed, Fleming, Hay, & Citi, 1993). The close intercellular adhesion at
tight junctions then forms a permeability seal between adjacent epithelial
cells. This allows the formation of the blastocoel cavity and the generation
of the blastocyst. During blastocyst expansion, small, strongly adhesive junctions called desmosomes are assembled between adjacent cells in the
trophectoderm. These junctions are multiprotein complexes containing
the desmosomal cadherins, desmocollins, and desmogleins (Fleming,
Garrod, & Elsmore, 1991). Adherens junctions and desmosomes functionally synergize to maintain epithelial polarity and structure. Thus, in the
sequence of developmental events generating adhesion in the embryo,
E-cadherin is a central player in a pathway that enables the progressive formation and organization of different types of anchoring junctions and the
establishment of the first forms of tissue-like organization during
development.

2. ADHESION REGULATES CELL SHAPE
There are many studies demonstrating the importance of adhesion for

morphogenesis in other experimental models (Lecuit, Lenne, & Munro,
2011). However, relatively little work has been performed in the preimplantation mouse embryo to elucidate how adhesion controls cell shape. The first
discernible changes accompanying the increased intercellular adhesion during compaction are morphological. From the late 8-cell stage, cells lose their
previously spherical shape and flatten into a tightly packed mass with indistinguishable membranes (Fig. 1). The formation of adherens junctions was


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Melanie D. White and Nicolas Plachta

widely believed to be responsible for this global change in morphology since
perturbations of E-cadherin using antibodies or gene deletion approaches
resulted in cells displaying a rounder morphology. However, recent work
has demonstrated that in addition to its localization to adherens junctions,
E-cadherin also accumulates in long cellular protrusions identified as
filopodia, which appear specifically during the stages of embryo compaction
(Fierro-Gonzalez et al., 2013). Cells extend these filopodia from their junctions onto the apical membrane of neighboring cells and adhere to them via
E-cadherin trans interactions (Fig. 4). The filopodia then maintain tension to
elongate the cell’s membrane over its neighbors, bringing them into close
apposition, and facilitating compaction (Fierro-Gonzalez et al., 2013).
Whereas E-cadherin localized at adherens junctions play an important role
in keeping cells together, the pool of E-cadherin in the recently discovered
filopodia helps cells establish tight interactions at their apical membranes in
order to compact the embryo.
Physically disrupting these filopodia with laser ablation causes cells to
revert to a more spherical shape. Moreover, interfering with the molecular
components present in these filopodia, which include E-cadherin,
α-catenin, β-catenin, or Myo10, prevents compaction. Expressing a mutant
form of E-cadherin lacking the extracellular domain or treating embryos
with an E-cadherin function-blocking antibody also disrupts filopodia formation and embryo compaction, suggesting that the adhesive function of

Figure 4 E-cadherin-dependent filopodia control embryo compaction. Two cells
labeled with a mCherry protein targeted to the cell membrane extend filopodia over
their nonlabeled neighbors during compaction at the late 8-cell stage. Dashed box
shows filopodia at higher magnification.


Adhesion in the Early Mouse Embryo

7

E-cadherin is critical. Only around 60% of cells in each embryo were found
to form filopodia and there is no direct association with subsequent cell fate
so it remains to be revealed what determines whether a cell will employ this
newly described mechanism of adhesion during preimplantation
development.

3. ADHESION CONTROLS CELL POLARITY
The initiation of adhesion at the 8-cell stage also directs establishment of
the first forms of cellular polarity in the developing mouse embryo. During this
time, the actin cytoskeleton is reorganized to establish an apicobasal polarity
together with the formation of an apical microvillous pole (Handyside,
1980) and a polarized distribution of cytoplasmic organelles and cytoskeletal
elements (Fleming & Pickering, 1985; Houliston, Pickering, & Maro,
1987; Johnson & Maro, 1984). Some classical cell polarity proteins such as
Ezrin, Pard6b, and the aPKCs (PKCζ and PKCλ) are then localized to the apical domain while Par-1, Jam-1, and Na/K ATPase are found at basolateral
cell–cell contacts (Barcroft, Moseley, Lingrel, & Watson, 2004; Louvet,
Aghion, Santa-Maria, Mangeat, & Maro, 1996; Pauken & Capco, 2000;
Thomas et al., 2004; Vinot et al., 2005; Wang, Ojakian, & Nelson, 1990).
Polar cells remain on the outside of the embryo and differentiate into

trophectoderm, whereas apolar cells are enclosed inside the embryo and form
the pluripotent ICM (Dyce, George, Goodall, & Fleming, 1987). Adhesion
does not appear to be required for the initiation of polarization as cells isolated
from early mouse embryos can polarize in the absence of cell–cell contacts
( Johnson, Maro, & Takeichi, 1986; Ziomek & Johnson, 1980) or following
inhibition of E-cadherin function with antibodies (Houliston, Pickering, &
Maro, 1989; Johnson et al., 1986). However, the polarization of these disaggregated cells is delayed and also random in orientation. Deleting both
maternal and zygotic E-cadherin confirms that E-cadherin is required to
restrict the area of the apical domain and confine basolateral proteins, ensuring
correct segregation of apical and basolateral domains (Stephenson et al., 2010).
Together, these studies reveal that E-cadherin-mediated adhesion controls the
timing and axis of polarization in the preimplantation mouse embryo.

4. ADHESION DETERMINES CELL FATE
Polarization has been closely linked to cell fate. Disruption of the apical
proteins Par3 and PKCλ preferentially directs cells in the embryo toward an


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Melanie D. White and Nicolas Plachta

ICM fate (Plusa et al., 2005). By determining the orientation of polarity,
intercellular adhesion has a critical role in specifying cell fate. The maternal
and zygotic deletion of E-cadherin demonstrated that E-cadherin-mediated
adhesion is important to restrict trophectoderm fate (Stephenson et al., 2010).
More cells in the mutant embryos express the trophectoderm-specific marker
Cdx2, and the normal spatial allocation of trophectoderm cells to the outside
of the embryo and ICM cells to the inside is disrupted. Recent work has now
established how adhesion affects cell fate indirectly through polarity and

shown that it also has an additional direct role in fate determination (Fig. 5).
Differentiation of the outer cells of the morula into trophectoderm requires
expression of the transcription factors Cdx2 and Gata3, which in turn is driven

Figure 5 Adhesion and polarity determine cell fate in the preimplantation mouse
embryo. The apical polarity complex in outer cells sequesters components of the Hippo
signaling pathway preventing its activation. Unphosphorylated Yap can enter the
nucleus and drive expression of trophectoderm-specific genes. In inner cells, Amot
localizes to adherens junctions where it binds to Lats1/2 and the E-cadherin adhesion
complex via Nrf2. Lats 1/2 phosphorylates Amot, activating it and this complex phosphorylates Yap. Phosphorylated Yap is excluded from the nucleus and the Hippo pathway is activated, allowing transcription of inner cell mass specific genes.


Adhesion in the Early Mouse Embryo

9

by the transcription factor Tead4 and its coactivator, Yap1 (Ralston et al.,
2010). Tead4 knockout mice can specify an ICM but do not form the
trophectoderm (Nishioka et al., 2008; Yagi et al., 2007). The Hippo signaling
pathway regulates Yap1 subcellular localization by phosphorylation (Nishioka
et al., 2009). When Hippo signaling is active, the kinase Lats1/2 phosphorylates
Yap1, excluding it from the nucleus, promoting transcription of ICM-specific
genes, and repressing trophectoderm fate. In the absence of sufficient Hippo
signaling, Yap1 is free to enter the cell nucleus and induce transcription of
the trophectoderm-specific genes Cdx2 and Gata3. However, Yap1, Tead4,
and the members of the Hippo signaling pathway are expressed in all cells of
the preimplantation mouse embryo. So, how is Hippo signaling suppressed
in outer cells and activated in inner cells? The answer lies in sequestration of
key members of the pathway by proteins involved in polarity and adhesion.
Angiomotin (Amot) and Nf2 are required to activate Lats1/2 and switch

on the Hippo signaling pathway (Hirate et al., 2013). Nf2 is uniformly distributed through the membrane but in outer cells, Amot is sequestered by
components of the apical polarity complex and localized to the apical
domain (Hirate et al., 2013). Here, it is bound to actin and held in an inactive
state. Lats1/2 may also be sequestered by the polarity complex as it too has an
apical localization in outer cells (Cockburn, Biechele, Garner, & Rossant,
2013). The apical localization of Amot and Lats1/2 prevents activation of
the Hippo signaling pathway, unphosphorylated Yap1 enters the nucleus
and the cell reverts to a trophectoderm fate.
In inner cells that lack apical polarity, Amot can interact with Lats1/2 and
the E-cadherin adhesion complex at adherens junctions via Nf2 (Hirate
et al., 2013). This interaction activates Amot and stabilizes it at adherens
junctions where it switches the Hippo signaling pathway on. Phosphorylated Yap1 is excluded from the nucleus and ICM genes are transcribed.
When Nf2 is removed by maternal and zygotic deletion, the Hippo
pathway cannot be activated and the mutant embryos fail to generate an
ICM (Cockburn et al., 2013). Instead, inner cells express trophectodermspecific genes demonstrating that, regardless of their position in the embryo,
cells revert to a trophectoderm fate when the components of the Hippo
pathway are not correctly localized to adherens junctions.

5. EMERGING TECHNOLOGIES TO STUDY ADHESION
In vivo imaging of developing mouse embryos has recently been used
to track cell progeny and fate (Bischoff, Parfitt, & Zernicka-Goetz, 2008;


10

Melanie D. White and Nicolas Plachta

Kurotaki, Hatta, Nakao, Nabeshima, & Fujimori, 2007; Morris et al., 2010;
Plachta, Bollenbach, Pease, Fraser, & Pantazis, 2011). In coming years,
imaging technologies that allow tracking of the levels, stability and distribution of proteins controlling adhesion will enable visualization of the establishment of adhesion and how this influences downstream processes such as

polarity and fate. For example, photobleaching methods like fluorescence
recovery after photobleaching (FRAP) allow measurement of the overall
stability of membrane and cytoplasmic proteins regulating adhesion. FRAP
has been useful to elucidate how E-cadherin is positioned and maintained at
adherens junctions in different cellular contexts (de Beco, Gueudry,
Amblard, & Coscoy, 2009; Huang et al., 2011). However, it has not been
applied to study adhesion yet in the early mouse embryo. Methods like fluorescence correlation spectroscopy (FCS), based on the study of the fluorescent fluctuations in a small volume, can provide even more detailed
information about the biophysical properties of proteins (Digman &
Gratton, 2011). FCS has been utilized to measure the dynamics of proteins
related to cell adhesion, such as actin (Gowrishankar et al., 2012). More
recently, FCS has been applied to living preimplantation mouse embryos
to study the dynamics of nuclear gene-regulatory proteins (Kaur et al.,
2013). In the future, it will be important to establish its use for cytoplasmic
and membrane-bound proteins involved in adhesion to reveal their dynamic
behavior throughout development in different subcellular contexts. Superresolution microscopy approaches are already starting to yield impressively
detailed information about adhesion complexes in fixed cells (Guillaume
et al., 2013; Indra, Hong, Troyanovsky, Kormos, & Troyanovsky, 2013).
So far, these methods are restricted to thin specimens or sliced tissues, but
they may soon be applicable in more complex three-dimensional structures
such as the mouse embryo and may even allow data to be gathered at different time points, as has been achieved in living brain slices (Berning,
Willig, Steffens, Dibaj, & Hell, 2012).
Finally, the study of adhesion will be greatly advanced by the development
of engineered proteins. E-cadherin has been engineered to contain a stretchable F€
orster resonance energy transfer probe that can be used to directly measure the tensile forces transmitted through the cytoplasmic domain of
E-cadherin (Borghi et al., 2012). So far, this approach has only been used
in cultured epithelial cells, but it will be highly informative to visualize tension
across adherens junctions in the developing mouse embryo. Optogenetic control of Rho-family GTPases engineered to be light-sensitive has been used to


Adhesion in the Early Mouse Embryo

11

remodel actin and alter cell shape in vitro (Leung, Otomo, Chory, & Rosen,
2008; Levskaya, Weiner, Lim, & Voigt, 2009). Chromophore assisted light
inactivation can be used to selectively inactivate proteins (reviewed in
Sano, Watanabe, & Matsunaga, 2014). It will be revealing to use these technologies to express light-sensitive adhesion proteins in the preimplantation
mouse embryo, and specifically alter their activity through targeting of laser
stimulation to defined subcellular microdomains.

6. QUESTIONS FOR THE FUTURE
Recent studies have just begun to explain the connection between cell
adhesion, polarity, and fate in the early mammalian embryo. However,
many important questions remain unanswered and the development of several new technologies will enable them to be addressed in future work.
For example, it remains unclear whether, aside from its role in activating
the Hippo pathway, adhesion has any other signaling functions in the mouse
preimplantation embryo. E-cadherin has been shown to negatively regulate
the receptor tyrosine kinases EGFR, IGF-1R, and c-Met and inhibit cell
growth in vitro in an adhesion-dependent manner (Qian, Karpova,
Sheppard, McNally, & Lowy, 2004). The small GTPases Rho, Rac, and
Cdc42 are activated upon E-cadherin-mediated cell contact formation
in vitro (Betson, Lozano, Zhang, & Braga, 2002; Calautti et al., 2002;
Kim, Li, & Sacks, 2000; Kovacs, Ali, McCormack, & Yap, 2002; Noren,
Niessen, Gumbiner, & Burridge, 2001). Little is known about potential signaling functions for adhesion in the preimplantation mouse embryo. However, E-cadherin has recently been shown to be required to activate Igf1r
signaling at adherens junctions for trophectoderm formation (Bedzhov,
Liszewska, Kanzler, & Stemmler, 2012). It remains to be established whether
it also influences signaling from other receptor tyrosine kinases. Given the
in vitro evidence and the recent role for adherens junctions in modulating the
Hippo signaling pathway, it seems likely that adherens junctions may serve as
scaffolds where signaling proteins are recruited and regulated. Careful imaging studies will be required to determine if this is the case in the embryo.

In addition, it will be important to establish if adhesion is involved in
signaling through the E-cadherin-dependent filopodia that control compaction. A similar mechanism has been reported in Drosophila where filopodialike structures known as cytonemes are involved in spatial patterning of the


12

Melanie D. White and Nicolas Plachta

embryo (Roy, Hsiung, & Kornberg, 2011). Further research will ascertain
whether adhesion contributes to spatiotemporally regulated signaling outside
of adherens junctions in the preimplantation mouse embryo.
Most of our current knowledge about adhesion in the mouse embryo
relates to E-cadherin. However, it is likely that other proteins and mechanisms also play a role. When E-cadherin is disrupted, cells become rounder
but still maintain some adhesive properties (Fierro-Gonzalez et al., 2013;
Shirayoshi, Okada, & Takeichi, 1983; Stephenson et al., 2010). While some
of this remaining adhesion may be explained by residual expression of other
less known cadherin members (for example, P-Cadherin; Stephenson et al.,
2010), other proteins, such as GalTase (Bayna, Shaper, & Shur, 1988) may
also participate.
Adhesion is one of the main physical forces acting between cells of the
embryo. However, it remains largely unknown how the adhesion force
interacts with some of the other main forces acting in the embryo. In particular, it is now well recognized that cells behave like viscoelastic fluids and
the opposing forces of adhesion and cortical tension determine the degree of
cell–cell contact (Lecuit & Lenne, 2007). Cells have been shown to behave
like fluid objects, with a tendency to maximize their intercellular adhesion in
the same way that liquids maximize their intermolecular attraction and
simultaneously minimize their free surface energy (Foty, Forgacs,
Pfleger, & Steinberg, 1994; Heintzelman, Phillips, & Davis, 1978).
E-cadherin is likely to initiate this adhesive process by bringing cell surfaces
into contact and providing the first anchoring point. The adhesion may then

be reinforced by cells minimizing their cell–liquid interfacial tension as
recently proposed in the zebrafish embryo (Maitre et al., 2012). Research
addressing the overriding question of how adhesion and tension interact
to control cell–cell interactions and embryo patterning has begun in other
nonmammalian systems like the zebrafish (Maitre et al., 2012) and Drosophila
(Rauzi, Verant, Lecuit, & Lenne, 2008) embryos using laser ablations of the
actomyosin cortex or indirect measurements of tensile forces. Similar work
will likely be focused in the mammalian embryo in the future.
In summary, future work should provide a better understanding of how
adhesion is integrated with other key processes patterning the early mammalian embryo and controlling cell–cell interactions in vivo. The application of
new technologies permitting real time and quantitative studies will help
reveal how adhesion controls cell shape, fate, and position and vice versa
in the mammalian embryo.


Adhesion in the Early Mouse Embryo

13

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