EMBRYOGENESIS Trophectoderm development Alarcon and Marikawa 2018

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Trophectoderm Development
Vernadeth B Alarcon and Yusuke Marikawa, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI,
United States
© 2018 Elsevier Inc. All rights reserved.
Glossary
Blastocyst A mammalian embryo at the final stage of development that is competent to implant in the uterus.
Morphologically, it appears as a hollow sphere consisting of two tissue types or cell lineages, the trophectoderm and inner cell
mass. The trophectoderm is responsible for implantation and placentation, whereas the inner cell mass gives rise to the fetal
body and the extraembryonic tissues, amniotic sac and yolk sac.
Cavitation The process that forms a fluid-filled space in the blastocyst. It is mediated by the trophectoderm, which regulates the
directional transport of fluid from the external environment into the developing blastocyst.
Conceptus The products of the fertilized egg, namely the embryonic and extra-embryonic tissues, which form the fetal body
and tissues that do not become part of the body (e.g., placenta, amniotic sac), respectively. It is distinct from the term
“embryo,” which refers to tissue that forms the body. The blastocyst can also be called a conceptus.
Epithelium A type of tissue which covers or lines the surface of tissues or organs. It functions as a selective barrier, regulating
the movement of molecules between the internal and external environments of a tissue or organ. Epithelial cells are
characterized as having apical-basal polarity, and are attached to each other by complexes of proteins, including the tight
junctions. A simple epithelium, such as the trophectoderm, consists of a single layer of flat-appearing cells owing to being
stretched by the expanding blastocyst cavity.
Preimplantation The period of development between the fertilization of the egg and implantation in the uterus. It occurs in
the oviduct (fallopian tube) and ends when the conceptus arrives in the uterus.
Introduction
The trophectoderm (TE; from the Greek “trophe,” “ektos” and “derma,”meaning to feed, outside and skin, respectively) is a simple
epithelium that makes up the outer wall of the blastocyst (from the Greek “blastos” and “kystis,” meaning bud or sprout and
bladder) (Fig. 1A). It is the first tissue to differentiate during preimplantation development, and it is unique to a distinct group
Fig. 1 Preimplantation development. (A) Schematic representations from fertilization to blastocyst formation. Trophectoderm (TE) differentiates as
the outer layer that surrounds the inner cell mass (ICM) and blastocyst cavity. A photograph of a mouse blastocyst at around 3 days after fertilization
is shown on the right. (B) A photograph of a blastocyst that is hatching out of the zona pellucida is shown. (C) Immunofluorescent visualization of
the cell type-specific transcription factors (CDX2 for TE and OCT4 for ICM) in a blastocyst at around 4 days after fertilization. Scale bars ¼ 50 mm.
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of mammals called the eutherians, also known as the placental mammals (Eakin and Behringer, 2004). Some examples are human,
mouse, dog, and cattle. The hallmark feature of TE is its adaptation for invasion and implantation in the uterus, after which it
contributes to the formation of the placenta. The viviparous mode of reproduction of eutherians is made possible by the placenta,
which functions in the nutritive and respiratory exchanges between mother and fetus. However, prior to implantation, the TE plays
a different role. It transports fluid from the environment into the blastocyst, causing it to increase in diameter. Since the blastocyst is
encased by a glycoprotein layer called the zona pellucida, it must hatch from its confinement before the TE can interact with the
uterus. It has been observed that when human and mouse blastocysts are cultured outside the maternal body in a Petri dish in
a laboratory setting, their expansion due to fluid accumulation appears to promote the tearing of the zona pellucida (Fig. 1B).
The failure of hatching leads to the inability of the blastocyst to implant and the failure to produce pregnancy.
In this article, we will examine TE development based on studies conducted primarily in the mouse. It is a popular experimental
system because it is amenable to investigations of mechanisms at the cellular and molecular level and is a useful proxy for
human since the two species undergo similar morphological changes during the preimplantation period. We will first provide
a morphological overview of the emergence of the TE epithelium in the developing blastocyst. We will then discuss the HIPPO
signaling pathway and its regulation by cell polarity as the molecular basis of how cells are segregated to differentiate into the
TE cell type. Lastly, the current knowledge of TE development is applied to examples of clinical issues that are significant to human
reproduction and fertility.
Morphogenesis of the Trophectoderm Epithelium
Preimplantation development prepares the conceptus for attachment to the endometrium, the inner lining of the uterus. The
process takes place in the oviduct (fallopian tube), and it begins with the egg. At approximately 60 mm in diameter, the egg is so
tiny that it can only be seen with the aid of a microscope. Yet it is one of the biggest cells in the adult body. The spermmust penetrate
the zona pellucida to fertilize the egg and produce the conceptus (Fig. 1A). Consisting of only one cell at the start, the conceptus
cleaves or divides to increase cell number without growth, so that daughter cells are eventually reduced to the size of adult cells. At
approximately 2 days after fertilization, the conceptus appears as a ball of cells or morula (from the Latin “morus,” meaning
mulberry), consisting of eight cells. It is from this stage onward that the TE epithelium is gradually constructed. The morula has
a bumpy surface that alters dramatically during the process of compaction. Cells change from spherical to wedge shape owing
to increases in adhesion between neighboring cells, causing the bumpy surface to smoothen (Fig. 2). This marks the establishment
for the first time of the apical-basal polarity in cells. It is the specialization of the plasma membrane into distinct domains that face
the external environment (apical) and neighboring (basal) cells. By the 16-cell stage, the morula arranges into an outside layer of
cells surrounding a central cluster of cells (Fig. 3). The spatially segregated cells are the progenitors of the first two tissue types in the
blastocyst, the TE and inner cell mass (ICM), the latter of which will give rise to the fetal body. The outside cells retain polarity
(Fig. 3) and assemble intercellular complexes of tight junction proteins between the apical and basal domains, creating a boundary
that segregates the specific functions of the two domains. At 3 days after fertilization or approximately 32-cell stage, a fluid-filled
Fig. 2 Compaction. A series of snapshot images that span the period of 3 h at the 8-cell or morula stage, highlighting cell shape changes during
the process of compaction. An arrow points to the second polar body, a remnant of oocyte meiosis. Scale bar ¼ 50 mm.
Fig. 3 Establishment of apical-basal cell polarity. Outside and inside cells are generated by the 16-cell stage, as progenitors of TE and ICM,
respectively. Distinct apical and basal membrane domains are retained in outside cells.
Embryogenesis j Trophectoderm Development 327
cavity forms inside the morula, transforming it into the blastocyst. The outside cells have matured into the TE epithelium,
functioning as a selective barrier between the internal milieu of the blastocyst and the external environment. The blastocyst appears
as a hollow, ovoid structure, consisting of a single layer of flat TE cells surrounding the aggregate of ICM cells in one side and the
fluid-filled cavityin the other side (Fig. 1A). TE cells adjacent to the ICM and adjacent to the cavity are designated as the polar TE and
mural TE, respectively. At 4 days after fertilization, the expanding blastocyst arrives in the uterus, hatches, and is ready for
implantation. A time-lapse movie of mouse development, showing progression from the 2-cell stage to the blastocyst stage
(Kono et al., 2014), can be viewed online in the PubMed database (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404313,
see under Supplementary Material, Movie 1: control): images of the conceptuses were captured every 15 minutes over a period
of 3 days of culture in a Petri dish.
The blastocyst cavity is essential for hatching and for promoting interactions between the TE epithelium and the endometrium
by increasing the surface area of the conceptus. The key to generating the cavity is the TE cell polarity that enables directional
transport of fluid, and it involves several steps (Fig. 4A and B). First, the basal domain serves as the target site for the exocytosis
of membrane vesicles that generate microlumens (small spaces) between the outside cells. Second, distinct ion pumps are
differentially distributed in the apical and basal domains that enable the directional transport of sodium ions from the external
environment into the microlumens. As sodium ions accumulate, osmotic pressure elevates, drawing in water and expanding the
microlumens, which coalesce to create a single fluid-filled cavity. Finally, to retain fluid in the cavity, an impermeable seal is created
by the tight junctions between the TE cells (Fig. 4B and C). The seal also enables blastocyst expansion during hatching, and the
increase in surface area enhances contact with the endometrium. Establishment of the apical-basal cell polarity is essential for
proper morphogenesis of TE, as the experimental disturbance of cell polarity in the mouse conceptus causes the abnormal
distribution of tight junction components and the absence of an impermeable seal (Alarcon, 2010). Consequently, blastocyst cavity
formation fails, and the conceptus appears as a morula.
Molecular Mechanisms of Trophectoderm Development
Both the TE and ICM have identical genetic composition, since they originate from the same fertilized egg. However, which genes are
transcriptionally activated or repressed are profoundly different between them. Genes that are specifically activated in the TE are
responsible for various functions unique to TE, such as blastocyst cavity formation, implantation, and trophoblast development.
In contrast, ICM expresses genes that are involved in the maintenance of pluripotency, i.e., the developmental capability that
generates various tissues to make the fetal body. Hundreds of genes are specifically expressed in the TE by the time the blastocyst
cavity is expanded. Some of the TE-specific genes start their expression at the 16- to 32-cell stages before cavitation occurs, and they
play critical roles in the development of functional TE. An example is Cdx2 gene, which encodes a transcription factor, i.e.,
DNA-binding protein that activates or represses transcriptions of other genes (Fig. 1C). When the fertilized egg is experimentally
created from sperm and oocyte that completely lack the Cdx2 gene, the resulting morula initially forms the blastocyst cavity but
fails to maintain it due to impaired epithelial tight junctions (Strumpf et al., 2005). Moreover, the Cdx2-deficient conceptus has
reduced expressions of other TE-specific genes (e.g., Eomes) and also abnormal ectopic expressions of ICM-specific genes (e.g.,
Oct4 and Nanog) in the outside cells. Cdx2 is one of the key regulators of TE development that coordinate differential gene
expressions, specifically by up-regulating transcription of other TE genes while repressing transcription of ICM genes.
One of the fundamental questions in elucidating the mechanisms of TE formation is how differential gene expressions between
the TE and ICM are achieved. Although the details of such mechanisms are still under investigations, a critical role is played by
HIPPO signaling (Cockburn et al., 2013; Hirate et al., 2013). HIPPO signaling is an intracellular signal transduction machinery,
consisting of over a dozen of interacting protein components, which regulate transcription of genes that control proliferation
and differentiation in various cell types (Fig. 5A). When HIPPO signaling is turned “on,” LATS (kinase protein), in association
with NF2 (membrane-associated protein) and AMOT (scaffold protein), phosphorylates YAP (transcriptional co-activator).
Fig. 4 Formation of the blastocyst cavity. (A) A series of snapshot images that span the period of 3 h around the 16- to 32-cell stages, highlighting
the emergence and expansion of the blastocyst cavity. An arrow points to a microlumen. (B) Schematic representations, depicting a sequence of
cellular and molecular events that lead to cavity formation. (C) Immunofluorescent visualization of ZO1 protein (green), a key component of tight
junction, in the blastocyst. Nuclei (blue) are also stained. Scale bars ¼ 50 mm.
328 Embryogenesis j Trophectoderm Development
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404313
Phosphorylated YAP is degraded or retained in the cytoplasm, and therefore is functionally inactive. In contrast, when
HIPPO signaling is turned “off,” YAP remains unphosphorylated and translocates into the nucleus to associate with TEAD
(DNA-binding protein), and together they function as a transcriptional activator. In the mouse embryo before the formation of
the blastocyst cavity, HIPPO signaling is turned on in inside cells, but is turned off in outside cells (Fig. 5A and B). As a result,
YAP accumulates in the nucleus only in outside cells, and forms a complex with TEAD to activate transcription of TE-specific genes,
such as Cdx2. Experimental inactivation of HIPPO signaling components causes abnormal expression patterns of TE- and
ICM-specific genes. For example, when YAP or TEAD is deficient, expressions of TE-specific genes are abolished and ICM-specific
genes are activated in outside cells (Nishioka et al., 2008, 2009). On the other hand, loss of NF2, LATS, or AMOT results in ectopic
expression of TE-specific genes in inside cells (Nishioka et al., 2009; Cockburn et al., 2013; Hirate et al., 2013). Therefore, the
differential regulation of HIPPO signaling between inside and outside cells is the key to attain cell type-specific gene expression
patterns.
The differential activity of HIPPO signaling in relation to cell position is regulated by the apical-basal cell polarity. Whereas
inside cells are completely surrounded by neighboring cells, outside cells have the surface that is exposed to the external
environment, which allows establishment of the apical-basal polarity. The exposed surface becomes the apical membrane domain,
whereas the area adjacent to the neighboring cells becomes the basal membrane domain (Fig. 3). Each membrane domain is
populated with unique sets of protein components in a manner similar to the apical-basal polarity found in most epithelial cell
types. The components localized to the apical domain are aPKC, PAR3, PAR6, and CDC42, whereas the basal domain components
include PAR1, LGL, and SCRIB. aPKC and PAR1 are kinases that modulate the activities of proteins through phosphorylation,
whereas the others act as scaffolds to tether proteins to a specific membrane domain. Localization and activity of these polarity
proteins are crucial to turn off HIPPO signaling in outside cells (Fig. 5A). For example, when the apical-basal polarity is impaired
by experimental disruption of the apical domain (e.g., removal of PAR6) or the basal domain (e.g., removal of PAR1), YAP does not
translocate into the nucleus and TE-specific genes are not expressed (Alarcon, 2010; Hirate et al., 2013; Korotkevich et al., 2017).
Although the molecular connection between the apical-basal polarity and HIPPO signaling has not yet been fully delineated,
a potential mechanisminvolves localized distribution of AMOT. In outside cells, the apical membrane domain sequesters and
possibly inactivates AMOT, leading to the turning off of HIPPO signaling (Hirate et al., 2013). Further investigations are underway
to identify other key mechanisms by which the apical-basal polarity influences HIPPO signaling.
Clinical Relevance of the Trophectoderm
The preceding sections are largely based on studies using the mouse as a eutherian model. However, the functional significance and
embryological origin of TE are likely to be similar in human. The following section focuses on three specific topics that involve TE
and bear clinical significance for human reproduction and fertility.
Early Pregnancy Loss
In human, early pregnancy loss is a common condition characterized by spontaneous abortion or miscarriage of the conceptus
before 12 weeks of gestation (Macklon et al., 2002; Larsen et al., 2013). A number of mechanisms have been proposed to cause
the miscarriage. One possibility is the abnormal development of the two tissues of the blastocyst, which would result in miscarriage
near the time of implantation. For instance, TE development is compromised, and it differentiates into inferior trophoblasts.
Normally, healthy trophoblasts secrete the hormone called human chorionic gonadotropin (hCG), which promotes a chain of
Fig. 5 HIPPO signaling and cell type-specific gene expression. (A) Schematic representations of HIPPO signaling that is differentially regulated
between outside and inside cells. Nuclear localization of YAP leads to transcriptional activation of TE-specific genes. (B) Immunofluorescent
visualization of YAP (green) and actin filament (red; highlighting cell boundaries) in a cross section of a morula at the 16- to 32-cell stage. Scale
bar ¼ 50 mm.
Embryogenesis j Trophectoderm Development 329
molecular events that are essential for the maintenance of the endometrium during the establishment of pregnancy. Since hCG is
produced only by the trophoblasts and is excreted in the maternal blood and urine, it is the molecule detected by pregnancy tests.
When trophoblasts are defective and fail to produce sufficient hCG, the endometrium degenerates and is shed from the uterus along
with the conceptus, thereby terminating the pregnancy. The requirement for proper interactions between trophoblasts and
endometrium may serve as a natural screen to select for good quality conceptus. However, TE may also be damaged by exposures
to developmental toxicants, resulting in pregnancy loss.
Hydatidiform Mole
In human, the abnormal behaviors of TE are linked to pregnancy-related disorders collectively referred to as gestational
trophoblastic disease. The most common type is hydatidiformmole (HM; “hydatid” is from the Greek “hydatidos,”meaning watery
vesicle), also known as molar pregnancy or mole. It is a non-viable pregnancy owing to an abnormal blastocyst that overexpresses
paternal-derived (i.e., sperm-borne) genes and whose TE gives rise to aberrant placental overgrowth. The ICM may give rise to little
or no fetal tissues. According to the United States National Cancer Institute, females in the extreme ends of reproductive age, namely
adolescents and perimenopausal women, are at increased risk for HM, affecting 1–3 out of 1000 pregnancies in developed
countries.
The placental overgrowth of HM is usually benign, though in rare cases it becomes malignant. Such tumors are unique since they
arise from the TE of the conceptus and not from tissue of the maternal body. Patients present with an abnormally high level of hCG
secreted by the molar trophoblasts compared to the trophoblasts of a normal pregnancy. Most women can now be cured because of
effective anti-neoplastic medications, such as methotrexate, and can have a normal pregnancy after experiencing a molar pregnancy.
HM is classified as complete (CHM) or partial (PHM) based on pathology and genetics (Seckl et al., 2010). CHM is a pregnancy
without an embryo and only placental tissues form. The trophoblastic villi of the placenta tend to accumulate fluid and swell,
appearing as bunches of grapes. CHM forms from the TE of an unusual blastocyst, the product of an egg that lost its nucleus
and is fertilized by two sperm or one sperm whose genome duplicated. While the resulting karyotype is numerically normal
(diploid), it is exclusively paternal in origin, and is 46, XX or 46, XY. 46, YY has not been observed since a conceptus with such
genetic makeup probably perishes. In rare cases, CHM originates from a conceptus that is biparental but with an abnormal maternal
genome that functions like the paternal genome.
PHM, on the other hand, consists of placental overgrowth with patches of swollen trophoblastic villi and some fetal tissues. The
blastocyst, in this case, is the product of an apparently normal egg that is fertilized by two sperm. The karyotype is triploid with two
sets of paternal-derived genomes in addition to the one set of maternal-derived genome, and is 69, XXX or 69, XXY or 69, XYY.
The occurrence of HM demonstrates that parental genomes are not functionally equivalent owing to the process called
imprinting. While paternal-derived and maternal-derived genomes contain the same genes, a parent puts its imprint or biochemical
mark on certain genes according to the sex of the parent. The imprint serves to regulate gene expressions, by activating (turning on)
or inactivating (turning off) them. Genes required for early placental development are turned on in the paternal-derived genome,
and are overexpressed in HM due to the two sets of paternal genomes. In contrast, genes required for early embryonic development
are turned on in the maternal-derived genome, and are either absent or underexpressed in HM. The observations with HM are
supported by experiments with mouse embryos that were manipulated to contain exclusively paternal genomes or exclusively
maternal genomes (McGrath and Solter, 1984).
Trophectoderm Biopsy
TE biopsy is the sampling of TE cells from the blastocyst produced by in vitro fertilization (IVF) to analyze for the presence of genetic
abnormalities (e.g., chromosome defect and single gene defect) or to determine sex (Hyde and Schust, 2015). The procedure is used
to aid in the selection of blastocysts for transfer into the uterus of patients undergoing assisted reproductive services.
According to the Centers for Disease Control and Prevention, many people experience problems of fertility in the United States.
Such problems are the inability to conceive after 1 year of sexual intercourse with the same partner without the use of contraceptives
or the inability to sustain a pregnancy to live birth. Factors that contribute to them originate from either the female or male, or both.
Fertility problems are not necessarily symptomatic of sterility, and assisted reproductive technologies (ART) may enable affected
individuals to achieve conception and the successful birth of a baby. It is estimated that almost 2% of all babies born annually
in the United States are conceived using ART.
IVF is the most commonly used ART. Eggs and sperm are retrieved from the patients, and are combined in a Petri dish. This
allows fertilization to take place, and the resulting conceptus is transferred into the uterus for implantation. However, IVF often
yields several conceptuses, and patients may decide for them to undergo preimplantation genetic diagnosis (PGD) or screening
(PGS) to select only the desired conceptus for uterine transfer. TE biopsy has become a common method for collecting a small
number of TE cells for PGD/PGS. During the biopsy, a portion of the mural TE, i.e., the TE covering the blastocyst cavity is aspirated
into a pipette, a glass tube with a narrow bore, and is separated from the blastocyst with the aid of a laser device. The collected TE
cells are then processed for genetic analyses (e.g., chromosome numbers and structures, and DNA sequences). Although the TE does
not become part of the fetal body, it is asuitable proxy for testing since its genetic composition is the same as the ICM.
Due to the hole created in the TE epithelium by the biopsy, the fluid in the cavity leaks out and the roof of the cavity collapses.
The biopsied blastocyst is cryopreserved to await the results of the genetic analyses. If a blastocyst is selected for transfer, it is thawed
330 Embryogenesis j Trophectoderm Development
during which the wound in the TE epithelium closes, and the cavity re-expands. Since the blastocyst consists of more than 100 cells,
it is resilient to the removal of a few TE cells and is capable of continuing development to produce a baby.
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Embryogenesis j Trophectoderm Development 331
	Trophectoderm Development
	Introduction
	Morphogenesis of the Trophectoderm Epithelium
	Molecular Mechanisms of Trophectoderm Development
	Clinical Relevance of the Trophectoderm
	Early Pregnancy Loss
	Hydatidiform Mole
	Trophectoderm Biopsy
	References