Hippo Signaling in Embryogenesis and Development

Trends in Biochemical Sciences
Review
Hippo Signaling in Embryogenesis

and Development
Zhengming Wu1 and Kun-Liang Guan1,*
Hippo pathway components are structurally and functionally conserved and Highlights
are notable for their role in controlling organ size. More diverse functions The evolutionary conserved Hippo path-
of the Hippo pathway have been recognized, including development, tissue way functions to limit tissue and organ
growth and its deregulation contributes
homeostasis, wound healing and regeneration, immunity, and tumorigenesis.
to human cancer.
During embryogenesis, different signaling pathways are repeatedly and cooper-
atively activated, leading to differential gene expression in specific developmen- Hippo pathway crosstalks with numer-
tal contexts. In this article, we present an overview on the regulation and function ous developmental signaling pathways.
of the Hippo pathway in mammalian early development. We introduce the Hippo Hippo signaling plays critical roles in early
pathway components and major upstream signals that act through this pathway embryonic development as low Hippo
to influence embryogenesis. We also discuss the roles of Hippo pathway in activity is required for trophoblast differ-
tissue specification and organ development during organogenesis. entiation and high Hippo activity permits
inner cell mass formation.
Hippo pathway positively or negatively

Hippo Pathway and Its Role in Organ Size Control regulates development of multiple
tissues/organs.
The Hippo pathway is the most recently discovered major signaling pathway identified to play a
role in limiting tissue and organ growth [1,2]. Subsequent studies demonstrate that Hippo is
highly conserved in mammals and controls development and tissue/organ homeostasis, whereas
dysregulation of the Hippo pathway contributes to human diseases, such as cancer [3,4]. Core
components of the Hippo pathway include a kinase cascade and a transcription module [5].
Genetic and biochemical studies have established that the Hippo kinase cascade functions to
inhibit the transcription module that represents the major functional output of the Hippo pathway,
thereby inhibiting cell and tissue growth. Unlike many developmental pathways that are defined
by specific morphogens/hormones and their corresponding receptors, the Hippo pathway
responds to diverse signals, such as hormones, cell–cell or cell–matrix contact, mechanic
cues, cellular energy status, and various stress, to control cell growth, differentiation, tissue/
organ development, and homeostasis. As such, the Hippo pathway can receive and integrate
a wide range of environmental and physiological signals to modulate transcription and cellular
activity.
In addition to a key role in tissue and organ homeostasis in adults, a large number of recent
studies have established the fundamental function of this pathway in mammalian embryonic
development. Although many reviews on the Hippo pathway have been published, most focus
on the signaling mechanisms, cancer implication, or a specific tissue type [5–7]. This review
1
aims to provide the current landscape of the Hippo pathway in early development with an empha- Department of Pharmacology and
Moores Cancer Center, University of
sis of mouse genetic studies. Here, we summarize the Hippo pathway components, mechanisms
California San Diego, La Jolla,
of regulation, and upstream regulatory signals, particularly those important in development. This CA 92093, USA
review mainly focuses on the functions of the Hippo pathway in early developmental stages in a
chronological order from zygotes to organogenesis. We discuss the functional crosstalk between
Hippo and other developmental signals as well as the therapeutic potential of manipulating the
Hippo pathway in regenerative medicine. We aim to provide readers with a comprehensive *Correspondence:
view of the Hippo pathway in early development and key open questions in the field. kuguan@ucsd.edu (K.-L. Guan).
Trends in Biochemical Sciences, January 2021, Vol. 46, No. 1 https://doi.org/10.1016/j.tibs.2020.08.008 51

© 2020 Elsevier Ltd. All rights reserved.
Core Components of the Hippo Pathway Glossary

A large number of signals act through various mechanisms to control Hippo pathway activity. The Adherens junctions (AJs): the
core components of the Hippo pathway are highly conserved and consist of a kinase cascade connection structure between
and downstream transcription effectors (Figure 1). In mammals, the kinase cascade includes neighboring cells in epithelial or
endothelial tissues. They are formed by
mammalian STE20 like kinase 1/2 (MST1/2), mitogen-activated protein kinase kinase kinase adherens molecules and connect to intra
kinase (MAP4K) family, and large tumor suppressor kinase 1/2 (LATS1/2), while the downstream cellular actin filaments or intermediate
effectors are further grouped into Yes associated protein 1 (YAP1, also known as YAP)/WW filaments.
Blastocyst: a structure formed at the
domain containing transcription regulator 1 (WWTR1, also known as TAZ) transcription coactivators
end of the cleavage stage of mammals.
and DNA-binding protein TEA domain transcription factor 1/2/3/4 (TEAD 1/2/3/4). It is composed of an inner cell mass
(ICM), which subsequently forms the
At the top of the kinase cascade, protein phosphatase PP2A in the striatin-interacting embryo, and an outer layer called
trophoblasts.
phosphatase and kinase (STRIPAK) complexes (see Glossary) limits the activity of MST1/2
Blastomere: totipotent cells produced
or MAP4Ks by direct dephosphorylation [8] (Figure 1). When stimulated by upstream signals, by the cleavage of the zygote. This term
the STRIPAK-mediated dephosphorylation is relieved, thereby MST1/2 or MAP4Ks are mainly refers to the two/four/eight-cell
phosphorylated and activated, possibly due to autophosphorylation or by the upstream TAO embryos.
Inner cell mass (ICM): cells that lie
kinase [9,10]. Salvador family WW domain containing protein 1 (SAV1) and MOB kinase activator
inside the blastocyst, which will develop
1A/B (MOB1A/B), which are regulatory subunits of MST and LATS, respectively, are then phos- into the embryo. The ICM is surrounded
phorylated by the active MST1/2 and recruit LATS1/2 for activation by MST [11–13]. Besides by trophoblasts, which are the outer
MST1/2, MAP4K proteins can also phosphorylate and activate LATS1/2 [14,15]. Neurofibromin layers of the blastocyst.
Morula: a 16-cell embryo formed by cell
2 (NF2), a FERM domain protein, is a critical upstream regulator that promotes the activation of division of a zygote in the early stage of
Hippo kinase cascade by interacting with upstream signals and Hippo pathway components animal embryo development.
[16,17]. YAP/TAZ is then phosphorylated by LATS1/2, resulting in their binding with 14-3-3 NuRD repressor complex: a
and cytoplasmic sequestration [18]. The LATS-phosphorylated YAP/TAZ can undergo further repressive transcription complex with
both ATP-dependent chromatin
phosphorylation by casein kinase 1δ/ε and this phosphorylation recruits SCF E3 ligase, leading remodeling and histone deacetylase
to protein ubiquitination and degradation [19,20]. YAP/TAZ can also be phosphorylated by activities. Mi2 and HDAC1/2 are the two
other kinases, however, YAP/TAZ inhibition by LATS-dependent phosphorylation represents core subunits.
Palmitoylation: a covalent modification
the major and most widely used mechanism in Hippo signaling.
of amino acid to cysteine (most
common), serine, or threonine residues
YAP/TAZ do not have any DNA binding domain and act as a coactivator. The output of the Hippo of proteins. Palmitoylation generally
pathway is mainly dependent on their binding to TEAD family members, which share a highly serves as a membrane attachment
signal. However, TEAD palmitoylation
conserved DNA binding domain and a YAP/TAZ binding domain [21]. One unique feature of
does not serve as membrane targeting,
TEAD is that it can be auto-palmitoylated and this palmitoylation is critical for TEAD function but rather is important for TEAD function.
[22]. In the absence of YAP/TAZ, TEAD interacts with vestigial like family member 4 (VGLL4), a Rho family of GTPases: a group of
protein containing Vg motif with transcriptional repression activity, to form a default repression small (~21 kDa) GTP binding proteins
with GTPase activity that belong to the
complex [23]. Once shuttled into the nucleus, YAP/TAZ binds TEAD to displace VGLL4, thus
Ras superfamily. Rho GTPase family
forming a transcriptional activation complex, which is responsible for the induction of the majority members play major roles in actin
of Hippo target genes. dynamics, cell morphology, and
migration.
SMAD: a group of structurally related
Upstream Regulators of the Hippo Pathway in Embryogenesis proteins that are the main downstream
Unlike classical signaling pathways that are defined by specific ligands and receptors, the effectors of the TGF-β receptor
Hippo pathway works as an integrator of signals from the biochemical, physical, and architectural superfamily. There are three SMAD
environment. As this review focuses on embryogenesis, we will only discuss the Hippo pathway’s subtypes: receptor-regulated SMAD
(R-SMAD), common partner SMAD
regulatory factors relevant in early development. Besides Hippo, embryonic development is also (Co-SMAD), and inhibitory SMAD
regulated by various other signaling pathways; the crosstalk between Hippo and these signaling (I-SMAD).
pathways should not be overlooked. Striatin-interacting phosphatase
and kinase (STRIPAK) complexes:
protein complexes containing both
Cell Polarity and Adhesion
kinases and phosphatases. In these
Cell polarity, the asymmetric distribution of constituents in a cell, is an essential feature of life. The complexes, striatin serves as a key
molecular basis of cell polarity is a set of evolutionarily conserved proteins called the PAR-aPKC scaffold protein to tether both kinases
system [24]. Researchers have revealed how cell polarity cooperates with junction-associated and PP2A phosphatase. STRIPAK
52 Trends in Biochemical Sciences, January 2021, Vol. 46, No. 1

scaffolding angiomotin (AMOT) family proteins to regulate the Hippo pathway (Figure 1). When complexes are evolutionarily conserved
and have critical roles in protein (de)
cells are nonpolar, AMOT is phosphorylated on serine 176 (S176) and distributed in adherens
phosphorylation, particularly in
junctions (AJs) [25] and interacts with NF2 and LATS kinases to facilitate the phosphorylation dephosphorylation and inactivation of
of YAP/TAZ [26,27]. When cells exhibit polarity, however, AMOT is sequestered from basolateral the STE20 family of kinases.
AJs to apical domains by cell polarity regulator Par-aPKC system [25], where AMOT interacts
with F-actin and suppresses YAP/TAZ activity [28].
Mechanical Force
During embryogenesis, the Hippo pathway is constantly regulated by mechanical force from the
extracellular matrix (ECM) and neighboring cells (Figure 1). Specifically, high stiffness of ECM
induces nuclear localization of YAP/TAZ, whereas low ECM stiffness promotes YAP/TAZ cytoplasmic
localization [29,30]. Most studies have indicated that integrin and its downstream SRC/FAK kinases
are involved in the initial events in response to matrix stiffness [31]. The Rho family of GTPases and
Figure 1. The Mammalian Hippo Pathway and Its Regulation by Cell–Cell and Cell–Matrix Contact. MST1/2, MAP4Ks, and LATS1/2 kinases are activated by
phosphorylation. YAP/TAZ are inhibited by LATS-dependent phosphorylation, which promotes YAP/TAZ cytoplasmic localization and degradation. STRIPAK inhibits MST and
MAP4Ks by dephosphorylation. Extracellular matrix acts via integrin to modulate the Hippo pathway. The cell–cell contact signal is detected by adherens junctions (AJs) and tight
junctions and mediated by AMOT family protein to stimulate Hippo signaling. In the absence of nuclear YAP/TAZ, VGLL4 binds TEAD to repress transcription. Abbreviations:
AMOT, angiomotin; LATS1/2, large tumor suppressor kinase 1/2; MAP4K, mitogen-activated protein kinase kinase kinase kinase; MOB1, MOB kinase activator 1; MST,
mammalian STE20 like kinase 1/2; NF2, neurofibromin 2; SAV1, salvador family WW domain containing protein 1; STRIPAK, striatin-interacting phosphatase and kinase; TAZ,
WW domain containing transcription regulator 1; TEAD, TEA domain transcription factor 1/2/3/4; VGLL4, vestigial like family member 4; YAP, Yes associated protein 1.
Trends in Biochemical Sciences, January 2021, Vol. 46, No. 1 53

the cytoskeleton are key mediators of mechanical cues to the Hippo pathway [29,32,33]. However,
the detailed mechanism of how the cytoskeleton controls the activity of YAP/TAZ is still unknown.
G Protein-Coupled Receptors (GPCRs)

Strict spatial and temporal control of soluble factors is the key feature of embryonic development.
GPCRs have been linked to Hippo regulation [34–38]. Based on the types of coupled G proteins,
GPCR signaling can either activate or inhibit the Hippo pathway [34,39] (Figure 2). Mechanistically,
Figure 2. Crosstalk between the Hippo Pathway and Other Developmental Cues. Stimulation of GPCRs can either
positively or negatively affect YAP/TAZ activity in a manner dependent on the type of heterotrimeric G proteins coupled to the
receptor. Notch intracellular domain (NICD), the effector of Notch signaling, enhances YAP/TAZ activity by promoting protein
stability. TAZ binds SMAD, the effector of TGF-β signaling, to promote its nuclear translocation. YAP/TAZ are stimulated by
Wnt ligands. Abbreviations: GPCR, G protein-coupled receptor; LATS1/2, large tumor suppressor kinase 1/2; TAZ, WW
domain containing transcription regulator 1; TEAD, TEA domain transcription factor 1/2/3/4; TGF-β, transforming growth
factor beta; YAP, Yes associated protein 1.

Rho-GTPases and the F-actin cytoskeleton relay GPCR signaling to Hippo [34]. Rho also acts in
part via STRIPAK to inhibit the Hippo kinase cascade [8].
Wnt
Wnt signaling is known for its critical role in embryonic development [40]. The destruction com-
plex, including Axin, adenomatous polyposis coli (APC), and glycogen synthase kinase 3 beta
(GSK3β) degrades β-catenin, the key effector of the Wnt pathway, in the absence of Wnt ligands.
Under Wnt stimulation, cytoplasmic β-catenin accumulates and enters the nucleus to activate
transcription [40,41]. Studies have revealed YAP activation by Wnt ligands, but the underlying
mechanism is still controversial (Figure 2). Some reports indicate that YAP/TAZ are released
from the destruction complex and hence stabilized upon Wnt stimulation [42,43]. One study sug-
gests that APC interacts with Sav1 and LATS to facilitate the phosphorylation of YAP/TAZ [44].
Yet another report shows that Wnt signaling inhibits YAP phosphorylation via the conventional
Hippo pathway [45]. However, it is clear that YAP activation also leads to suppression of Wnt
signaling.
Notch
The Notch pathway functions in development and homeostasis. When cells are in contact with
each other, the Notch receptor interacts with its ligands in the neighboring cells. This interaction
leads to cleavage of Notch receptor and the release of Notch intracellular domain (NICD),
which translocates into the nucleus to induce target genes [46,47]. The crosstalk between
the two signaling pathways has been demonstrated (Figure 2): NICD is reported to enhance
YAP/TAZ activity by promoting protein stability and YAP/TAZ can activate the transcription of
Notch receptors and its ligand, Jagged canonical Notch ligand 1 (JAG1) [48,49].
Transforming Growth Factor (TGF)

The TGF-β superfamily is involved in a large number of biological events in embryogenesis [50].
TGF-β ligands bind to their specific Ser/Thr kinase receptors to activate SMAD and gene
transcription [50]. TAZ is reported to bind to SMAD, promoting its nuclear translocation under
TGF-β stimulation [51] (Figure 2). Conversely, TGF-β signaling can be inhibited via YAP/TAZ-
mediated SMAD cytoplasmic sequestration [52].
Hippo Pathway in the Preimplantation Stage

Embryogenesis in mammals is a complicated process that can be divided into several develop-
mental stages. In the next few sections, we review the role of the Hippo pathway in embryogenesis,
with a focus on genetic data from mouse models (Figure 3).
During embryogenesis, the preimplantation stage refers to the time from fertilization to implantation.
The preimplantation stage takes around 10 days in human and 4.5 days in mice. It begins with
the formation of a zygote, which then undergoes several initial cell divisions to form a solid ball of
cells called a morula. A morula undergoes additional cell division and morphogenesis to form a
blastocyst. The preimplantation stage ends when the blastocyst implants in the uterus. Here,
we will discuss the role of the Hippo pathway in zygote pluripotency, maternal–zygotic transition,
and trophoblast differentiation.
Zygote and Blastomere

A zygote is totipotent, meaning it has the ability to produce all the different cell types in an
organism and the extra embryonic tissues. YAP/TAZ has been shown to have a role in totipotency.
Zygotes and blastomeres without maternal YAP/TAZ perish before the blastocyst stage [53]. In
the four/eight-cell blastomeres, YAP/TAZ inhibits the stem factor SRY-box transcription factor 2

Figure 3. Functions of the Hippo Pathway in Embryogenesis and Development. This diagram shows the YAP/TAZ subcellular distributions and their functions at
different embryonic stages. In zygotes, YAP/TAZ is crucial for preventing the premature expression of SOX2 and abnormal ICM differentiation. In morula and blastocyst,
cells are specialized depending on Hippo pathway activity in response to their position and polarity. The exterior cells with high YAP/TAZ activity develop into trophoblasts,
while the inner cells with low YAP/TAZ activity develop into ICM. YAP/TAZ is required for organogenesis in all three germ layers. The color of cell outlines and text boxes
represent certain germ layers (gray, blue, green, and red for trophectoderm, ectoderm, mesoderm, and endoderm, respectively). The text in colored background denotes
phenotypes of altered Hippo signaling at each indicated embryonic ‘E’ stage. Abbreviations: diff., differentiation; E, embryonic day; ICM, inner cell mass; SRY-Box
Transcription Factor 2; TAZ, WW domain containing transcription regulator 1; TE, trophectoderm; YAP, Yes associated protein 1.
(SOX2) expression and the formation of inner cell mass (ICM), the cells of origin for all three
germ layers of endoderm, ectoderm, and mesoderm [54]. Knockout of maternal YAP/TAZ results
in the premature expression of the ICM marker SOX2 prior to the 16-cell stage and eventually
inhibits the expression of trophoblast factor caudal type homeobox 2 (CDX2) [54]. This abnormal
ICM differentiation and the inhibition of trophectoderm differentiation may explain the failure of
blastocyst formation in zygotes and blastomeres without maternal YAP/TAZ.
Maternal–Zygotic Transition
After fertilization, in mammals, transcription of the newly formed embryo genome is inactivated.
Development strictly depends on the maternal RNA and proteins in the ovum [55]. The start of
embryonic transcription, called ‘zygotic genome activation (ZGA)’, occurs later during the preim-
plantation period and, in the meantime, zygotes begin to eliminate the contribution of maternal
gene products by degrading the maternally supplied mRNA. The event containing both ZGA
and degradation of maternal products is called the ‘maternal to zygotic transition (MZT)’. After
MZT, the developmental process is controlled by zygote genomes [56].
The very early stage of development is controlled by maternal RNA and protein. Conventional
YAP knockout mice (YAP–/–) from YAP–/+ parents are unable to achieve true YAP inactivation in
early embryos, as the mother will produce oocytes with YAP protein. Oocyte-specific knockout

can solve this problem and produce early embryos devoid of YAP. Maternal YAP has been
found to play an important role in MZT. When fertilized by wild type spermatozoa, the resulting
maternal Yap1-knockout embryos (YAP♀–/♂+) display a prolonged two-cell stage and develop
into the four-cell stage at a much slower rate when compared with wild type maternal controls
(YAP♀+/♂+) [57]. Transcriptomes of four-cell embryos derived from YAP♀–/♂+ and YAP♀+/♂+
show significant differences in thousands of genes. Many maternal transcripts are upregulated
in YAP♀–/♂+ compared with YAP♀+/♂+, implying the evasion of maternal mRNA degradation.
Meanwhile, early zygotic genes targeted by YAP/TAZ are downregulated, including Rpl13 and
Rrm2, suggesting the failure of zygote gene activation in the YAP♀–/♂+ embryo [57]. These data
suggest a critical role of YAP in MZT.
Trophectoderm (TE) Specification

In early embryogenesis, blastocysts are composed of the outer epithelial TE and the ICM
(Figure 3). TE cells form extraembryonic tissues, including placenta, supporting the embryo
proper. ICM further gives rise to primitive endoderm and epiblast. Although historical studies
established the blastocyst atlas in the 20th century, the mechanism of fate decision for the initial
embryonic totipotent cells to develop into the TE or ICM lineage was not available until the
2000s [58].
Hippo signaling activity, which is dependent on cell polarity, is a critical step for this first cell fate
specification (Figure 3). In the outer polar cells, Hippo signaling is suppressed: dephosphory-
lated YAP/TAZ translocate into nucleus, interact with TEAD, and directly induce TE-specific
transcriptional factors CDX2 and GATA binding protein 3 (GATA3), which are required to
repress pluripotent genes and promote differentiation into TE [53,59–61]. Perhaps, cell polarity
and less cell contact play a role in Hippo inactivation in the outer layer cells during this very first
cell specification in mammalian embryonic development. But in the inner apolar cells, the Hippo
pathway is active: LATS1/2 phosphorylates and promotes YAP/TAZ in the cytoplasm, thus
preventing YAP/TAZ to induce target genes. The requirement of differential Hippo pathway
activity for TE and ICM patterning is transient [62].
Consistently, loss of LATS1/2 results in a developmental bias toward the TE-like lineage [53,62].
By contrast, TEAD4 knockout embryos show severe downregulation of CDX2 expression and
aberrant high expression of ICM-specific transcription factors like POU class 5 homeobox 1
(POU5F1) and Nanog in all blastomeres [59]. Besides Hippo core components, NF2 (Merlin) is
also involved in establishing TE fate. Nuclear YAP localization and CDX2 expression can be
observed in both outer and inner cells when NF2 is mutated [63]. Collectively, the Hippo pathway
plays a major role in controlling TE differentiation.
In addition to CDX2, Hippo signaling also affects the expression of SOX2, which is a key transcrip-
tion factor in ICM. Enforced cytoplasmic YAP in outer cells is sufficient to induce aberrant SOX2
expression [64]. A recent study has shown that YAP/TAZ-TEAD4 directly suppresses SOX2 gene
expression [54].
ICM Specification
In the ICM, the activation of LATS kinases induces the cytoplasmic translocation of YAP/TAZ,
thus relieving the transcriptional repression of SOX2 and promoting the differentiation of ICM
[54]. Before the blastocyst is implanted, the ICM will further differentiate into epiblasts and
hypoblasts; the former will develop into the three germ layers and the latter will become the
yolk sac. Epiblasts need to form an entire individual from a small number of cells, thus they are
highly pluripotent. YAP-TEAD plays an important role in the formation of epiblasts: TEAD can

activate expression of pluripotent genes and MYC simultaneously [65]. Cells that fail to activate
YAP-TEAD signal will be eliminated by cell competition, which ensures the formation of uniform
epiblasts with naïve pluripotency [65].
Based on their ability to maintain a pluripotent state upon MEK inhibition, embryonic stem cells
(ESCs) can be grouped into naïve pluripotent or primed pluripotent ESCs [66]. Usually, mouse
ESCs are naïve pluripotent, similar to the preimplantation epiblasts, whereas human ESCs are
in a primed pluripotent state similar to postimplantation epiblasts [66].
Some studies report that YAP/TAZ are required for mouse ESC self-renewal and differentiation
in vitro [67]. YAP reduction leads to the loss of pluripotency in ESCs. Knockout of MST1/2 causes
resistance to differentiation induced by Leukemia Inhibitory Factor (LIF) withdrawal [68]. Mecha-
nistically, YAP/TAZ bind to TEAD and activate the transcription of POU5F1 and Nanog, which
are the key transcriptional factors in ESCs. YES1, a LIF-stimulated Src family tyrosine kinase,
can activate YAP via tyrosine phosphorylation [69]. However, other groups report that YAP is es-
sential for differentiation [43,70]. Cells lacking YAP/TAZ maintain aberrant pluripotency and show
impaired induction of lineage-specific genes during differentiation, as loss of YAP/TAZ compen-
sates for lack of Wnt signaling and opposes ESC differentiation [43]. Conversely, overexpression
of YAP in ESCs disrupts self-renewal and triggers differentiation by upregulating lineage markers
[70]. Furthermore, bioinformatics analyses have indicated that YAP–TEAD is not involved in the
transcription factor network of naïve pluripotency [71,72]. Further studies are needed to elucidate
the precise functions of YAP/TAZ in ESCs.
In human ESCs, besides interacting with the TEAD family, YAP/TAZ can also form a regulatory
complex with SMAD2/3 and POU5F1, cooperating with the NuRD repressor complex to buffer
pluripotency gene expression while suppressing differentiation genes [73]. A mechanism for YAP/
TAZ activation in ESCs/induced pluripotent stem cells (iPSCs) has been proposed. A-kinase
anchoring protein 13 (AKAP13), a Rho GEF highly expressed in ESCs/iPSCs, stimulates YAP/
TAZ activity by modulating the cytoskeleton, thereby maintaining the survival and pluripotency
of ESCs [74]. A common regulator of YAP in mouse and human ESCs is Ras association domain
family member 1 (RASSF1A). When ESCs differentiate, RASSF1A is induced and functions to
prevent YAP from binding to TEAD, thus abolishing POU5F1 transcription [75].
Finally, it is worth mentioning that although ESCs/iPSCs are considered to be pluripotent rather
than totipotent, extra-embryonic cell differentiation protocols from ESCs/iPSCs are emerging
[53,76–79]. The potential of totipotency in ESCs/iPSCs is likely to be underestimated. In
conventional in vivo chimera experiments, ESCs are often injected into the center of the
morula/blastocyst. But it has been shown that the location and polarity of cells in the morula/
blastocyst is critical for embryonic/extra-embryonic differentiation. The exterior cells develop
into trophoblasts, while the inner cells develop into ICM. Therefore, the results of these chimera
experiments should be interpreted carefully.
Hippo Pathway in Gastrulation and Neurulation Stage

Gastrulation and neurulation stages follow the preimplantation stage and are characterized by the
formation of a multilayered structure known as the gastrula, as well as the folding process from
the neural plate into the neural tube. It takes place around the 17th day after fertilization and
ends at day 26 in human, and from mouse embryonic day 6 (E6) to E9 in mice.
Even though YAP plays an important role during preimplantation, conventional YAP–/– embryos
from YAP+/– parents do not show obvious developmental defects until E8.5 in mice [3]. By

contrast, knockout of maternal YAP or YAP/TAZ causes a failure of blastocyst formation [54,57].
This suggests that maternal YAP in oocytes from YAP+/– mothers is sufficient to support zygotes
through development events like MZT. In addition, conventional YAP/TAZ knockout embryos
from YAP+/–/TAZ+/– parents die before morula stage, which implies that the expression of zygotic
TAZ compensates for the effects of YAP in conventional YAP–/– embryos [53]. Mouse embryos
lacking YAP arrest development around E8.5 and display defects in yolk sac vascular develop-
ment, chorioallantoic fusion, and embryonic axis elongation [3]. Histologic analysis shows these
defects are not due to problems with tissue specification, but rather, are due to the requirement
for YAP in morphogenesis and proliferation. These studies indicate that YAP has unique functions
in vasculature which cannot be compensated for by TAZ.
Vascularization begins in yolk sac and placenta. Loss of YAP/TAZ in mice endothelial cells (ECs)
leads to embryonic lethality, supporting a critical role of YAP/TAZ in vascularization [80–82]. YAP/
TAZCDH5-CreERT2 mice show impaired vasculature [82]. Vascular endothelial growth factor
(VEGF)-VEGF receptor ® signaling, the primary factor for vascularization, activates YAP/TAZ in
ECs by regulating the Src family of kinases, Rho GTPase, the cytoskeleton, and Lats1/2 activity.
This mechanism is critical in EC migration and angiogenesis mediated by VEGF [82–85]. YAP/
TAZ is also responsible for some trafficking proteins induced by VEGF, participating in a positive
feedback loop that helps VEGFR2 translocate from the Golgi to the plasma membrane [82,86].
Organogenesis Stage
Organogenesis begins from 3 to 8 weeks in humans and around E9.5 in mice. Organ develop-
ment continues until birth in humans, with the exception of some organs like the mammary
gland, which occurs after birth. In this section, we will review the importance of the Hippo pathway
in organogenesis in chronological order (see also Figure 3). Generally, the Hippo pathway plays
an important role in cell survival, cell migration, and 3D structure formation.
Cardiac Development
Accumulating data show that the Hippo pathway is closely involved in cardiac development. Loss
of Hippo pathway components SAV1 early in development using SAV1Nkx2.5-Cre mice leads to
substantial cardiomegaly [87]. Similar phenotypes can be observed in MST1/2Nkx2.5-Cre and
LATS2Nkx2.5-Cre mice [87]. The change of myocardium thickness and heart size is due to
overproliferation of cardiomyocytes rather than hypertrophy [87]. Consistently, loss of YAP in
early development(YAPNkx2.5-Cre) or cardiac muscle (YAPTnnt2-Cre) results in severe myocardium
hypoplasia and embryonic lethality [88,89]. Despite the change of heart size, ectopic apoptosis
is not observed. Instead, cardiomyocyte proliferation is severely reduced [88]. The dramatic
myocardial overgrowth and cardiomegaly in embryos of active YAP conditional transgenic mice
further support the function of Hippo pathway in regulating cardiomyocyte proliferation [88–90].
Craniofacial Development
At E10.5 YAP/TAZ deletion in neural crest (YAP/TAZWnt1-Cre) causes overt craniofacial defects.
Hemorrhages in the branchial arch regions are also observed. Embryos undergo fetal demise
prior to E11.5. Histologic analysis shows that maxillary and mandibular branchial arches, which
neural crest normally migrate and develop into, are deficient [91]. Mechanistically, PAX3, a critical
transcription factor in the premigratory neural crest, binds to YAP/TAZ and loss of YAP/TAZ leads
to the downregulation of neural crest-specific genes such as Mitf [91]. Furthermore, YAP is also
required for smooth muscle differentiation of neural crest by coordinating with Notch signaling.
YAP can interact with the NICD, recruited to the enhancer of the Notch ligand Jagged by the
DNA binding protein Rbp-J independent of TEAD, thus promoting further Notch signaling and
smooth muscle differentiation [92].

Lung Development
From E12.5 to E15.5, YAP is reported to regulate proximal–distal patterning in lung development
[93]. Proximal cells express YAP in cytoplasm while distal counterparts express YAP in the
nucleus [93]. Lungs without YAP expression (YAPShh-Cre) are highly hypoplastic and show severe
disruption in branching morphogenesis, resulting in dilated cyst-like structures, where distal cells
expand at the cost of the proximal progenitors, which normally develop into airways [93]. TGF-β
plays an essential role in lung development. YAP activity is required for epithelial progenitor cells to
properly respond to TGF-β cues and initiate the differentiation program [93], in which YAP pro-
motes Sox 2 expression to specify the airway epithelial differentiation transcriptome. Besides
the Hippo pathway, many other signaling pathways are involved in the patterning of lung. In
in vitro differentiation protocols, modulation of WNT signaling is sufficient to give rise to organoids
with proximal and distal traits, suggesting that the Hippo pathway is likely a regulator of morpho-
genesis, rather than the lineage specification determinant [94].
Eye Development
The retina is originated from the two-layered optic cup in the embryo. The outer layer develops
into retinal pigment epithelium (RPE), which supports the neural inner cells called neural retina
(NR) [95,96]. The requirement of YAP activity during eye organogenesis was first found in
zebrafish, where knockdown of YAP results in reduced eye size [97]. Mice harboring conditional
retina knockout of YAP (YAPrx-Cre) display hypopigmentation and protrusion of retina-like epithelia
and the overall morphology of eyes is severely impaired [98]. Histologic analysis in E12.5 shows
pigmentation loss and organization change, suggesting a trans-differentiation of RPE to NR [98].
Furthermore, progressive degeneration is also observed in NR as YAP maintains NR polarity via
crumbs polarity complex [98]. Notably, mutation in TEAD1 preventing binding to YAP causes
Sveinsson chorioretinal atrophy in human, supporting a conserved role of YAP in retinal
development.
At E14.5, deletion of YAP (YAPnestin-Cre) in the lens leads to severe atrophy due to lens fiber (LF)
defect and the hypocellularity in the lens epithelium (LE). Anatomically, the lens is composed of
LE and LF cells. LE cells are progenitor cells and LF cells are fully differentiated cells that constitute
the majority of a lens [99,100]. YAP cooperates with tight junction and polarity complex proteins
to maintain self-renewal of LE cells. YAP-null LE cells exit from the cell cycle and differentiate into
LF cells [101].
Brain Development
In E14.5, YAP plays a critical role in neocortical astrocytic differentiation and proliferation. In the
developmental neural system, YAP is selectively expressed in neural stem cells (NSCs) and astro-
cytes while it is repressed in neurons. YAP astrocyte conditional knockout mice (YAPGFAP-Cre)
display less neocortical astrocytes, whereas YAP-deficient NSCs (YAPnestin-Cre) show normal
self-renewal and neural differentiation ability. Mechanically, YAP is involved in the stabilization of
SMAD1 induced by BMP2, to promote astrocytic specification [102,103].
Kidney Development
At E15.5, YAP/TAZ also play important roles in kidney development. Knockout of YAP(YAPsix2-Cre)
in cap mesenchyme, which develops into nephrons, leads to dramatic reductions in Henle’s loop,
glomeruli, and proximal tubule formation [104]. Conventional TAZ knockout mice are viable, but
display renal cysts with dilatation of Bowman’s capsules and proximal tubules [105]. These results
suggest that YAP and TAZ play distinct roles during kidney development. Kidneys without YAP
display early defects in nephron induction and stereotypical morphogenesis. CDC42 is an activator
of YAP/TAZ during kidney development; CDC42 knockout mouse kidneys show phenotypes
similar to YAP knockout [104].

Bile Duct Development

Outstanding Questions
YAP is required for bile duct development through E18.5. Hepatoblasts are progenitors for both
Is YAP and TAZ activity required for
hepatocytes and intrahepatic biliary epithelial cells (BECs) in embryonic liver [106]. Conditional maintaining zygote totipotency in vivo?
YAP knockout (YAPAlbumin-Cre) in biliary cells leads to the loss of tubular and nontubular structures
formed by wild type BECs [107]. Knockout of NF2 (NF2Albumin-Cre) or LATS1/2 (LATS1/2Albumin-Cre) Do YAP and TAZ play redundant roles
in embryogenesis in addition to tissue
in the liver leads to a bias towards BECs at the expense of hepatocytes [107–109]. Furthermore, specificity?
an in vitro differentiation assay shows that hepatocyte differentiation is compromised when LATS
activity is abolished. Consistently, RNA-Seq analysis shows an enrichment of BEC differentiation Is Hippo signaling essential in controlling
germ layer specification?
gene signature [110]. In an in vitro differentiation protocol, activation of TGF-β and Notch signaling
is sufficient to transform hepatoblasts into BECs. Thus, the crosstalk between Hippo signaling What are the upstream cues regulating
and TGF-β/Notch signaling is possibly responsible for the differentiation bias towards BECs in YAP/TAZ activity during organ
development and size control?
the absence of LATS kinases [111].
Is manipulating Hippo pathway activity
Concluding Remarks enough to induce certain tissue/cell
development?
Extensive genetic studies in the last decade, primarily in mouse models, have revealed fundamen-
tally important roles of the Hippo pathway and its effectors YAP/TAZ in mammalian development. How can the Hippo pathway activate
YAP/TAZ have a broad function in early development in most, if not all, tissues and organs. These different target gene transcription in
different tissues during embryogenesis?
functions of YAP/TAZ in development are not simply due to their role in tissue size (cell number)
control as YAP/TAZ can promote either lineage-specific differentiation or stem cell maintenance/
self-renewal in a context-dependent manner. Another general conclusion is that YAP/TAZ often
collaborate with other morphogens/development cues to control development. Moreover, in
addition to TEAD, YAP/TAZ can interact with other DNA binding factors to regulate developmental
programs. Although not discussed in this review, it should be noted that the Hippo pathway also
plays a key role in wound healing and tissue homeostasis in adults.
Despite the tremendous progress made regarding the function of Hippo in early development,
many key questions remain (see Outstanding Questions). Given the significant functional overlap
between YAP and TAZ, many genetic studies with a single deletion of YAP or TAZ may not
necessarily uncover the true function of the Hippo pathway. Moreover, due to the essential role
in TE differentiation, the function of the Hippo pathway in germ layer specification is difficult to
study. Techniques such as cell type-specific inducible knockout in early embryos may be helpful
to address these fundamental questions. The knowledge gained about the role of the Hippo
pathway in development may be translated for future tissue and organ engineering in vitro.
Disclaimer Statement
K-L.G. is a cofounder and has equity interest in Vivace Therapeutics. The terms of this arrangement have been reviewed and
approved by the University of California, San Diego, in accordance with its conflict of interest policies.
Acknowledgments
This work was supported in part by the National Institutes of Health grant (CA217642).
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