Current Biology

Review

Coordination of Morphogenesis and Cell-Fate

Specification in Development
Chii J. Chan1,*, Carl-Philipp Heisenberg2, and Takashi Hiiragi1,*
1European Molecular Biology Laboratory, 69117 Heidelberg, Germany
2Instituteof Science and Technology Austria, 3400 Klosterneuburg, Austria
*Correspondence: chii.chan@embl.de (C.J.C.), hiiragi@embl.de (T.H.)
http://dx.doi.org/10.1016/j.cub.2017.07.010

During animal development, cell-fate-specific changes in gene expression can modify the material properties
of a tissue and drive tissue morphogenesis. While mechanistic insights into the genetic control of tissue-
shaping events are beginning to emerge, how tissue morphogenesis and mechanics can reciprocally impact
cell-fate specification remains relatively unexplored. Here we review recent findings reporting how multicel-
lular morphogenetic events and their underlying mechanical forces can feed back into gene regulatory path-
ways to specify cell fate. We further discuss emerging techniques that allow for the direct measurement and
manipulation of mechanical signals in vivo, offering unprecedented access to study mechanotransduction
during development. Examination of the mechanical control of cell fate during tissue morphogenesis will
pave the way to an integrated understanding of the design principles that underlie robust tissue patterning
in embryonic development.

Introduction signals in vivo and long-term cell-lineage tracking during

The fields of physical biology and mechanobiology have bur-
geoned in the past decade [1–3], revealing that mechanical
forces play an integral role in tissue morphogenesis. In recent
years, substantial progress has been made towards understand-
ing how mechanical cues, either extrinsically induced by the
cellular microenvironment or intracellularly generated, can be
transduced into biochemical signals that regulate cell prolifera-
tion, migration and differentiation [3–5]. Understanding the
crosstalk between tissue-scale mechanics and cell-fate specifi-
cation is essential to uncover the key design principles that regu-
late robust tissue patterning during development. Furthermore,
these principles may provide a mechanistic framework for future
tissue engineering efforts, with clear implications for disease
modeling and regenerative medicine [6–8].
Extensive studies in the past have focused on elucidating
how differential gene expression can modify the physical
properties of cells and influence tissue patterning [9,10]. While
these studies provide insights into how differential adhesive or
contractile properties may arise in different cell types to
mediate cell sorting and tissue shaping [11,12], how genetic
cascades link cell-fate specification to tissue morphogenesis
remains unclear. Even less well understood is how morphogen-
esis and tissue mechanics can conversely feed back into
cell-fate decisions and regulate developmental programs. In
this review, we specifically discuss the potential upstream
roles of tissue geometry and forces in controlling cell-fate
specification during embryonic development. We first summa-
rize our current understanding of the molecular mechanisms
underlying cellular mechanotransduction in vitro and in vivo.
We then describe how changes in 3D tissue architecture,
cellular rearrangements and cytoskeletal flow can shape
morphogen gradients and tissue polarity to critically influence
cell-fate determination. Finally, we review new biophysical tools
that allow the measurement and manipulation of mechanical
development.
Impact of Tissue Stress on Cell-Fate Specification
Cells can ‘sense’ forces through mechanosensing and mecha-
notransduction, and subsequently control their differentiation.
The terms ‘mechanosensation’ and ‘mechanotransduction’
have been used extensively, and sometimes interchangeably,
in different biological contexts. In this review we specifically
define mechanosensation as the physical mechanisms by which
a cell senses mechanical cues, such as forces or stiffness, from
its microenvironment. Meanwhile mechanotransduction involves
the conversion of these mechanical cues to biochemical signals
downstream of mechanosensation. While such transduction
may affect various cellular components at a post-translational
level, here we focus on how it triggers gene expression changes
to impact fate specification.
Cellular Mechanisms of Mechanotransduction
In general, cellular mechanotransduction can be classified into
chemical or physical transduction [13]. Chemical transduction
relies on a series of chemical signaling cascades bridging the
plasma membrane and the nucleus. When the cells experience
mechanical strain, focal adhesion proteins such as zyxin and
paxillin can alter their binding kinetics and shuttle between the
cytoplasm and nucleus to modulate transcriptional activity.
Force-induced phosphorylation of E-cadherin-bound b-catenin
at intercellular junctions results in its translocation to the nucleus
and activation of transcription of downstream effectors [14–16].
Mechanotransduction can also involve the nuclear translocation
of transcription factors from the cytoplasm, such as NF-kB or
MAL, in response to fluid shear [17] and mechanical stress
[18,19], respectively. Recently, the stretch-activated channels
Piezo1 and Piezo2 were identified as key mechanosensors regu-
lating calcium signaling by sensing changes in membrane
tension [20–22] and they can potentially play a role in neural

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Current Biology
Review
stem cell differentiation [23]. Another well-studied signaling
cascade involved in mechanotransduction is Yap/Taz signaling,
where mechanical tension arising from physical stretching of
cells or increased stiffness of the extracellular matrix (ECM) ac-
tivates F-actin remodeling and Yap/Taz nuclear localization
[24,25]. While ECM stiffness is known to direct differentiation
or self-renewal of embryonic or somatic stem cells [26–30],
ECM viscosity can also influence cell-fate specification, for
example during the osteogenic differentiation of mesenchymal
stem cells [31]. Collectively, these studies highlight the diverse
set of mechanical cues that cells can sense and biochemically
transduce to regulate differentiation.
Physical mechanotransduction relies on direct force transmis-
sion from the cell surface to the nucleus through physical
coupling between the nuclear membrane and the extracellular
space by cytoskeletal components. The nucleus has been pro-
posed to act as a mechanosensor, whereby changes in nuclear
shape can evoke transcriptional changes by locally altering the
spatial accessibility of chromatin to transcriptional regulators
[13]. Importantly, the physical links between the cytoskeleton
and nuclear membrane proteins allow the entire cell to function
as a single mechanically coupled system [32–36]. A key molec-
ular player involved in such physical transduction is the nuclear
lamina component lamin A [37,38]. In stiff tissue, tension from
the ECM transmitted via the cytoskeleton to the nucleus can
enhance lamin A transcription and stability of lamin A/C, leading
to activation of Yap, serum response factor (SRF) and retinoic
acid signaling pathways [37]. A recent study on mouse epidermal
stem cells also revealed that stress transmitted through the actin
cytoskeleton can induce enrichment of the nuclear membrane
protein emerin on the outer face of the nuclear membrane, lead-
ing to Polycomb-mediated gene silencing and precocious differ-
entiation [39].
Mechanotransduction in Development
During embryonic development, forces are generated and trans-
mitted across multiple scales. The interplay between tissue me-
chanics and biochemical signaling involves multiple feedback
interactions, rendering the assessment of mechanotransduction
in vivo at the organismal level non-trivial. To date, mechanotrans-
duction has been identified in only a few developmental contexts
where the underlying genetics, cell-fate changes and mechanics
are sufficiently well characterized.
In Drosophila embryos, tissue deformations caused by germ-
band extension upregulate the expression of the transcription
factor Twist via nuclear translocation of b-catenin (Figure 1A)
[14]. Furthermore, both static and pulsed mechanical deforma-
tions of mesodermal cells have been shown to promote Folded
gastrulation (Fog) signaling and apical myosin II accumulation,
which is required for mesoderm invagination prior to germ-
band extension [40,41]. Interestingly, mechanical strains occur-
ring during the early phases of gastrulation in zebrafish and fly
embryos both lead to nuclear translocation of b-catenin, thereby
triggering the expression of transcription factors required for
early mesoderm identity [15]. In addition, tissue compression reg-
ulates tooth formation in mouse embryos, where morphogen-
induced condensation of mesenchymal cells beneath the dental
epithelium leads to odontogenetic cell-fate specification [42].
Mechanical cues also play a key role during mouse embry-
onic development. In pre-implantation stage mouse embryos,
asymmetrical inheritance of less-contractile apical domains re-
sults in a polarized daughter cell that exhibits lower cortical
contractility than its apolar sister cell (Figure 1B) [43]. The
difference in contractility leads to subsequent internalization of
the apolar cell. The outer polar cell adopts the trophectoderm
fate with Yap localized to the nucleus, while the apolar cell
adopts the inner cell mass fate with Yap restricted to the cyto-
plasm [44]. Notably, reducing cortical contractility leads to a
loss of Yap in the nuclei of externally positioned cells, pointing
to the possibility that tension in the outer cells may trigger the
Yap nuclear localization required for trophectoderm cell-fate
specification.
Fluid flow also plays a critical role in driving cell-fate specifica-
tion in development. For example, during development of the
circulatory system, haemodynamic forces can induce an endo-
thelial-to-hematopoietic cell-fate switch through Runx1, a mas-
ter transcription factor for hematopoiesis (Figure 1C) [45,46]. In
zebrafish heart valve morphogenesis, intracardiac and pericar-
diac fluid flow influence cardiac chamber differentiation
[47,48], while klf2a, an endocardial flow-responsive gene, con-
trols fibronectin synthesis and cardiac valve development [49].
Recent work demonstrated that Notch signaling is sensitive to
shear stress and plays a role in arteriovenous differentiation
[50,51]. Another morphogenetic force that shapes cell differenti-
ation is hydrostatic pressure. For example, the sustained stretch
of undifferentiated pulmonary mesenchymal cells by fluid pres-
sure leads to SRF activation and smooth muscle differentiation
in lung organ cultures (Figure 1D) [52]. During lymphatic vascula-
ture development, increased interstitial fluid pressure leads to
stretch and proliferation of endothelial cells [53]. Pressure me-
chanotransduction in urothelial cells mediated by Piezo1 leads
to ATP release, activating afferent nerves of the bladder to indi-
cate bladder fullness [54,55]. Interestingly, osmotic pressure can
modulate plasma membrane tension, which can influence endo-
cytosis of signaling molecules involved in cell differentiation [56].
There is also growing evidence from plant studies that organ
shape changes generate mechanical signals that directly impact
cell-fate determination (Figure 1E). In Arabidopsis shoot meri-
stems, differential growth generates anisotropic mechanical
stress, leading to tissue folding [57]. It was shown that the
expression of SHOOT MERISTEMLESS, a key regulator of meri-
stematic identity, is correlated with tissue curvature, and its
expression can be induced by mechanical stress through an
auxin-independent mechanotransduction pathway. Since tissue
folding naturally occurs during morphogenesis, such ‘shape-to-
gene’ feedback may serve to constrain the gene regulatory
network and ensure developmental robustness. In the following
section we discuss the influence of tissue geometry on cell-fate
specification.
Impact of Geometry and Cytoskeletal Forces on Tissue
Patterning
Establishment of well-defined forms and patterns during devel-
opment requires that cells recognize their position within the em-
bryo and differentiate accordingly. Two current pre-eminent
concepts for tissue patterning are based on the positional infor-
mation and reaction–diffusion models [58,59]. In brief, positional
information involves the graded distribution of a morphogen
across the tissue, from which the cells interpret their position
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A B
Compression of Trophectoderm fate
stomodeal cells Germ band extension Apical domain

Nuclear
β-catenin

Twist Higher contractility

Inner cell mass fate
Anterior midgut

C D

Shear stress Hydrostatic pressure

Haemodynamic flow

Nitric oxide
Mechanical stretching
of mesenchymal cells
Fluid pressure
Runx1 in lung airway
Smooth muscle
differentiation

Endothelial–hematopoietic
cell fate transition

E
Tissue folding

Mechanical stress
Shoot
meristem
STM and
meristematic identity

STM is upregulated at curved regions of

boundary domains
Current Biology

Figure 1. Examples of mechanotransduction during embryonic and adult organ development.

(A) Tissue-scale compression in Drosophila embryos caused by germ-band extension triggers Src42A-dependent nuclear translocation of b-catenin in anterior
stomodeal cells, thereby activating the transcription of Twist, which is crucial for subsequent midgut differentiation. (B) In early mouse embryonic development,
asymmetrical inheritance of the apical domain results in a polarized daughter cell that exhibits lower cortical contractility than its apolar sister cell. The difference
in contractility leads to internalization of the apolar cell. The outer polar cell adopts the trophectoderm fate with nuclear Yap localization while the apolar cell
adopts the inner cell mass fate with cytosolic Yap localization. (C) During the formation of the hematopoietic system, shear stress can induce nitric oxide
production and upregulation of Runx1 transcriptional factor, thereby inducing endothelial-to-hematopoietic cell-fate switch. (D) Mechanical stretching of the lung
drives myogenesis in airway smooth muscle, which is crucial for bronchial development. This process is mediated by the activation of the transcription factor
SRF. (E) In Arabidopsis, strong mechanical stresses arising from tissue folding at the shoot apical meristem can induce the expression of STM, a master regulator
of meristematic identity.

to make fate choices. The morphogen concentration therefore
provides positional coordinates along the embryonic axis. The
reaction–diffusion model involves a self-amplifying local signal
and a long-range inhibitor that is stimulated by the former. Inter-
actions between these two components lead to the generation of
periodic patterns of diffusible morphogens and consequently
cell fates. While the significance of both models has long been
recognized in developmental biology, these models are so far
based purely on biochemical signaling. Here we discuss the
new possibility that cytoskeletal forces, cell adhesion and polar-
ity, and tissue-scale mechanical signals can confer positional in-
formation and control gene expression and fate at the cellular
level, thereby allowing cells to coordinate embryonic patterning.
Cytoskeletal Forces Drive Tissue Patterning
Forces generated by motor proteins can couple with biochem-
ical signals to generate morphogen gradients and patterns

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A Cytoskeletal force and advection

Advection of aPAR by
actomyosin-dependent cytoplasmic flow

Stably polarized
C. elegans zygote

pPAR binds to
posterior membrane

B Position sensing via polarity C Multicellular rearrangement

FGF trapped in lumen

Lateral line
primodium

Differential cell contact Apical domain Trophectoderm fate Luminal signaling leads to enhanced differentiation

D Geometric constraints

TGF-β localized at apical surface

Sonic
hedgehog

Edge Center
Mechanical
buckling
BMP4

Local increase in morphogen

induces villus cluster NOGGIN

Current Biology

Figure 2. Cytoskeletal forces and geometric cues can mechanically feed back to morphogen signaling.
(A) Mechanochemical patterning in C. elegans zygote is established through the advection of anterior PAR proteins (red) by actomyosin-driven cytoplasmic flow.
The binding of posterior PARs (blue) to the posterior membrane results in the amplification and stabilization of polarity by a reaction–diffusion mechanism. (B) In
8-cell early mouse embryos, contact asymmetry induces apical domain formation at the contact-free cell surface. Upon division, the daughter cells that inherit the
apical domain adopt the trophectoderm fate while the apolar daughter cells may or may not become trophectoderm, depending on their eventual position.
(C) Luminal signaling can impact cell-fate acquisition in a multicellular context. In migrating zebrafish lateral line primordium, concerted apical constriction leads
to the formation of a microlumen. Local trapping of FGF in these microlumens leads to enhanced signaling and restricted cellular differentiation in the neighboring
cells. (D) Left: In the mouse gut, mechanical buckling of the epithelium due to growth leads to a local build-up of Shh signals in the villi, which then restricts
progenitor cells to the base of these structures. Right: Geometric confinement of human embryonic stem cells leads to the spatial ordering of germ layers
recapitulating human gastrulation. In the presence of BMP4, differential localization of TGF-b receptors at the colony edge (apical) and center (basolateral)
triggers differential signaling and generates a reaction–diffusion mechanism that involves the BMP inhibitor Noggin.

[60,61]. In Caenorhabditis elegans zygotes, actomyosin contrac-
tion induces large-scale, anterior-directed cortical flows, which
in turn transport polarity and cell-fate-determining proteins to
the anterior half of the cell [62]. Importantly, if contraction itself
causes flows that carry regulators of contraction, such as parti-
tioning defective (PAR) proteins, this will lead to positive feed-
back and subcellular patterning (Figure 2A) [63]. Following
division, asymmetrical cortical contractility between sister cells
in turn induces chiral cortical actomyosin flows, thereby driving
skewing of the spindle orientation at the 4-cell stage and estab-
lishing the left–right body axis [64]. From the 2- to 4-cell stage,
such chiral actomyosin flows can also displace the midbody, a
remnant of the previous division, to the future ventral side of
the embryo, thereby influencing spindle orientation and estab-
lishing dorsoventral axis patterning [65]. These studies clearly
highlight the role of differential cell-contact signaling in partition-
ing cell fates along the body axis.
Microscopic fluid flow can also drive cell differentiation and
embryonic patterning through chiral machineries in tissues. Dur-
ing gastrulation in several vertebrate species, posteriorly tilted
cilia in the ventral node rotate in a clockwise fashion to induce
leftward extracellular fluid flow [66]. This unidirectional flow has

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been proposed to trigger asymmetrical transport of morpho-
gens, such as Sonic hedgehog (Shh) and retinoic acid, ultimately
leading to left–right asymmetrical gene expression throughout
the entire body [67]. Alternatively, the unidirectional flow has
been suggested to trigger a mechanosensitive response in the
cilia at the edge of the node, which in turn activates subsequent
asymmetrical signaling [68,69].
Position Sensing at the Cellular Level
For early mouse embryogenesis, the inside–outside model pro-
poses that cell fate depends on the position of a cell at the morula
stage, with cells on the inside forming the inner cell mass and
cells on the outside taking up the trophectoderm fate [70]. How-
ever, the underlying mechanism remained elusive. A recent study
shed new light in this direction by showing that, at the eight-cell
stage, contact asymmetry induces apical domain formation on
the contact-free cell surface (Figure 2B) [71]. Upon division, the
daughter cells that inherit the apical domain adopt the trophecto-
derm fate, while the apolar daughter cells may or may not
become trophectoderm, depending on whether they are pushed
out to the surface due to mechanical constraint. This work there-
fore identified the presence of a contact-free cell surface and api-
cal polarization as crucial determinants distinguishing the
‘outside’ from ‘inside’ positions, thereby directing cell fate.
Tissue Tension Orients Cell Division and Polarity
There is growing evidence that tissue-scale tension can orient
cell division patterns and polarity during development [72,73],
which can influence cell-fate determination. The mechanosens-
ing mechanism for Hertwig’s rule describes how astral microtu-
bule length and spindle positioning are influenced by cell shape
to result in division along the long axis of the cell. A recent study
in Drosophila epithelia further revealed that the tricellular junc-
tions (TCJs), to which the dynein-associated protein Mud
localizes and pulls on the astral microtubules, can translate infor-
mation about interphase cell shape anisotropy into a ‘stable’
biochemical cue, thereby determining spindle orientation during
mitotic rounding [74]. Mechanical stress has also been shown to
induce tissue-level planar cell polarity (PCP) in development. In
the Drosophila wing disc, global anisotropic tension originating
from the contraction of the wing hinge results in the reorientation
of intercellular junctions, cell elongation and tissue flow, which
collectively establish long-range PCP along the proximal–distal
axis [75,76].
Generation of cell-fate diversity from oriented cell division is
also observed in vertebrates. In Xenopus embryos, the deep
and superficial cells at the blastula stage show differential
gene expression for primary neurogenesis. The distinct fate of
deep versus superficial cells was proposed to be generated by
oriented cell divisions along the apicobasal axis at the surface
of the embryo, giving rise to a superficial cell with an inherited
apical domain marked by atypical protein kinase C (aPKC) and
an apolar deep cell [77]. Similarly, in the early zebrafish gastrula,
cell-division orientation is determined by cell-shape changes,
which are in turn regulated by tissue tension and geometrical
constraints imposed by the overall tissue surface area [78].
This leads to the robust formation and segregation of surface
epithelial and deep cells.
Cellular Rearrangements Shape Tissue Pattern
Active cell sorting mediated by changes in cell adhesion and
cortical tension can also drive tissue patterning. In the inner
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Review
cell mass of mouse blastocysts, cells undergo active cell sorting
to form two distinct tissues, the epiblast and the primitive endo-
derm [79,80]. Such sorting behavior was suggested to arise from
the progressive polarization and epithelialization of the future
primitive endoderm facing the blastocoel, leading to the spatial
separation of primitive endoderm cells from the epiblast cluster
[81]. Differential cortical tension and/or adhesion in the inner
cell mass have also been proposed to contribute to active cell
sorting during lineage segregation [80], although these mechan-
ical properties remain uncharacterized.
In the zebrafish neural tube, cell movements mediated by
changes in cell–cell adhesion appear to correct the initially noisy
Shh signaling pattern to organize the neural progenitor cells into
strictly demarcated domains [82]. Changes in tissue architecture
can also feed back directly onto the activity of signaling centers.
During zebrafish lateral line formation, fibroblast growth factor
(FGF) activity within the tissue controls the frequency at which
rosette-like mechanosensory organs are deposited [83]. Here,
the lateral line primordium, consisting of about 100 cells, mi-
grates along the length of the developing body. During this pro-
cess, concerted apical constriction of discrete groups of cells
leads to the formation of rosette-like structures and the assem-
bly of a central microlumen that traps the secreted FGF
(Figure 2C). The local increase in FGF ligand concentration in
turn creates a positive feedback loop by maintaining the apical
constriction process and rosette formation [84]. Luminal
signaling therefore represents an excellent mechanism by which
cellular rearrangement can feed back to locally restrict and
enhance molecular signaling and direct cell differentiation within
migrating tissues.
Geometric Constraints Modulate Spatial Patterning of
Cell Fate
Recent studies have highlighted the extensive interplay between
tissue geometry and signaling. Physical deformation of the
morphogenetic field in three dimensions can generate graded
signaling activities to pattern tissues. For example, during verte-
brate gut formation proliferating intestinal stem cell progenitors,
which are initially evenly distributed throughout the epithelium,
become restricted to the base of the villi following tissue buckling
[85,86]. Physical buckling due to continued growth under me-
chanical constraint causes the epithelial Shh signal to become
locally concentrated at the villi tips (Figure 2D, left) leading to vil-
lus cluster formation. Local maxima of Shh signals then induce
reciprocal signaling by bone morphogenetic proteins (BMPs)
within the cluster, repressing intestinal stem cell identity in the
overlying epithelium and restricting stem cell proliferation to
the base of the villi. Another example comes from post-implanta-
tion stage mouse embryos, where mechanical constraints from
the maternal uterine tissues have been proposed to lead to the
elongated cylindrical shape of the embryos and thinning of the
basement membrane at the distal tip [87]. This induces the for-
mation of distal visceral endoderm cells that play a role in embry-
onic anterior–posterior axis patterning. Interestingly, a recent
study demonstrated that the distal visceral endoderm can still
form in the absence of external mechanical constraints [88].
The discrepancy may result from differences in the culture con-
ditions or methods of expression studies.
Geometric cues can also trigger self-organized patterning dur-
ing embryonic development. Recent in vitro studies using human
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A Measuring forces and stiffness in vivo

Microdroplet
ΔP
Tension sensor
module F-actin

Pressure
sensor

FRET biosensor
Micropipette aspiration
Droplet-based sensor

High stiffness

Pressure

Tension
2D epithelium

Force inference
Brillouin microscopy

B Spatio-temporal control of forces in vivo

Optogenetics Magnetic tweezer

Blue laser

Inhibition of apical constriction

blocks mesoderm invagination Magnetic liposomes

C Ex vivo reduced systems

Organoid technology
Micropatterning

Intestinal organoid
Polarity inversion
controlled by
substrate stiffness

3D hydrogel with tunable

mechanical properties e.g.
Cell doublets on 2D micropattern
ligand density, matrix stiffness

Current Biology

Figure 3. Techniques to study morphogenetic feedback mechanisms in vivo.

(A) FRET biosensor: FRET tension sensor with known spring stiffness is inserted between F-actin molecules. The length of module increases with the tension
between the molecules, resulting in lower FRET efficiency. Quantifying the FRET efficiency generates a spatiotemporal map of endogenous forces in vivo.
Micropipette aspiration: Negative aspiration pressure is applied through a micropipette in contact with the mouse embryo blastomeres. Using the Young-Laplace
equation, the surface tension of the blastomeres can be measured during mouse compaction development. Droplet-based sensor: Microdroplets coated with
fluorescent surfactant molecules are inserted into the tissue. With the known surface tension and 3D deformation of the droplets, the anisotropic stress of the
(legend continued on next page)

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‘gastruloids’ (3D aggregates of embryonic stem cells) grown on
micropatterned substrates demonstrated that spatial confine-
ment of human embryonic stem cells can trigger spatial
patterning similar to that observed during gastrulation [89,90].
Mechanistically, transforming growth factor b (TGF-b) receptors
were found to be differentially localized to the apical and baso-
lateral surfaces of the cells at the edges and center of the colony,
respectively, thereby triggering different signaling responses
across the colony in the presence of the BMP4 ligand
(Figure 2D, right). BMP4 subsequently activates Noggin, a
secreted BMP4 inhibitor, and generates self-organized gene
expression patterning through a reaction–diffusion mechanism
[90]. In this case, the geometric cues that allow the cells to
‘sense’ the edge of the tissue come from the boundary condition
that provides the initial receptor pre-pattern.
In Vivo Techniques to Study Morphogenetic Feedback
Mechanisms
The study of how tissue morphogenesis and mechanics may in-
fluence cell-fate specification during development requires
simultaneous imaging and/or perturbation of tissue forces,
morphology and cell fate in vivo. In this section, we review
recent technological progress in these areas and discuss
complementary approaches of ex vivo assays that can disen-
tangle the contribution of each parameter governing cell-fate
specification.
Non-Invasive Approaches to Measure Tissue Stress and
Stiffness In Vivo
A number of approaches have been developed to quantify
forces in living tissues and embryos [91,92]. Here, we limit
our discussion to selected techniques that allow non-invasive,
spatiotemporal mapping of tissue mechanics at develop-
mental timescales. One technique that offers high spatiotem-
poral resolution of intracellular or intercellular tensions in living
tissues is the use of Forster resonance energy transfer (FRET)-
based tension sensors [93,94]. The FRET signal intensity de-
pends on the extension of a molecular spring between the
donor and acceptor fluorophore, which can be anchored to
molecular components of adherens junctions, such as a-cate-
nin and E-cadherin (Figure 3A). Once the spring constant is
calibrated, the FRET signal provides an effective readout
about molecular tension at the cell–cell contact during devel-
opment. Other tools include a-actinin tension sensors, which
have been developed to reveal lineage-dependent intercellular
tension during Xenopus laevis embryonic development [95].
Similarly, b-spectrin tension sensors allow visualization of
tissue stresses mediated via b-spectrin during C. elegans
embryogenesis [96].
Micropipette aspiration has recently been used to quantify sur-
face tension of blastomeres during early mouse embryogenesis
(Figure 3A) and revealed the critical role of cortical contractility
in oocyte meiosis, compaction and lineage segregation
[43,97,98]. The technique involves the local aspiration of a part
of a cell or tissue by applying a constant negative pressure.
The surface tension of the sample can then be calculated based
on the Young-Laplace equation [97]. One major limitation of
micropipette aspiration is that it cannot be used to measure force
generation by cells within the interior of 3D tissues. To address
this issue, droplet-based sensors have been developed
(Figure 3A): cell-sized oil microdroplets coated with adhesion
ligands are injected into 3D tissues to serve as force transducers
in vivo. Mechanical stresses exerted by the surrounding cells will
deform the microdroplet. As a result, the tissue stress can be
deduced from the known interfacial tension and shape deforma-
tion of the microdroplet [99]. Additionally, pressure sensors made
of polyacrylamide microbeads have been developed to quantify
mechanical stress in multicellular spheroids [100], offering the
exciting prospect of tracking tissue or luminal pressure in devel-
oping embryos.
Perhaps the least invasive technique is the direct ‘imaging’ of
forces in vivo using the force inference method [101,102].
Assuming that the tissue is in a quasi-static equilibrium state
where the deformation of cells is sufficiently slow, one can
extract mechanical information about the tissue by analyzing
the shape of the cells, which is governed by the local balance
of the forces that the cells exert on one another (Figure 3A).
While this method allows quantification of relative cell–cell junc-
tional tension and cellular pressure up to a prefactor, cross-
validation with other techniques can help provide an absolute
measure of forces. So far, force inference methods have
been limited to 2D systems, such as epithelial monolayers in
Drosophila embryos and pupae [76,102–104]. However,
promising work extending the approach to 3D systems has
recently been reported for early mouse and zebrafish embryos
[105,106].
With substantial evidence supporting the role of ECM stiffness
in directing cell-fate specification in vitro, a label-free bioimaging
technique known as Brillouin microscopy has been developed to
map tissue stiffness in vivo [107]. By analyzing the frequency shift
of the scattered light when it interacts with sound waves in the
sample, one can obtain a spatiotemporal map of cell or tissue
stiffness in situ (Figure 3A). While previous studies have demon-
strated its feasibility in measuring intracellular or cell mechanical
properties with submicron resolution [108–110], recent studies
also provide proof-of-principle applicability in measuring the me-
chanical properties of ECMs in whole organisms [111]. Future
studies combining Brillouin microscopy with fluorescence imag-
ing of cell-fate reporters and mechanoreceptors will likely reveal
new insights into the role of ECM stiffness and cell-fate specifi-
cation during development.

tissue can be determined. Pressure sensors made of polyacrylamide microbeads yield the isotropic pressure distribution in 3D tissues. Force inference: Image
analysis of a 2D epithelium generates a segmented cell shape and angles between cell–cell junctions. Using force-balance equations at the vertex points, the
junctional tension and intracellular pressures of all cells can be inferred. Brillouin microscopy: Pressure waves are generated in the sample following illumination
with light of very high frequency. By measuring the frequency shift of the scattered light, the mechanical stiffness of the sample can be measured. (B) Left: Using
laser light to spatiotemporally modulate protein activities governing cell contractility, tissue tension and geometry were shown to influence mesoderm invagi-
nation in Drosophila. Right: Using a magnet, cells loaded with magnetic liposomes are mechanically towed and the impact of ectopic stress on gene expressions
can be monitored. (C) Left: 2D micropatterning of cell doublets revealed the role of matrix stiffness in polarity orientation and epithelial-to-mesenchymal cell-fate
transition. Right: Using a 3D hydrogel with tunable ECM parameters, high matrix stiffness is shown to enhance intestinal stem cell expansion while a soft matrix
leads to differentiation of these cells and organoid formation.

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Current Biology
Review
Visualizing Cell Fate Specification and Tissue
Morphogenesis
To study the interplay between tissue morphogenesis and
cell-fate specification in development, it is essential to monitor
cell-fate changes in live cells. Recent advances in imaging tech-
niques have allowed accurate reconstruction of cell lineages
and monitoring of gene expression dynamics in multicellular
organisms. The low phototoxicity associated with light-sheet mi-
croscopy ensures minimal photodamage when imaging living
embryos [112]. In addition, the improved computational frame-
work opens up the prospect of automated image segmentation
and cell tracking in real time [113]. Meanwhile, label-free
nonlinear microscopy offers an alternative approach to trace
cell lineages in deep tissues with high spatiotemporal resolution
[114]. To further elucidate the decisive role of mechanical forces
on cell-fate specification during development, it will be essential
to simultaneously monitor cell fate, cellular/tissue stress and
morphology. This will require future development and integration
of advanced microscopy, biophysical tools and cell-fate re-
porters, as demonstrated in recent studies [78,82,104,115,116].
Spatiotemporally Controlled Functional Perturbations
A powerful approach to decipher the causal links between tissue
stress and gene expression is functional perturbation. Recent
technological advances have made it possible to manipulate tis-
sue forces in a spatiotemporally controlled manner within the
embryo. One such approach is optogenetics, which makes use
of pulses of laser light to inhibit or stimulate protein activity
with spatial and temporal precision [117–119]. By optically
manipulating the activity of plasma membrane phosphoinositi-
des and cell contractility (Figure 3B, left), local inhibition of apical
constriction was shown to be sufficient to globally arrest meso-
derm invagination during ventral furrow formation in Drosophila
[120]. Using the same approach, local activation of the GTPase
RhoA was found to be sufficient for inducing cleavage furrow
formation in rounded interphase cells, in a manner uncoupled
from usual spatiotemporal restrictions [121]. The possibility of
inducing de novo assembly or disassembly of actomyosin net-
works allows for the sufficiency of mechanical forces in regu-
lating cell-fate specification to be tested in vivo. Given the recent
demonstration of compensatory networks that buffer against
deleterious mutations [122], it will be important to investigate
mechanisms governing development not only using permanent
gene deletion, but also using spatiotemporally and quantitatively
controlled manipulations of gene expression and protein distri-
bution in vivo. An example of this approach is the use of GFP
nanobodies to abolish Dpp gradient formation in the Drosophila
wing imaginal disc, which has provided new insights into the
mechanisms of tissue size control [123].
Another approach to physically manipulate forces in vivo is the
magnetic tweezer, a tool extensively used for cells in vitro
(Figure 3B, right). The application of magnetic forces on injected
magnetic nanoparticles or liposomes in living tissues has been
shown to induce the expression of Twist and reveal the role of
b-catenin in mechanotransduction in Drosophila and zebrafish
embryos [14,15]. The same technique was used to study tumor
progression in adult mice, where mechanically induced stress
can trigger the tumorigenic b-catenin pathway [124]. Recently,
the magnetic tweezer has been successfully applied to ferrofluid
microdroplets to measure tissue viscosity and fluidity in zebra-
fish embryos [125]. It will be interesting to apply ectopic forces
at such mesoscopic scales and study their downstream effects
on signaling and cell-fate specification.
Ex Vivo Reduced Systems
Given that tissue morphogenesis is inherently a non-linear, self-
organizing process involving multiple feedback mechanisms,
ex vivo and in vitro experimental systems have been engineered
in which the function of each mechanical cue can be assessed
independently. One such experimental model employed in early
mouse embryology is the analysis of single blastomeres isolated
from 8-cell stage embryos (1/8-cell) [70]. Such a reduced system
is able to develop into mini-blastocysts and recapitulate the line-
age segregation of inner cell mass and trophectoderm under
spatially simplified conditions [126]. This approach allows the
unambiguous identification of symmetry-breaking cues, such
as apical–basal polarity [71] and differential cortical contractility
[43], in driving the first lineage segregation in early mouse devel-
opment. The use of minimal tissue models, such as cell doublets
on confined micropatterns, also reveals the role of centriole re-
positioning in driving polarity inversion during epithelial-to-
mesenchymal cell-fate transition (Figure 3C, left) [127].
Over the past decades, 3D organoids have served as excellent
in vitro model systems to study organ development [6,7]. While
these studies employ primarily chemical cues, new findings
have revealed the roles of biophysical cues in controlling cell
fate and tissue organization. For example, intestinal stem cells
cultured on a stiff matrix with fibronectin-based adhesion display
enhanced expansion through a Yap-dependent mechanism,
while those grown on a soft matrix with laminin-based adhesion
differentiate and form organoids (Figure 3C, right) [30]. Studies of
neuroepithelial cyst formation using 3D hydrogels have identified
key mechanical parameters determining dorsoventral patterning
during neural tube morphogenesis [128]. Further, the use of
bone-marrow-like ECM has revealed the role of matrix stiffness
in directing hematopoietic stem cell fate decisions [129]. Inter-
estingly, advances in microdevice fabrication technology have
led to the concept of ‘development-on-chip’. For example, it
was demonstrated that neural tube development can be recapit-
ulated in microfluidic systems with spatiotemporally tunable
chemical environments [130]. In the future, the combination
of microfluidic platforms with 3D hydrogels equipped with
tunable geometric/mechanical properties may represent an
integrated approach to study the interplay between mechanics
and biochemical signaling in cell-fate specification and tissue
patterning.
Future Directions
In this review, we discussed recent evidence for tissue-shaping
events and tissue stress as important upstream regulators of
developmental signaling pathways. An emerging theme for
studies of mechanotransduction in development is the extension
from 2D epithelial systems to 3D tissue morphogenetic pro-
cesses. Mechanical constraints arising from tissue bending or
buckling can generate long-range tissue stress that propagates
to individual cells and triggers potential mechanotransduction
pathways. Dynamic cellular rearrangements and global shape
changes of tissues can also direct the trapping and positioning
of signaling centers, leading to spatial restriction of cell lineages.
Current Biology 27, R1024–R1035, September 25, 2017 R1031

Importantly, the interplay between mechanical signals and
cellular polarity can lead to the establishment of multicellular
patterning.
Several outstanding questions and challenges remain
regarding the coordination of tissue morphogenesis, mechanics
and cell-fate specification during development. How do individ-
ual cells sense the global change in tissue shape and size and
transduce these physical signals into differential gene expres-
sion in a highly robust manner? Usually there is considerable
delay between the primary events of mechanosensation,
involving the translation of mechanical signals into biochemical
ones, and downstream transcriptional events. This implies that
cell-fate specification during development may arise from a
combination of physical force transduction and biochemical
signaling induced by tissue morphogenesis. How can we
disentangle these two clearly distinct processes that act in
concert during development? For example, tissue strain also
drives cellular movement, which makes it difficult to distinguish
the direct impact of mechanical stress on differentiation
from secondary induction due to new contacts formed between
the signaling and responding cells. Can we always devise
the necessary experimental approach to test not only for
requirement but also for sufficiency of a particular mechano-
transduction pathway, and in a quantitative manner? Here,
computational simulation may offer one approach in identifying
key parameters sufficient for recapitulating developmental pro-
cesses in vivo [131].
Importantly, tissue morphogenesis characterized by feedback
interactions between cell contact, mechanics, polarity and gene
expression acts across multiple scales (organism, organ, multi-
cellular, cellular and subcellular). It is therefore important to
remember that the cell-fate switch itself can change cell/tissue
mechanical properties, such as cell adhesion and contractility,
thereby modifying tissue-scale forces that eventually feed back
into cell-fate specification. How can we then specifically test
for the causal role of tissue mechanics in controlling develop-
mental fate changes, and vice versa? One approach is to
spatiotemporally manipulate cell signaling, which might link
mechanics to cell-fate specification. Dissecting the specific
instructive or permissive roles of mechanical forces in driving
cell-fate specification will continue to be an important and
challenging goal in the field of developmental biology in the years
to come.
ACKNOWLEDGEMENTS
We thank François Graner and members of the Hiiragi lab for insightful discus-
sions. We sincerely apologize for not being able to include citations to all rele-
vant works in the field due to space limitations. C.J.C. is supported by the
German Research Foundation (DFG) and Marie Curie Actions COFUND (EC
Grant Agreement No. 664726) EMBL Interdisciplinary Postdoc (EIPOD).
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