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Gutsy moves in mice: cellular and
molecular dynamics of endoderm
morphogenesis
Manuel Viotti1, Ann C. Foley2 and Anna-Katerina Hadjantonakis3
rstb.royalsocietypublishing.org
1
Genentech Incorporated, South San Francisco, CA 94080, USA
Department of Bioengineering, Clemson University, Charleston, SC 29425, USA
3
Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
2
Review
Cite this article: Viotti M, Foley AC,
Hadjantonakis A-K. 2014 Gutsy moves in mice:
cellular and molecular dynamics of endoderm
morphogenesis. Phil. Trans. R. Soc. B 369:
20130547.
http://dx.doi.org/10.1098/rstb.2013.0547
One contribution of 14 to a Theme Issue
‘From pluripotency to differentiation: laying
the foundations for the body pattern in the
mouse embryo’.
Subject Areas:
cellular biology, developmental biology,
genetics, molecular biology, evolution
Keywords:
gastrulation, gut morphogenesis, visceral
endoderm, definitive endoderm, epithelial-tomesenchymal transition, mesenchymal-toepithelial transition
Author for correspondence:
Anna-Katerina Hadjantonakis
e-mail: [email protected]
Despite the importance of the gut and its accessory organs, our understanding
of early endoderm development is still incomplete. Traditionally, endoderm
has been difficult to study because of its small size and relative fragility. However, recent advances in live cell imaging technologies have dramatically
expanded our understanding of this tissue, adding a new appreciation for
the complex molecular and morphogenetic processes that mediate gut formation. Several spatially and molecularly distinct subpopulations have been
shown to exist within the endoderm before the onset of gastrulation. Here,
we review findings that have uncovered complex cell movements within the
endodermal layer, before and during gastrulation, leading to the conclusion
that cells from primitive endoderm contribute descendants directly to gut.
1. Introduction
The endoderm is one of three definitive germ cell layers generated at gastrulation.
Cells descended from the endoderm will constitute the epithelial lining of the respiratory and digestive tracts, as well as the endocrine glands, and auditory and
urinary systems. Although a large body of work exists concerning the specification and formation of the endoderm in different model systems, in the mouse,
recent studies have called for a rethinking of the cellular dynamics that drive
the initial phases of gut endoderm formation. It is now clear that, rather than
respecting a strict embryonic–extraembryonic segregation of cell lineages, the
gut endoderm is composed of cells derived from both definitive endoderm
(DE) and so-called extraembryonic endoderm, and that these two cell populations
are coordinately integrated into a single tissue, the gut endoderm, through a
process of dispersion and intercalation.
2. A panoply of endoderm
At the time of implantation into the maternal uterus (at around embryonic day (E)
4.5), the late blastocyst stage mouse embryo comprises three tissue lineages: the
pluripotent epiblast and two extraembryonic lineages, the primitive endoderm
(PrE; figure 1 and table 1) and the trophectoderm. Shortly after implantation
(E4.5–E5.0), the embryo elongates to form a radially symmetric egg cylinder,
which concomitantly cavitates at its centre (reviewed in this issue [1]). At this
point, the embryo comprises a bilaminar cup-shaped epithelium. The outer
layer consists of the PrE-derived visceral endoderm (VE) that encapsulates the
proximally positioned extraembryonic ectoderm (ExE), and the distally positioned, pluripotent epiblast that will go on to form the embryo proper as well
as some extraembryonic mesoderm (figure 1).
As is the case in several vertebrates, in the mouse there are two recognized
sources of endoderm tissue, one arising from the PrE and the other from the pluripotent epiblast [2,3]. Until recently, the canonical thinking dictated that the PrE
gave rise only to extraembryonic lineages such as the parietal endoderm and VE
of the early postimplantation conceptus, whereas endodermal cells that ingressed
through the streak made up the embryonic endoderm that comprises the gut.
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
mid
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parietal endoderm (PE)
trophectoderm
early
blastomeres
primitive endoderm
(PrE)
visceral endoderm (VE)
inner cell mass
(ICM)
definitive endoderm (DE)
mesoderm
ectoderm
primitive
streak
P
A
P
D
mesoderm
progenitors?
mesoderm
progenitors
DE
progenitors?
DE
progenitors?
R
A
P
L
primitive
streak
Figure 1. Schematic depicting early lineage relationships in the mouse embryo. By embryonic day 3.5 (E3.5), the blastula-stage embryo has given rise to the
trophectoderm (green) and the inner cell mass (ICM; magenta). The ICM gives rise to both the pluripotent epiblast (red) and primitive endoderm (PrE; blue).
The PrE later differentiates into the both the parietal (PE) and visceral (VE) endoderms (dark and light blue, respectively) and the epiblast gives rise to the
three embryonic lineages, definitive endoderm (DE; purple), mesoderm (orange) and ectoderm (red).
At early postimplantation (E5.5–E6.5), the VE epithelium
comprises several subpopulations, generally encompassing:
the extraembryonic VE (exVE), a cuboidal epithelium overlying the ExE, and the embryonic VE (emVE), a squamous
epithelium overlying the epiblast [4–9]. While exVE and
emVE cells have different morphology and patterning abilities,
it is important to note that these names (ex versus em) primarily relate to the position of the VE relative to the conceptus, but
that all VE cells stem from the PrE. A further segmentation
occurs within the emVE, its most distal cells forming the
distal VE (DVE) at E5.5 [10]. Between E5.5 and E6.5, cells of
the emVE undergo positional rearrangements and become
further patterned, resulting in the establishment of the anterior
VE (AVE) [11]. DVE and AVE express specific factors that
are involved in patterning the underlying epiblast along its
proximal–distal (P–D) and anterior–posterior (A–P) axes
[2,12–14]. Additional marker-specific subpopulations of VE
cells have been reported, although such regions have typically
not been given names by their position [11,15,16].
3. The displacement model of gut endoderm
formation
About a quarter of a century ago, elegant fate-mapping experiments using single-cell iontophoretic injection of horseradish
peroxidase resulted in the first conceptual reconstruction of
the cellular dynamics involved in endoderm formation in
mouse embryos [17,18]. In this set of studies, cells of the epiblast and VE were individually labelled, embryos incubated
for various time periods and then the position of their cellular
descendants noted. Derivatives of epiblast cells from the region
in the vicinity of the anterior primitive streak (APS) were likely
to end up on the embryo’s surface, often in the area of the DE,
and at later stages appeared within the gut tube. By contrast,
descendants of VE cells overlying the midline, which are
referred to as axial VE cells, often appeared in extraembryonic
regions of the conceptus. This gave rise to the notion that epiblast cells around the APS ingress (EMT) at the primitive
streak (figure 2), and subsequently egress (MET) to emerge at
the surface inserting into the VE at the distal tip of the
embryo. As increasing numbers of cells undergo this process,
the newly formed sheet of DE cells would form a contiguous
epithelium with the VE, which would gradually and uniformly
displace the latter to proximal regions, from where it would
exclusively contribute to extraembryonic tissues. Additional,
fate-mapping analyses using orthotopic grafting and other
cell labelling techniques supported the notion that cells in the
posterior epiblast near the APS end up on the embryo’s surface
and give rise to gut endoderm cells [14,19] (figure 2). Few
studies have looked directly at the fate of VE cells, but one in
particular appeared to support the displacement model. VE
Phil. Trans. R. Soc. B 369: 20130547
epiblast
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Table 1. Endodermal cell populations of the early mouse embryo.
PrE
PE
blastula-stage tissue layer, precursor to parietal and visceral endoderm
PrE-derived cell population that lies adjacent to the mural trophectoderm. Fated to contribute to the
endoderm layer of the parietal yolk sac as well as the deposition of Reichert’s membrane
VE
PrE-derived epithelial sheet that encapsulates the extraembryonic ectoderm proximally and epiblast
distally. Fated to form the endoderm layer of the visceral yolk sac, as well as part of the gut tube
distal visceral endoderm and
anterior visceral endoderm
DVE and AVE
two molecularly distinct subdomains of the VE, one located at the distal tip of conceptus (DVE), and
the other demarcating the prospective anterior of the conceptus (AVE)
extraembryonic VE
exVE
subset of VE cells situated in the extraembryonic region of the postimplantation conceptus, overlying
embryonic VE
emVE
the extraembryonic ectoderm. Fated to give rise to the endoderm of the visceral yolk sac
subset of VE cells in the embryonic region of the early postimplantation conceptus, overlying the
DE
epiblast. Probably fated to give rise to some yolk sac endoderm as well as part of the gut tube
epiblast-derived cell population of cells that contributes to the gut endoderm
definitive endoderm
gut endoderm
precursor of all endodermal tissues in the adult organism. It is an epithelium composed of
emVE- and DE-derived cells
cells at the distal tip of the conceptus at E5.5 were labelled with
DiI and examined after 24 h. These labelled cells showed a
marked directional movement towards the anterior of the
forming embryo, ending up in the AVE. While these cells did
not themselves cross over into the extraembryonic region,
this finding strongly suggested that cells ‘ahead’ of those
labelled must have migrated outside of the embryo proper
[20]. Later, it was shown that VE cells abruptly stop their forward movement once they have reached the embryonic/
extraembryonic border, and thereafter begin to migrate laterally [9]. Whether the organization of the VE epithelium
overlying the ExE provides an impenetrable physical barrier,
or a repulsive signal, to promote the cessation of anteriorward
AVE migration remains an open question.
In addition to fate-mapping studies, several other observations appeared to support the notion that VE is displaced
by the forming DE. Morphological analysis of cells on the
embryo’s surface nicely fits in with the displacement
model. Initially, heavily vacuolated cuboidal cells, typically
associated with VE, surround the entire conceptus, but
after gastrulation, cells with this shape are confined to extraembryonic regions while cells on the surface of the
embryonic region are squamous, classically associated with
the DE lineage [7]. Analysis of the localization of pan-VEmarkers also fit the displacement model. At first, VE markers
such as Ttr, Afp, Sox7 or Hnf4a are present in a layer of cells
surrounding the entire conceptus, but when gastrulation is
complete, their domain of expression is restricted to proximal
regions [4,21,22]. This supported the notion that the VE is
displaced proximally as a uniform cellular sheet.
4. Proposing cell dispersal as a morphogenetic
mechanism for gut endoderm
The displacement model remained in place for nearly two
decades until recent advances in live cell imaging techniques
allowed, for the first time, direct imaging of cell movements throughout the VE during gastrulation. These studies
revealed surprising new details about how the gut endoderm is formed. In particular, generation of the Afp–GFP
transgenic mouse strain permitted labelling of the entire VE
and this allowed global imaging of the cellular dynamics
occurring within the VE layer during gut endoderm morphogenesis [23]. In this strain, green fluorescent protein
(GFP) is expressed under the enhancer/promoter sequence
of alpha-fetoprotein (Afp). Afp is a VE-specific marker in
early postimplantation embryos, and is also expressed at
later stages of development in a series of tissues, including
endoderm derivatives. At gastrula stages, all VE cells in
Afp–GFP embryos are labelled with GFP. Additionally,
owing to the perdurance of the GFP, immediate descendants
of VE cells are labelled as well, even if the transgene is no
longer being transcribed. This renders the Afp– GFP strain
both a marker for pre-gastrula VE and a short-term cell
lineage tracer for VE-derived cells.
Live imaging of Afp–GFP embryos resulted in a surprising
finding [21]. In accordance with the displacement model, upon
gastrulation, one might have expected an initially uniform
sheet of GFP-positive cells covering the entire conceptus to be
gradually displaced proximally by a new sheet of GFP-negative cells emerging from the distal tip of the embryo. This
was not observed. Instead, GFP-negative areas, corresponding
approximately to the size of single cells, began appearing
within the region of the emVE. Over time, the GFP-negative
areas grew in size, diluting the GFP-positive areas initially to
cohorts and eventually to single cells. Further experiments
suggested that single epiblast-derived cells probably intercalated into the emVE, the descendants of which remained in
the embryonic region of the conceptus rather than being displaced to the extraembryonic region. Time-lapse movies
revealed that, over time, cells of the emVE epithelium
became dispersed to the point of ending up as single cells
among a majority of DE cells. This was achieved through widespread DE cell intercalation at VE cell interfaces over a short
period of time. Furthermore, time-lapse movies revealed that
the progeny of VE cell divisions appeared to move apart,
suggesting an intrinsic repulsive mechanism operating
between VE descendants, or an underlying differential cell–
cell adhesion. These studies also revealed that emVE-derived
cells could later be found in the gut tube, and were present
until at least the 15 somite stage, raising the possibility that
Phil. Trans. R. Soc. B 369: 20130547
visceral endoderm
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primitive endoderm
parietal endoderm
3
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primitive streak
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primitive streak
epiblast
mesoderm
epithelial cell
(epiblast)
Phil. Trans. R. Soc. B 369: 20130547
ingression
(EMT)
mesenchymal
(mesoderm) cell
time
mesenchymal
(mesoderm) cell
partially polarized
definitive endoderm progenitor?
mesenchymal
(mesoderm) cell
egression
(MET)
epithelial cell
(visceral endoderm)
epithelial cell
(definitive endoderm)
epithelial cell
(visceral endoderm)
embryo surface
visceral endoderm (VE)
gut endoderm
definitive endoderm (DE)
mesoderm
Figure 2. Relating the cellular dynamics of gut endoderm morphogenesis to epithelial – mesenchymal transitions. During gut endoderm formation, cells undergo
both epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions in rapid succession. At the primitive streak, DE progenitors ( purple) exit the
epithelial pluripotent epiblast (blue) and enter the mesenchymal wings through the process of ingression. The ‘wings’ consist of DE precursors and mesoderm
(orange). Once they have reached their final position, DE cells egress from the mesenchymal wings into the newly formed endodermal epithelium that is composed
of DE cells and visceral endoderm cells (VE; blue).
cells from an extraembryonic lineage persist within the gut of
the fetus, and possibly even the adult. These observations
were confirmed using other fluorescent protein reporter lines,
as well as using genetic fate mapping with a pan-VE Ttr-Cre
line and lacZ- and GFP-based Cre reporters [24].
These data suggest a new model that can account for both
these new findings as well as previous fate-mapping and
gene expression data. In what we refer to as the cell dispersion model, the epiblast-derived DE does not displace
the emVE as a sheet. Instead, the DE initially migrates
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beneath the emVE and gradually causes the emVE to disperse as increasing numbers of DE cells egress into it.
(a) Pre-gastrulation and ingression through the
primitive streak
(b) Migration of definitive endoderm progenitors
The concept that DE emerges at the distal tip of the conceptus
resulted from fate-mapping studies that showed that DE precursors reside near the APS [17 –19]. The primitive streak is a
dynamic structure: it starts out as a small region of cellular
ingression in the most posterior part of the epiblast (at the
epiblast – ExE border), which then gradually elongates distally and anteriorly until reaching the embryo’s distal tip.
Thus, the position of the APS and fate of cells emanating
from it are likely to change over time. Furthermore, DE of
the mid- and hindgut is unaffected in Foxh1 and Foxa2
(Hnf3b) mutant embryos, both of which fail to specify the
APS [35]. Therefore, while stage-specific fate maps suggest
a fairly localized origin for the DE, in fact these cells might
have origins all along the primitive streak.
While live imaging data of fluorescent reporter strains visually demonstrate dispersal of the emVE in lateral regions of the
embryo, these data do not capture the cellular dynamics occurring in the midline in real-time [21]. Analysis of sequentially
staged Afp–GFP embryos reveals that emVE cells overlying
the midline become displaced anteriorly by epiblast-derived
cells fated to form axial mesoderm structures (node and notochord) [21]. Hence, in accordance with a displacement model,
some epiblast-derived cells presumably originating in the APS
probably insert directly into the endodermal layer from the
distal tip of the conceptus, although reporter-based studies
and earlier fate maps [9,20] suggest that these cells spread
only anteriorly and not laterally. This suggests that the midline
emerges by intercalation and VR displacement, whereas the
Phil. Trans. R. Soc. B 369: 20130547
Before gastrulation, the emVE encapsulates the epiblast, where
the precursors of the DE lineage reside. The anterior part of the
emVE epithelium, the AVE, expresses inhibitors of Nodal and
Wnt3, restricting the activity of these genes to posterior regions
[25,26]. In the posterior of the embryo, high levels of Nodal
and Wnt3, in addition to bone morphogenetic protein 4
(BMP4), elicit the formation of the primitive streak, marking
the onset of gastrulation [27]. Posterior epiblast cells become
fated to the mesoderm and DE lineages, ingress through the
primitive streak and undergo an epithelial-to-mesenchymal
transition (EMT). Cells lose epithelial morphology and adopt
a mesenchymal shape by breaking down apical–basal cell
polarity, cell–cell junctions, as well as constituents of the basement membrane, freeing them to migrate away from the
vicinity of the primitive streak. Fibroblast growth factor
(FGF) signalling mutants [28–30] as well as embryos lacking
Snail [31], Eomes [32] or Brachyury [33,34] have cells that
ingress at the primitive streak but cannot correctly migrate
away from it, as a result of incomplete EMT. These mutants
invariably fail to establish both mesoderm and DE. It is therefore largely accepted that all epiblast cells that will give rise to
DE lineage undergo EMT at the primitive streak.
5
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5. A stage-by-stage description of cellular
dynamics that drive endoderm formation
gut endoderm forms through intercalation and dispersal. A
key open question pertains the mechanism by which the
majority of DE cells enter the gut endoderm.
After ingression at the primitive streak, mesoderm progenitors emerge as a new mesenchymal layer sandwiched
between inner epiblast and VE on the embryo’s surface,
and are referred to as the wings of mesoderm. This mesenchymal tissue layer migrates from both left and right sides of
the primitive streak in a posterior-to-anterior direction, effectively circumnavigating the conceptus, but stopping short of
the midline. As a consequence, the most proximal cells in the
wings are more advanced in their migration because they
emerged from the streak before the more distal cells. A key
component of the dispersal model argues that the DE does
not migrate into the emVE layer as a sheet, but rather DE
cells must co-migrate with mesoderm progenitors in the
wings of mesoderm and then intercalate into different regions
of the overlying emVE. The intercalation of single DE cells joining the endodermal epithelium from the wing of migrating
mesoderm can be defined as an egression, because it is the reciprocal mechanism to ingression, where single cells leave an
epithelium [36]. A study on Mixl1 mutants argued that at gastrulation DE and mesoderm movements occur independently
of one another [14]. Mixl1 mutants do not make DE, and yet
mesoderm is capable of migrating. The observation is per se
not in disagreement with a dispersal model: if DE cells fail to
be specified, they cannot intercalate into the emVE.
One question that arises is whether cells in the migrating
wings of mesoderm are bipotential. Experiments in other
organisms [37–39] and mouse cell lines provide evidence
for a mesendodermal population, but the existence of true
bipotential cells that can give rise to either mesoderm or
endoderm remains unresolved in the mouse [27,40]. The
early lineage-tracing experiments using single-cell iontophoresis of horseradish peroxidase showed that in some cases a
single labelled posterior epiblast cell could result in cells of
both mesoderm and gut endoderm [17,18]. Investigations of
Nodal mutants have provided several insights into this
matter. In vivo, the levels of Nodal signalling regulate the
selective allocation of both mesoderm and endoderm [41].
Mice lacking Nodal or its co-receptor Cripto fail to form a
primitive streak and lack both mesoderm and gut endoderm
[42 –44]. Hypomorphic mutants with moderate levels of
Nodal signalling can form some mesoderm and endoderm,
whereas lower levels of Nodal signalling result in a loss of
endoderm, but not mesoderm [45]. By contrast, elevated
Nodal signalling caused by loss of the repressor DRAP1
resulted in an expanded primitive streak and production of
excess mesoderm [46]. These findings suggest that the
levels of Nodal signalling play an important role in regulating the selective allocation of mesoderm and endoderm
from a common progenitor population. Further support for
the existence of a bipotential mesendodermal population
comes from studies on the Wnt/b-catenin pathway. Upon
lineage-specific removal of b-catenin, mesoderm-like cells
are present in the endoderm layer of mutant embryos
suggesting a transfating of endoderm to mesoderm [47],
and deletion of b-catenin specifically in Sox17-expressing
cells prevents endoderm formation [48]. Nonetheless, based
on marker expression, it has been suggested that DE and
mesoderm cell lineages might already be predetermined
before gastrulation [49], though it should be noted that
markers indicate the state, not fate, of cells.
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We have already described how cells fated to contribute to
the mesoderm and endoderm enter the mesodermal wings
6
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6. Egression of definitive endoderm cells into the
overlying embryonic visceral endoderm
by undergoing EMT at the primitive streak. These cells
migrate anteriorly until, shortly thereafter, certain cells in
their outer edges begin to express DE makers and intercalate
into the emVE. As they leave a mesenchymal population and
join an epithelial layer, they undergo a mesenchymal-to-epithelial transition (MET). This rapid EMT– MET cycle raises
the question of whether DE cells undergo a complete transition between epithelial and mesenchymal states, or rather
whether they might loosen their epithelial characteristics temporarily to migrate, then re-epithelialize during egression. An
incomplete or partial EMT and MET have been described in
other contexts [55,56], and further work will be needed to elucidate these details in mouse gut endoderm morphogenesis.
It is not known how Foxa2 and Sox17, the evolutionarily
conserved DE transcription factors expressed in egressing
cells [50], control cell egression. Mutants in either one of
these factors exhibit severe reductions of the DE lineage in
the gut endoderm [57 –59]. Evidence is accruing to suggest
that Foxa2, as well as Sox17, regulate factors necessary for
epithelialization, such as cell polarity, cell –cell junctions
and basement membrane components. An embryo chimaera
study focusing on the cell dynamics in the axial midline has
reported that Foxa2 mutant cells are capable of inserting into
the VE layer, but fails to acquire epithelial polarity or establish new adherens or tight junctions with neighbouring
emVE cells, and consequently regress back into deeper
(mesoderm) layers [49]. Accordingly, proteins involved in
cell adhesion during epithelialization have been proposed
as direct targets of Foxa2, including Claudin4, Flrt2, Flrt3,
Pcdh19 and Itga3 [60].
While experimental evidence has yet to connect Sox17 to
cell polarity or cell –cell junctions, Sox17 has been shown to
bind promoter regions and directly regulates the expression
of genes encoding basement membrane factors in some endodermal cell types [61 –63]. Furthermore, a study on mouse
blastocyst stage embryos revealed a requirement for Sox17
in basement membrane formation in the PrE [64].
Another recent study has highlighted the importance of
DE epithelialization as a requisite intermediate step in the
differentiation of DE progenitors. The Rho GTPase Rhou controls F-actin distribution and cytoskeletal organization in
epithelial cells. Knockdown of Rhou impairs endoderm differentiation in tissue culture as well as in the embryo [65]. It will
be interesting to determine if and how Rhou and other factors
regulating cell morphology could function downstream of
Sox17 and/or Foxa2.
Loss of Foxa2 leads to absence of anterior DE (which
would make the foregut) and axial mesoderm [59,66], but formation of mid- and hindgut is largely unaffected [35]. Sox17
mutants appear to display the converse phenotype. Foregut
endoderm forms, whereas the regions of the prospective
mid- and hindgut are devoid of DE cells [58]. Furthermore,
the Afp–GFP transgenic reporter reveals the emVE only
shows limited dispersal in anterior regions and no dispersal
in mid- and posterior regions [50], suggesting that DE cells
fail to egress in the mid- and posterior regions. Nonetheless,
we have also noted that Sox17 mutants properly specify axial
midline structures [50]. Interestingly, Sox17 is localized in the
majority of gut endoderm cells, but is absent from axial midline structures as well as the gut endoderm area around the
anterior intestinal portal [50]. Chimaera studies have shown
that cells lacking Sox17 cannot contribute to mid- or hindgut,
but are capable of contributing to foregut [58]. Conversely,
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If a mesendodermal state indeed exists within the mouse
embryo, it is worth considering how it might tie in with a dispersal model of endoderm morphogenesis. Bipotentiality
could be restricted to stages immediately after ingression
through the primitive streak, or extend on through migration
of cells within the wings of mesoderm. While the levels of
Nodal, seen by any individual cell as it passes through the
primitive streak, are thought to affect its decision to become
mesoderm or DE, we do not know if this is sufficient for the
fate choice or if it merely primes cells to one or the other fate,
and subsequent environmental cues seal their fate. DE progenitors express endoderm markers just prior to and during
intercalation into the emVE [50]. It is conceivable that a signal
from the emVE instructs adjacent cells to upregulate DE factors.
Only cells in the outer layers of the wings of mesoderm making
direct contact with the emVE show expression of DE markers
[50]. Accordingly, ultrastructural examinations of cells in the
wings of mesoderm have revealed that cells adjacent to the
emVE are more closely opposed to the basement membrane
and more tightly packed than their counterparts that lie closer
to the epiblast [17,51,52], perhaps hinting at an intrinsic heterogeneity within the mesoderm, or a subpopulation primed to
join the surface endoderm by virtue of its position.
Recently, miR-335 has been implicated in the fate choice
between mesoderm and DE [53]. This intragenic microRNA
is encoded in a mesoderm-specific transcript (Mest) and
targets the 30 UTRs of Sox17 and Foxa2. Overexpression of
miR-335 reduces Sox17 and Foxa2 levels and blocks endoderm
differentiation in embryonic stem cells (ESCs) and ESCderived embryos. Conversely, inhibition of miR-335 leads to
increased Sox17 and Foxa2 protein accumulation and endoderm formation [53]. Yet, when, where and how cells are
instructed to express miR-335 remain open questions.
Mouse ESCs that are treated with Activin A in many respects
provide an in vitro model of primitive streak formation and gastrulation-like EMT. Molecular studies in this model suggest that
EMT at gastrulation involves a gene-regulatory feedback loop
consisting of Brachyury, Foxa2 and Sox17 [54] such that upon
Activin A treatment, cells changed their molecular identity, heterogeneously expressing previously silenced Brachyury, Foxa2
and Sox17. Interestingly, Brachyury depletion decreased Sox17
and Foxa2 expression at both transcript and protein levels,
suggesting that expression of Brachyury might be a prerequisite
for a cell to enter a mesendodermal state. Supporting this mechanism, chromatin immunoprecipitation sequencing experiments
revealed direct binding of Brachyury to regulatory sequences of
Sox17 and Foxa2. By contrast, overexpression of Sox17 in
embryonic bodies resulted in a significant downregulation of
Brachyury, pointing to a negative feedback loop. How the
post-EMT heterogeneity of cells is achieved remains an open
question. Further studies will be needed to address the question
of how cells in the wings of mesoderm sort into mesodermal and
DE populations, and specifically whether a predetermination to
one lineage is established at the primitive streak, or if migrating
cells become programmed by a positional cue, possibly
emanating from the emVE.
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anterior
anterior
7
ventral view ~E8.0
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yolk
sac
visceral endoderm (VE)
gut endoderm
definitive endoderm (DE)
midline
Figure 3. Congregation of VE cells around midline structures. By the end of gut endoderm formation, most of the endodermal epithelium consist of DE cells that
have migrated through the streak. Cells from the original visceral endoderm (VE) layer are dispersed as single cells throughout this epithelium except at the midline
where VE cells encircle the node and form a continuous line of cells on either side of the midline head process (white and blue cells).
cells lacking Foxa2 can colonize the hindgut, but not the foreand midgut [67]. Taking these data into account, one might
envision a mechanism whereby MET of DE cells is controlled
by Sox17 in the mid- and posterior areas of the emVE (which
will give rise to mid- and hindgut), and by Foxa2 in the
anterior (which will give rise to foregut). Additionally,
Foxa2 regulates the epithelialization of cells in the axial
midline [49].
As is the case with almost all endoderm markers, both
Sox17 and Foxa2 are expressed in the extraembryonic
and embryonic endoderm lineages, at different stages of
development and even adulthood [27]. They are both first
expressed in the PrE [54], the first epithelium formed in the
embryo, and absence of Sox17 leads to a disorganized PrE
epithelium in mouse blastocysts [64]. It is therefore tempting
to speculate that Sox17 and Foxa2 are key regulators of endodermal tissue epithelialization throughout development and
in adult homeostasis.
emVE cells downregulate cell–cell junctions to accommodate egression of DE cells [21]. This response by the emVE
reveals a tight-knit coordination with egressing DE cells,
especially taking into account the multifocal egression of DE
progenitors into different areas of the emVE. The preferential
egression into emVE cells with relaxed cell–cell junctions
rather than already egressed DE cells might account for the
full dilution of the emVE to single cells by the end of gut endoderm morphogenesis. If a bias to egress into neighbouring
emVE cells did not exist, one could expect to find small cohorts
of emVE-derived cells in the gut endoderm. The communication signals between cell donor tissue (DE) and the
epithelial acceptor tissue (the emVE) remain to be elucidated,
and might have implications in other contexts of epithelial reorganization and metastasis.
7. Gut endoderm patterning and gut tube
formation
In lateral regions of the gut endoderm, emVE-derived cohorts
of cells become diluted until they are single cells surrounded
by cells of the DE lineage. By contrast, in the axial midline
region, emVE-derived cells remain organized around the
node and notochord (figure 3), suggesting a distinct morphogenetic behaviour is likely to have occurred and that the
egression mechanism described above may not apply to midline regions [21]. One could envision axial mesoderm
precursors inserting into the distal tip of the embryo, pushing
emVE cells aside, which consequently remain organized
around the emergent midline and node. As the node expands
anteriorly forming the notochord, those axial mesoderm cells
could displace a portion of emVE cells anteriorly up to, but
not beyond, the embryonic –extraembryonic junction. This
would be in concordance with the lineage-tracing and live
cell imaging experiments, where pre-gastrulation axial
emVE or AVE cells were labelled and the majority of their
descendants ended up in anterior regions of the conceptus,
but respected the embryonic/extraembryonic border by
remaining in the embryonic region [9,17,18,20,68]. Another
portion of axial emVE cells could be pushed aside, thereby
explaining the manner in which emVE cells lie on either
side of the notochord in Afp–GFP embryos [21].
By the late bud stage (approx. E7.5), no more DE cells
egress onto the surface, and the resulting gut endoderm is
a monolayered epithelium. Different regions of the gut endoderm already have distinct molecular characteristics, and
have thus started transitioning from a naive epithelium to a
patterned tissue. Over time, broad regions of gene expression
become refined and result in three distinct domains: foregut,
midgut and hindgut. Gut endoderm patterning is, at least in
part, directed by environmental cues, for example from the
neighbouring mesoderm [69–72]. Evidence from two in
vitro studies of mouse ESC differentiation into endoderm
suggests that the gut endoderm is partly patterned by the
underlying basement membrane [73,74]. In particular, FGFinduced PI3K/Akt1 signalling was shown to modulate
regional extracellular matrix deposition along the A–P axis,
which in turn dictates positional cell-type specification [73].
These findings are substantiated by the observed transfating
of mesoderm to endoderm in zebrafish embryos upon
deletion of fibronectin [74].
Phil. Trans. R. Soc. B 369: 20130547
posterior
posterior
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8
b
at
cal
n
tio inter
a
l
tru rm
gas dode
(en
on
lati )
u
r
n
t
gas itiatio
n
(i
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t
cys
to
las
)
ion
)
on
tati rmed
s
e
o
g
f
d
e
mi t tub
(gu
time
Figure 4. Distribution of primitive endoderm (PrE) and cellular descendants until midgestation. PrE and derivatives (green) in mouse embryos from E4.5 to E8.75.
Note dispersed VE cells in the distal regions of E7.5 and E8.25 embryos, and presence of VE-derived cells in the gut tube of E8.75 embryos.
Sox17 visceral endoderm nuclei
Xenopus
zebrafish
mouse
cyc/sqt (Nodal)
Nodal
Wnt
FGF
maternal VegT (T-box)
Xnrs (Nodal)
Gata4/5/6
ps
Bix1/4 (Mix)
Eomes
Eomes
ps
Gata5/fau
Bon & Mezzo (Mix)
mixer (Mix)
Cas (Sox)
xSox17
epiblast/ectoderm
Foxa2
Sox17
definitive endoderm (DE)
mesoderm
visceral endoderm (VE)
Sox17 expression
Eomes
Gata4/5/6 Mixl Foxa2
Sox17
epi
epi
mes
en
mes
en
time
epi
endoderm
(VE)
epiblast (epi)
mesoderm (mes)
endoderm (en)
(VE + DE)
Figure 5. Conservation of a gene-regulatory network driving endoderm specification in vertebrates. A conserved set of molecular players directs endoderm formation
across vertebrate phyla. Smad-dependent Nodal signalling activates transcription mediated by conserved members of the Bix/Mix, Sox, Forkhead, Gata and T-box
families of transcription factors. Expression of Sox17 (red) and Afp – GFP (green) in the mouse embryo. Endodermal precursors initially migrate from the streak in the
mesenchymal layer but only begin to express Sox17 shortly before they egress into the endodermal epithelium. Below, a cartoon showing the acquisition of Sox17
expression by cells in the mesenchymal layer and their subsequent egression into the gut endoderm epithelium.
With the onset of gut tube formation, first a foregut pocket,
constituting a local involution of the gut endoderm, and then a
hindgut pocket emerge. Over time, these two pockets extend
and finally meet, forming the gut tube. The gut tube exhibits
extensive contribution of emVE-derived cells up until (at
approx. E8.75) the 15-somite stage [21] (figure 4).
8. A universal cell dispersal mechanism driving
endoderm morphogenesis?
The core components of the gene-regulatory network
involved in endoderm specification are largely conserved
across species [40]. The main transcription factors are orthologues of Foxa2, Sox17, T-box family members, Mix-like
proteins and Gata factors (figure 5). These factors are regulated by Nodal, Wnt and FGF signalling inputs [75]. But
are the cellular dynamics of endoderm morphogenesis conserved, and to what extent might the dispersal model
proposed in the mouse be applicable to other vertebrates?
The formulation of a model for gut endoderm morphogenesis in the chick has had a remarkably similar history to
the mouse. During gastrulation, the chick embryo needs to
reorganize its extraembryonic endodermal tissues (called
hypoblast and endoblast) around newly generated DE. A
series of fate-mapping experiments initially suggested an
equivalent to the murine displacement model, namely that
DE precursors ingressed at the primitive streak and inserted
into the extraembryonic endoderm tissues, gradually displacing them to the periphery of the embryo where they would
contribute primarily to extraembryonic tissues [76–80]. These
fate-mapping experiments were performed with techniques
such as carbon particle and vital dye staining that proved sufficient to look at mass movements, but did not provide
sufficient resolution to address the movements of all cells
and their individual dynamics.
A lineage-tracing experiment using [H3] thymidine
revealed that the hypoblast does not move away from the
primitive streak as a uniform epithelium [81]. Instead,
during gastrulation, the gut endoderm is a mosaic layer
Phil. Trans. R. Soc. B 369: 20130547
primitive endoderm and descendants
Downloaded from http://rstb.royalsocietypublishing.org/ on May 13, 2017
Table 2. Endoderm dynamics and fate across vertebrates.
9
Xenopus
chick
rabbit
mouse
extraembryonic and embryonic endoderm cells intermingle
yes [89]
yes [85,86]
yes [81]
?
yes [21]
comigration of mesoderm and DE progenitors
extraembryonic endoderm component to gut endoderm
no
no [37,88]
no
yes [85,86]
yes [82]
yes/? [83,84]
yes [87]
?
yes [21]
yes [21,90]
arguing for conservation of the mechanism across species
(table 2). Further studies will be necessary to determine the
cell dynamics of the murine dispersal model across vertebrates and, notably, whether endoderm tissues in the
adult human contain an extraembryonic component. In
worms and flies, different germ layers contribute to the gut
tube [91], making a comparison difficult with vertebrates.
9. Concluding remarks
The advent of high-resolution live imaging techniques, which
enable the visualization of entire populations of cells in realtime, is adding new insights to our understanding of the cellular dynamics that underlie critical morphogenetic events in
vertebrate development. Nowhere is this more striking than
the recent finding that so-called extraembryonic VE cells contribute to the gut endoderm. This finding opens up exciting
possibilities with several important questions that remain to
be answered. Do VE cells persist in the gut of the adult, or
are they lost as embryonic development proceeds? If they do
indeed persist, to which organs or tissues do VE descendants
contribute? Do they behave any differently than their DE
neighbours? What is their potential role in the adult? The
next generation of live cell imaging tools that will allow for
longer and more temporally regulated expression of lineage
labels will allow us to investigate the fate of emVE-derived
cells persisting within the gut endoderm.
Acknowledgements. We thank Laina Freyer and Sonja Nowotschin for
reading and commenting on this review.
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