RES EARCHREVIEW SUMMARY◥DEVELOPMENTAL BIOLOGY

The primitive streak and cellular principles ofbuilding an amniote body through gastrulation
Guojun Sheng*, Alfonso Martinez Arias*, Ann Sutherland*by embryonic divisions that occur shortly after fertilization. These cells are transformed
into the recognizable outline of an organism
through the process of gastrulation, which
endows them with lineage and spatial identities in the context of an emerging coordinate
system. In many amniote embryos (such as
those of reptiles, birds, and mammals), gastrulation has been associated with a transient
structure called the primitive streak. Humandevelopment also follows this pattern. In humans, the primitive streak forms ~14 days after
fertilization. The appearance of the primitive
streak breaks the radial symmetry of the epiblast (a sheet of epithelialized pluripotent cells)
and has been suggested to symbolize the emergence of human individuality. As such, many
countries have established a legal limit of
14 days for the in vitro culture of fertilized human eggs—this is known as the “14-day rule.”
In recent years, pluripotent stem cells have become a promising in vitro model for studying
the cellular and molecular mechanisms associated with early human development. Interpretation of developmental features observedin these in vitro models requires proper understanding of animal gastrulation in general andof the amniote primitive streak in particular.

ADVANCES: In this Review, we offer a phylogenetic and ontogenetic overview of the primitive streak and its role in mediating amniote
gastrulation, and we discuss the implicationsof embryonic stem cell–based models of earlymammalian embryogenesis on the function ofthis iconic structure. We provide evidence thatthe primitive streak is not a conserved featurein amniote development and that the mammalian and avian primitive streaks have evolved
independently through different supracellularmechanisms that led to their morphologicalemergence. We argue that, in addition to mediating the emergence of germ layers from thepluripotent epiblast, gastrulation is principallya process in which an embryo acquires a coordinate system to organize its primary cellfates and the primordia of organs and tissues
relative to each other in space. We highlightthat in amniotes this process is regulated bya set of conserved signaling and transcriptional networks through a small collection ofcellular behaviors, the tissue-level effects ofwhich are governed by boundary conditions.
We suggest that changing boundary conditions,in the form of evolution of extraembryoniclineages such as the trophectoderm and primitive endoderm, have played a key role in thetransformation of the blastopore, characteris-

Epiblast

A
Fertilization

Implantation

A: Anterior
P: Posterior
D: Dorsal
V: Ventral

Epiblast

D
V
P
D

P

A
V
Gastrulation

Spatial coordination of cell fates

Schematic view of how a human embryo acquires spatial coordination of its primary cell fates
through gastrulation. After fertilization, a human embryo implants into the uterus (between days 6 and 12)
and gastrulation begins soon afterward, with the primitive streak emerging at day 14. Gastrulation allocates
cell fates and spatial coordinates to epiblast cells, laying down the foundation of a human body.
Sheng et al., Science 374, 1213 (2021)

3 December 2021

OUTLOOK: Our model predicts that the mostfundamental feature of a primitive streak–like structure in early amniote development
is not its morphological manifestation but
rather its capacity to mediate coordinated cellfate specification events in space. Our model
also suggests that cell fate specification andtissue-level morphogenesis are regulated independently during gastrulation and thencoordinated during embryonic development
in vivo. In developmental models in vitro, thesetwo processes can be uncoupled and have beenshown to be influenced by different types ofbiomechanical parameters that mediate theircoordination. This modularity leads us to suggest that the in vitro models are useful forstudying gastrulation because their use without necessarily having to recapitulate embryonicstructures. Future analyses of early amniote
development, both in vivo and in vitro, would
benefit from putting less emphasis on theprimitive streak as a distinct embryologicalstructure and more on its roles as a conduit forsymmetry breaking and coordinated germlayer differentiation.
Research into human development has direct societal and ethical impacts. Current ethical oversight in human embryo research, the14-day rule, is effective in many countries andreflects an interdisciplinary consensus drawn
somewhat arbitrarily to determine the legal
rights of a human embryo. Our observations
suggest that use of the primitive streak as a key
developmental landmark for limiting ex vivo
culture of human embryos should be reassessed.
An alternative landmark, necessary for exerting
ethical oversight in human-related developmental and stem cell biology research, should
be selected through a consensual discussion
between different stakeholders to ensure scientific and ethical rigor.

▪

The list of author affiliations is available in the full article online.
*Corresponding author. Email: sheng@kumamoto-u.ac.jp (G.S.);
alfonso.martineza@upf.edu (A.M.A.); as9n@virginia.edu (A.S.)
Cite this article as G. Sheng et al., Science 374, eabg1727(2021). DOI: 10.1126/science.abg1727

READ THE FULL ARTICLE AT
https://doi.org/10.1126/science.abg1727
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CREDIT: (“FETUS IN THE WOMB” SKETCH) LEONARDO DA VINCI/ROYAL COLLECTION TRUST/© HER MAJESTY QUEEN ELIZABETH II 2021

BACKGROUND: Pluripotent cells are generated

tic of anamniote embryos, into the primitive
streak. Variability in the organization of thesetissues and the demarcation of embryonic andextraembryonic territories underpins the observed variation in the morphological appearance of the primitive streak in mammals andbirds and of primitive streak–related structuresin reptiles. Over the past few years, embryonicstem cells have been used as models to recapitulate several aspects of early mammalianembryogenesis. These studies have revealed
that the germ layers, and even a rudimentary body plan, can form in the absence of aprimitive streak.

RES EARCHREVIEW◥

DEVELOPMENTAL BIOLOGY

The primitive streak and cellular principles ofbuilding an amniote body through gastrulation
Guojun Sheng1*, Alfonso Martinez Arias2*, Ann Sutherland3*
The primitive streak, a transient embryonic structure, marks bilateral symmetry in mammalian andavian embryos and helps confer anterior-posterior and dorsal-ventral spatial information to early
differentiating cells during gastrulation. Its recapitulation in vitro may facilitate derivation of tissues
and organs with in vivo–like complexity. Proper understanding of the primitive streak and what it entails
in human development is key to achieving such research objectives. Here we provide an overview
of the primitive streak and conclude that this structure is neither conserved nor necessary for
gastrulation or early lineage diversification. We offer a model in which the primitive streak is viewed
as part of a morphologically diverse yet molecularly conserved process of spatial coordinate acquisition.
We predict that recapitulation of the primitive streak is dispensable for development in vitro.

1

International Research Center for Medical Sciences,
Kumamoto University, Kumamoto, Japan. 2Systems
Bioengineering, DCEXS, Universidad Pompeu Fabra, Doctor
Aiguader, 88 ICREA, Pag Lluis Companys 23, Barcelona,
Spain. 3Department of Cell Biology, University of Virginia
Health System, Charlottesville, VA, USA.

*Corresponding author. Email: sheng@kumamoto-u.ac.jp (G.S.);
alfonso.martineza@upf.edu (A.M.A.); as9n@virginia.edu (A.S.)

Sheng et al., Science 374, eabg1727 (2021)

an endodermal or mesodermal fate. Bioethicists
and government regulatory bodies have associated the primitive streak with human individuality and, as such, have established a legal
limit of 14 days—approximately when the primitive streak appears in a human embryo—for
the in vitro culture of fertilized human eggs
(known as the “14-day rule”) (4, 5). Until recently (May 2021), the International Society
for Stem Cell Research (ISSCR) (www.isscr.org)
has stated its commitment to this 14-day rule,with updated guidelines recommending caseby-case review by national and institutional
review boards (6). Placing such an emphasison the primitive streak for the emergence ofa human being raises questions about its rolein the establishment of the human body plan
and whether it represents a stereotyped andgeneralizable structure. To address these questions, the definition (or lack thereof) of gastrulation must be revisited.
William Ballard, an American embryologist who coined the word “pharyngula” as aphylotypic stage for all vertebrate animals,
stated in a discussion on gastrulation that “realproblems that require new observation and experimental proof are being glossed over” andthat “there has been no progress at all sincethe 1930’s in defining what gastrulation is orwhen it begins or ends” (7). Ballard also quoted
Jean Pasteels (8) “in that the vertebrate gastrula
does not have a definitive form. It is just an
abstract collective term for individuals undergoing the movements of gastrulation.” We
believe that these statements reflect a situation that has persisted to this day. Genetic
studies have identified a conserved network ofsignaling molecules and transcription factors
associated with gastrulation and have revealed
many common features of cell biology in different species that bridge the gap between
unicellular and supracellular behaviors (9, 10).

3 December 2021

An organismal view of gastrulation

Gastrulation (literally “formation of a smallgut”) and its embryological connotation wereintroduced by Ernst Haeckel in the context of
his gastraea theory (1), as part of his biogenetic
law that lent embryological support to theDarwinian notion of common descent of allliving organisms. A gastraea is a hypotheticalancestral organism characteristic of the gastrula stage that all metazoans presumably go
through ontogenetically, and gastrulation was
the process encompassing this developmental
stage as a universal “rite of passage” in Haeckel’s
recapitulation theory. A key piece of evidence
for Haeckel’s theory was the presence of an
endoderm-like, lineage-restricted cell layer
during embryogenesis of sponges (Porifera; aphylum of diploblastic metazoans; Fig. 1A)—
hence the names “gastraea” and “gastrulation”[from “gaster” (which means “stomach”), an
endoderm derivative]. Recently, this description has been disputed in light of data frommorphological and lineage-tracing analyses(13, 14), suggesting that sponges do not undergo
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G

astrulation is a stage of animal development that transforms the mass of cells
from early postfertilization cell divisions
into the recognizable outline of an organism. The term “gastrulation” was coinedby the German embryologist Ernst Haeckel
when discussing the development of sponges(1, 2). Later, combined with the germ-layer
theory, it acquired its common meaning as aphylogenetically conserved stage during which
the primary germ layers are defined and organized. Scrutinized at the cellular level, gastrulation was revealed to be associated withspecies-specific cell movements that, for vertebrates, lead to a conserved pharyngula stage
when embryos from different phylogenetic
groups resemble each other the most and whenorganogenesis begins. Over the years, the term
gastrulation has come to refer to a set of morphogenetic movements that serve to organize
the three germ layers in animal development.
Gastrulation in amniotic vertebrates (reptiles,
birds, and mammals; see Fig. 1A for phylogenetic relationships among major animal groups)
is thought to involve the primitive streak, a transient structure that forms along the midline of
the epiblast (the pregastrulation ectoderm),
first observed in a chick embryo by Russian
embryologist Christian Pander in 1817 (3). The
emergence of the primitive streak breaks anearly morphologically radial symmetry, outlines the anterior-posterior axis of the embryo,and serves as a channel for the continuous passage of mesenchymal cells toward the interiorof the embryo, where they are further assigned

Furthermore, pluripotent stem cells (PSCs) represent a model system for the study of earlymammalian development (11, 12), and the interpretation of such experiments raises questions about the nature of gastrulation and,more importantly, whether the behavior of
PSCs in vitro can identify fundamental mechanisms underlying the events happening
in vivo. Progress in human PSC research has
prompted discussions of whether PSC-derived
structures should be considered within thesame ethical realm as embryos, and the conventional wisdom is converging toward drawing a similar line on the basis of whether thesestructures assemble a primitive streak andundergo gastrulation. All of these issues suggestthat it is time to reassess gastrulation in lightof data from more recent experimental techniques and models, from which general rules
may emerge, with potential ethical implications.
Here we focus on the primitive streak inamniotic vertebrates and argue that gastrulation is not only a stage but, principally, a
process whereby an embryo acquires a coordinate system to organize primary cell fates inspace. We highlight that this process is regulated by a set of conserved signaling and transcriptional networks through a small set of cell
behaviors, the tissue-level effects of which aregoverned by boundary conditions. This discussion leads us to suggest that global control
by boundary conditions underpins the variability of the gastrulation process in different
amniotic clades and to propose a relationshipbetween gastrulation modes observed in amniotic and anamniotic (nonamniotic) vertebrates. Later, the process of organogenesis canalso lead to similar conclusions, as supported
by data from PSC models.

RES EARCH | R E V I E W

gastrulation as commonly seen in triploblastic
animals. Nevertheless, comparative genomics
and molecular phylogeny studies have shownthat most of the genes involved in epithelialorganization are present in sponge genomes(15, 16), as are some of the genes involved in germlayer formation in Bilateria (the protostomes
and deuterostomes, including all vertebrate speSheng et al., Science 374, eabg1727 (2021)

symmetry). (Middle) Triploblastic animals have three germ layers and two majoraxes [the anterior-posterior (A-P) and dorsal-ventral axes]. (Right) Simplified
view of cellular functions of each germ layer. This overall functional assignment
is conserved in all triploblastic animals. In diploblastic animals, some cellularfunctions that resemble those of the mesoderm are performed by either
ectoderm or endoderm cells. (C) Basic 3D organization of a postgastrulation
amniote embryo (left), with relationship of three germ layers and spatial
coordinates in the anterior-posterior and medial-lateral axes shown in transversesection view (middle). The medial-lateral axis in early development is transformed
into the dorsal (D)–ventral (V) axis in the adult (right). (D) Schematic viewof the relationship between the embryo and extraembryonic tissues in amniotes.
am, amnion; ch, chorion; ys, yolk sac; al, allantois. Dark gray, ectoderm; light gray,endoderm; gold, mesoderm.

cies) (14) and in the epithelial-to-mesenchymaltransition (EMT) (17). In Cnidaria, a phylum of
diploblastic animals most closely related to triploblastic Bilateria (Fig. 1, A and B), molecular
tool kits for epithelial organization and EMT
are present in genomes, and gastrulation generates mesendoderm-like cells that express
mesoderm transcriptional regulators and3 December 2021

mesoderm-specific lineage markers, despite theirlack of a genuine mesoderm germ layer (18).
These seemingly contradictory observations highlighted the need for a conceptual
framework for understanding the relationshipbetween the primitive streak and amniote gastrulation from the perspective of metazoan
phylogeny. The gastraea theory posited that
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Fig. 1. Animal phylogeny and amniote body plan. (A) Phylogenetic relationships among major animal groups. Gastrulation is traditionally viewed as aconserved process of achieving cell lineage diversification in metazoans(animals). The relationship between gastrulation in sponges and that in therest of the metazoans awaits further clarification (dashed arrow). Diploblastic
indicates two germ layers (ectoderm and endoderm); triploblastic indicates
three germ layers (ectoderm, mesoderm, and endoderm). Bilaterians are
animals with bilateral body symmetry. The presence of a primitive streak–likestructure during gastrulation is not conserved among amniotes (land-developing
vertebrates, including extant mammals, birds, and reptiles). The term
anamniotes collectively denotes vertebrate groups other than the amniotes.
w/o, without. (B) Germ layers and their biological functions. (Left) Diploblastic
animals have two germ layers and one major axis (the central axis of radial

RES EARCH | R E V I E W

functional diversification is fundamental tomulticellularity and that specification of cells
dedicated to nutritional acquisition (i.e., theendoderm) is a conserved process among all
metazoans (Fig. 1B). With this came notionsof conservation of gastrulation at the genetic
and morphogenetic levels, revealed in modern
biological terms as molecular regulatory networks and dynamic changes in supracellularorganization, respectively. Gastrulation, therefore, can be practically defined by traits that
describe cell fates (e.g., neurons, notochordcells, and enterocytes) and shapes (e.g., epithelium, mesenchyme, migration, and EMT), as
well as by their associated molecular characteristics (e.g., Brachyury for mesoderm andendoderm progenitors and Nanog for undifferentiated pluripotent cells). Depending on
the choice of experimental models and investigative tools, the particular aspect of gastru-

lation to emphasize has varied since Haeckel’stime. The core, unchanged, and unifying concept of gastrulation, in our view, is for it toserve as a conduit for diversification of cellularlineages and acquisition of a spatial coordinate system for subsequent functional integration in later development.
Central to our current understanding ofgastrulation is the primitive streak, which is
associated with a spatially oriented, dynamic
sequence of individual cells leaving the surface layer in the process of ingression, throughthe EMT. The primitive streak has been viewed
as iconic for gastrulation, although it is present only in specific amniotic vertebrates, suchas birds and mammals (19) (Figs. 1 and 2). In
reptiles (nonavian reptiles, in the context of
this article), gastrulation is associated withinvolution (i.e., rolling of a sheet of cells overa horseshoe-shaped blastopore, an orificecharacteristic of amphibian gastrulation) accompanied by ingression in the blastoporal(primitive) plate located posteriorly to theblastopore (19–22) (Fig. 3). Thus, all functional
cell types shared by amniotic vertebrates canbe generated regardless of the presence orabsence of a primitive streak, leading to thequestion of whether gastrulation-related morphological features are conserved among allamniotes. Furthermore, relevant to the effort
to recapitulate all, or part of, mammalian development in vitro is the question of whether
a conserved morphogenetic process similar
to the appearance of a primitive streak or ablastopore is required for the functional diversification of cell lineages.
A closer look at amniote embryos undergoing gastrulation, compared with embryosof anamniotes (nonamniote vertebrates), identifies three shared morphological features

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Fig. 2. Phylogenetic comparison of spatial organization in amphibian (frog),avian (chicken), and mammalian (human) embryos before gastrulationand hypothetical transitions in gastrulation morphogenesis during early
amniote evolution. Future anterior-posterior and dorsal-ventral axes are indicatedin pregastrulation embryos. (A) In the frog (Xenopus) embryo, the animal pole iswhere the oocyte nucleus is located before fertilization (it is also lighter because it
has less yolk than the vegetal pole). (B) In the chicken embryo, the cleaved pole
is where the oocyte nucleus is located and where cellularization of the fertilized egg
takes place. The uncleaved pole is a part of the oocyte that does not contributeto the cellularized embryo and contains mostly nutritious materials. (C) In thehuman embryo, the embryonic pole is the side where the inner cell mass is located inSheng et al., Science 374, eabg1727 (2021)

3 December 2021

a blastocyst. The abembryonic pole is the opposite side. The trophectoderm andprimitive endoderm are drawn but not labeled. Epiblast cells form the proamniotic
cavity, with upper cells giving rise to the amniotic ectoderm and lower cells(area represented by the dashed rectangle) giving rise to the major part of all
three germ layers. In the lower panels, dark gray indicates cells that will contribute
mainly to the ectoderm lineages; light gray denotes the marginal zone, which
will control the location of internalization, contribute to mesendoderm cells, andmediate boundary biomechanical cross-talk—which, in amniotes, will influence themorphogenesis of primitive streak–like structures. (D) Hypothetical evolutionarytransitions in gastrulation morphogenesis, leading to an amniote-specific modeof mesendoderm internalization.
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among all amniote clades: (i) Internalizationof mesoderm- and endoderm-fated cells takes
place in a circumferentially restricted manner (compare an anamniote embryo in Fig. 2A
with amniote embryos in Fig. 2, B and C). (ii)Pregastrulation ectoderm (epiblast) cells areorganized as a single-cell–layered epithelium.
(iii) The epiblast is divided into embryonic andextraembryonic territories, and gastrulation is
initiated at their boundary (Fig. 2, B to D). To
integrate these three features of amniote gastrulation in a simple model, the primitive streakin birds and therian mammals can be viewed
as an independent morphogenetic adaptationof a basal, reptile-like bimodal form of involution and ingression (Figs. 1 to 3) (19, 20). The
presence of a neurenteric canal (i.e., an opening of the epiblast that connects the amniotic
cavity and the yolk sac) in human and otherprimate embryos (23) and the rudimentary
chordal canal associated with head process
formation in the mouse embryo (24) can beconsidered to represent a residue of blastoSheng et al., Science 374, eabg1727 (2021)

proliferation of the epiblast before onset of gastrulation and by boundary
conditions reflected as embryo-specific biomechanical constraints on epiblast
cells (C). In reptilian (e.g., turtle) embryos, such morphogenesis results in theformation of a blastopore as the active center of internalization, whereas in chickand mouse embryos, a primitive streak–like structure forms, with different
morphogenetic origins. (D) Internalization of mesendoderm cells (small whitearrows; the dotted parts indicate cell movement after internalization, contributing
to the 3D organization of a postgastrulation embryo). M, medial; L, lateral.

poral involution that continues to play a rolein axial mesendoderm internalization. Thesefeatures and the proposed unifying model,
however, highlight only morphogenetic constraints placed on amniote gastrulation, whichby themselves may or may not be essential
for fulfilling functional roles of gastrulation
as a conduit for cell lineage diversification,
spatial coordinate establishment, and inter–or intra–germ-layer coordination during tissue and organ formation.
The first two features conserved among amniote vertebrates are also observed in certain
groups of anamniotes. For example, unlike
Xenopus, the main amphibian model, all salamanders (Urodela) (25–29) and caecilians(Gymnophiona) (30, 31) studied so far exhibita gastrulation process that is restricted to or
extremely biased toward the dorsal marginalzone. Similarly, such dorsally restricted gastrulation is also present in lungfish (the closest
relatives of Tetrapoda) (32, 33), dogfish (cartilaginous fish) (34), and lampreys (jawless

3 December 2021

vertebrates) (35), which suggests that the amniotes likely inherited this feature of gastrulation from an anamniote ancestor (see Fig. 1A
for phylogenetic relationship). The second feature, a unilaminar, epithelialized pregastrulation
ectoderm, is also present in certain anamniotegroups (e.g., urodeles) (36), which suggests a
preamniote origin. The third feature, that gastrulation is initiated at the boundary between
intraembryonic and extraembryonic territories,
is associated with the evolutionary invention of
the amnion and chorion in ancestral amniotes(Fig. 1D), where internal fertilization, intrauterine early embryogenesis, and land-based
fetal development necessitate the presence of
these protective layers. The amnion, composedof ectoderm- and mesoderm-derived cells, provides a protective liquid-filled environment forthe developing embryo and is the defining
feature of amniotic vertebrates. The chorion,
similarly composed of ectoderm and mesoderm cells, forms the external boundary of
the embryo (including all intraembryonic and
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Fig. 3. The primitive streak as a morphogenetic consequence of variableboundary conditions in the pregastrulation epiblast of an amniote
embryo. The primitive streak is neither conserved nor necessary for amniote
gastrulation. Ontogenetically, in the pregastrulation embryo, radial symmetry(A) (similar to the top view in Fig. 2) is transformed into bilateral symmetry whenmesoderm and endoderm precursors are induced asymmetrically in the marginalzone (B). Geometric organization of active internalization of mesendoderm
cells is influenced by planar morphogenesis (white arrows) and cellular

RES EARCH | R E V I E WSheng et al., Science 374, eabg1727 (2021)

of transcription factors and signaling molecules associated with gastrulation in both theamniotes and anamniotes. For example, interactions between bone morphogenetic protein (BMP), Nodal, and Wnt signaling lead to
Brachyury expression at the onset of gastrulation and serve as a gateway for the specification of different organ and tissue primordia.
However, this conservation at the molecularlevel contrasts with the morphogenetic-levelvariability (39) and leads us to search for common motifs at the level of cell behaviors as away to understand the origin of the variety ofgastrulation modes.
A cellular view of gastrulation

Vertebrate gastrulation is generally associatedwith the transformation of a cellular aggregate
into a bilaterally elongated structure with spatial information conferred in all three germlayers (Figs. 1 to 3). This is accomplished in a
species-specific manner. In amniotes, the initial step in this process is a mesenchymal-toepithelial transition (MET) of the early cells
to form the pluripotent epiblast epithelium(40), followed by ingression and concomitantspecification of the endoderm and mesoderm,with an orderly process of EMT placing thethree germ layers with respect to the anteriorposterior body axis (Fig. 1C). At the cellularlevel, the behaviors associated with EMT arequite well conserved between the mammalian and avian primitive streaks (41–45). The
underlying basement membrane is degraded,and epiblast cells apically constrict to become
flask-shaped and then detach from neighboring cells to exit the epithelium (46–48). At themolecular level, this involves down-regulationof E-cadherin expression (49) as well as decreased Rho activity and changes in organization of cytoskeletal components [particularly
microtubules, (41, 50) in the ingressing cell]without disturbing the overall epithelial natureof the tissue (41, 43). These changes in cell
behavior are associated with similar patternsof signaling and gene expression as well—including increases in Wnt, Nodal, and fibroblast growth factor (FGF) and FGF receptor 1(FGFR1) signaling—that lead to up-regulated
expression of Brachyury, Snai1 and Snai2,
and FoxA2 transcription factors.
Similarly, at the cellular level, behaviors
underlying formation and elongation of theprimitive streak are fairly well conserved, including cell shape changes and polarized rearrangement of cells along the mediolateralaxis. However, how these cellular level behaviors are deployed and choreographed to generate tissue shape changes are distinctly species
specific, likely due to biomechanical constraints
imposed by the particular embryonic environment. In birds, the mesendoderm precursors
that will form the primitive streak are induced
in a sickle-shaped region at the posterior margin

3 December 2021of the area pellucida (51) and undergo extensive, polarized rearrangements that result
in convergence on the midline and anterior
extension (52–54). Epiblast cells in the area
anterior to the forming primitive streak rearrange to extend perpendicular to the primitive
streak, leading to anterior-posterior contraction and lateral elongation of this region andlarge-scale counterrotational flows of cells inregions of the epiblast lateral to the forming
streak (52).
Studies of gastrulation in mammalian embryos reveal distinct tissue-level differencesin primitive streak initiation and elongationfrom the model established in the avian embryo. The mouse embryo, with an elongatedcup-shaped epiblast, has no morphologicalstructure equivalent to the sickle-shaped mesendodermal precursor region of the chick, andthe mouse primitive streak forms through progressive initiation of EMT rather than convergent extension of a precursor population (43).
In discoid embryos (such as those of rabbits
or pigs), in which the size and geometry of
the epiblast are more similar to those in chickembryos, the posterior margin expands posteriorly and becomes less dense cellularly
before primitive streak formation, in contrast
to the convergent extension of mesendoderm
precursors in the avian embryo (55, 56). In addition, the pronounced counterrotational flows
characteristic of the avian embryo are not seenin the rabbit embryo (55, 57). Our knowledge ofthe primitive streak in human embryos, primarily on the basis of anatomical descriptions
and comparisons with other primate embryos,
reflects similarity to other mammalian embryos, although additional features (such as
the neurenteric canal) have been discovered(58, 59). The differences between avian andmammalian primitive streak formation reflectdistinct patterns of biomechanical force generation, perhaps as a consequence of distinct
biomechanical architecture at the border of
intraembryonic and extraembryonic territories
that influences tissue-level behaviors in theseembryos. In addition to centrifugal tension
exerted by expansion of the area opaca (theregion of the epiblast that is physically attached to the yolk) in avian embryos (60, 61),
polarization and mediolateral intercalationof posterior marginal cells (52) initiate gradedtangential forces along the margin of the epiblast that lead to cell shape changes in themarginal cells and formation of supracellular
myosin cables in groups of 5 to 20 cells (53).
The forces associated with this tensile ring are
anisotropic, providing active tension posteriorly
and passive tension anteriorly, and modellingshows that they are sufficient to drive the posterior epiblast cells forward (53). Anteriorly, themarginal tension may provide a boundary that
directs the lateral movement of cells anterior
to the streak to generate the counterrotational
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extraembryonic cell lineages) and is the primary interface for fetal-environmental (including fetal-maternal) exchanges.
Except for birds, in which gastrulation andprimitive streak formation takes place after
oviposition (egg laying), all extant amniote
embryos initiate gastrulation during theirintrauterine development. Variations in amniote early development are manifested as
differences in oocyte size (e.g., 30 mm in thechick versus 0.1 mm in the human), cleavage
pattern (e.g., meroblastic cleavage with delayed
cytokinesis and incomplete cytoplasm partitioning in the chick versus holoblastic cleavage
with complete partitioning of zygotic cytoplasm
in the human), cell cycle duration (e.g., a rapidto-slow shift in birds versus a slow-to-rapid
shift in mammals), and cell number at theonset of gastrulation (e.g., 100-fold difference
between the chick epiblast and the mouse
epiblast). These variables lead to diverse
epiblast “landscapes” during the transition
from pluripotency to a rudimentary embryonic architecture with three germ layers. Furthermore, these preconditions are met with
the need to demarcate the pregastrulation
epiblast into the intraembryonic and extraembryonic territories to facilitate amniogenesis, and as a consequence, internalization of
mesoderm and endoderm cells is initiated at
the intraembryonic-extraembryonic boundary.
In embryos of eutherian animals (placental
mammals, including mice and humans), thegastrulation process comes under the additional influences of implantation and placentation, both of which exhibit pronounced
diversity and evolutionary adaptability. For
example, a horse embryo initiates trophoblastendometrium contact 1 month after fertilization, when the embryo has already reachedorganogenesis stages and placentation thereafter remains epitheliochorial (i.e., with superficial, noninvasive feto-maternal contacts). By
contrast, a human embryo starts to breach thematernal endometrium soon after blastocyst
hatching and completes the invasive implantation process by day 12 after fertilization, wellbefore the initiation of gastrulation or primitive
streak formation (37, 38). Functional differentiation of the trophectoderm lineage becomesa crucial early event for eutherian mammals,
and the onset and morphogenetic process ofgastrulation are affected to a greater or lesser
extent, reflecting physical and structural variations in eutherian feto-maternal interactions.
These variable and adaptable features ofamniote early development at the organismallevel challenge the notion that gastrulation isassociated with a specific structure and underscore the importance of looking for components of the process that are more conserved
across amniotes, potentially at the molecularand cellular levels. Genetic studies over the past
20 years have indeed revealed a conserved set

RES EARCH | R E V I E W

mations that generate a linear region of cell
ingression. Are these morphogenic constraints
a necessity for amniote gastrulation or merely
a problem to be solved? The variations in themode of gastrulation among amniote and anamniote vertebrates [as discussed above andin (65)] suggest the latter. In fact, recent data
show that manipulation of the extent andcellular behaviors of the mesendoderm in thechick embryo can transform the primitive
streak into gastrulation modes more similar
to those of amphibian or reptile embryos(62, 66). Data from the field of mammalianstem cell and regenerative biology appear tofurther support this line of argument.
in vitro models of mammalian gastrulation

Over the past few years, PSCs have been used
to generate a number of models of early mammalian development. PSCs are clonal derivatives from mammalian blastocysts [embryonicstem cells (ESCs)] or reprogrammed adult cells(induced PSCs). PSCs can be maintained inculture for many generations without losing
their ability to produce all cells of an organism,and they can be steered to differentiate into
any cell type by controlling the culture conditions. This differentiation is asynchronous,
exhibits large heterogeneities in gene expression (67, 68), and reveals the existence ofcell-intrinsic programs of gene expression associated with specific fates. In all cases, cells
go through patterns of gene expression thatmirror events observed in embryos and, earlyon, cell fate decisions in the early epiblast.
Thus, during differentiation into endodermal
and mesodermal derivatives in culture, cells
go through a sequence of events similar to
those of gastrulation—down-regulation ofFig. 4. Biomechanical properties at the embryonic-extraembryonic boundary
can influence epiblast morphogenesis before gastrulation. (A) An amniote
embryo after symmetry breaking but before gastrulation. Epiblast morphogenesis and, consequently, the morphology of a primitive streak–like structure
are influenced by embryonic-extraembryonic boundary conditions. (B) The
avian embryo has strong biomechanical anisotropy at the boundary andundergoes prominent planar rearrangement of epiblast cells, leading toprimitive streak formation. (C) The reptilian embryo has weak biomechanicalanisotropy at the boundary and undergoes limited, regional rearrangement,
Sheng et al., Science 374, eabg1727 (2021)

3 December 2021

pluripotency genes, MET, activation of Wnt
and Nodal signaling, transient expression ofBrachyury, and engagement into an EMT (69)—
before expressing specific fate determinants.
However, these changes occur without any
multicellular coordination or morphogenesis.
Despite the sequential events of MET, expression of Brachyury, and EMT, no primitive
streak–like structure is visible in the culture.
In an attempt to restrain the heterogeneities that arise in adherent culture, humanand mouse PSCs have been cultured on twodimensional (2D) micropatterned structures(70). Under these conditions, cells form tightepithelia, and exposure to BMP, Nodal, and
Wnt signals results in the emergence of proportionate concentric rings of gene expression
identified as the different germ layers andsome of their derivatives during gastrulation(70–72). In these arrangements, also known
as 2D gastruloids, radial symmetry can be
broken only by spatially controlled asymmetric flow of signal agonists and antagonists (73). These experiments have provided
insights into the mechanisms by which cells
interpret and respond to morphogens in perigastrulation stages. However, likely becauseof the confinement of cells on the substrate,
cellular growth and movement are impaired
and patterns of gene expression do not exhibit
the structural evolution that they do in theembryo. In the case of mouse PSCs, this experimental setting has been shown to recapitulate relationships between signals and fatesin the embryo that are known from genetic
studies, thus validating the patterns observedfor human ESCs (72). On the basis of movements
correlated with cells expressing Brachyury,
it has been suggested that there is a circularleading to blastopore formation. (D) The mammalian embryo has no or weakbiomechanical anisotropy at the boundary and undergoes no (in mice) or
limited (in rabbits) epiblast planar rearrangement. A primitive streak–likestructure still forms in the mammalian embryo, likely due to directional
EMT signal propagation and cell proliferation. In (B) to (D), the dashed line
denotes the midline, and the white arrows indicate the direction of global
movements of embryonic epiblast cells. In (B) and (C), the red arrows indicate
the direction and strength of the intercellular tension force between epiblast
cells located at the embryonic-extraembryonic boundary.
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flows of cells toward the posterior of the embryo (Fig. 4B). Furthermore, the marginal tensile
ring may well act to regulate gene expression
and to define the region of primitive streak
formation (62).
It is tempting to speculate that such a tensile ring could similarly provide the boundary
conditions to generate tissue-wide cell movement in mammalian embryos with discoid
epiblasts, including human embryos. However, the posterior expansion and decreasein cellular density observed in some of theseembryos (56, 63), as well as the lack of strong
posterior convergence movements, seem inconsistent with initiation of a tensile ring at theepiblast margin (Fig. 4D). Additionally, theeffects on primitive streak formation of inhibiting myosin activation through Rho kinase inhibition differ substantially between rabbit andavian embryos (52, 57), suggesting that the roleof actomyosin contractility during primitive
streak formation and gastrulation is not thesame in these two embryo types (Fig. 4). Forces
tangential to the margin of the epiblast are very
unlikely to play a role in mouse embryo gastrulation, given the cuplike geometry of the epiblast. In fact, it may be that the absence of suchtangential forces, as well as a less fluidlike epiblast, act as constraints for the behavior of
epiblast cells in mouse embryos, leading to formation of the primitive streak without global
cell movements. Additionally, although centrifugal forces on the epiblast may be generated
through expansion of the fluid-filled proamniotic cavity (64), there is no tension generated by
the extraembryonic tissues across the epiblast.
Thus, coordinated cell behaviors, acting
under the particular constraints of the embryonic environment, produce tissue-level defor-

RES EARCH | R E V I E WSheng et al., Science 374, eabg1727 (2021)

multicellular ensembles can pattern gene expression in space and, when allowed to grow,create patterns of structured gene expressionthat mimic the situation in embryos. Gastruloids also highlight the importance of boundary conditions in early morphogenetic events.
In this regard, a recent variation of the gastruloids demonstrated that spatial localizationof a Wnt/Nodal signaling center to one end ofan aggregate of naïve cells increases the spatial ordering of the fates emerging from thegastruloid (87). Mouse and human epiblasts
are organized very differently, owing to thearrangement of the extraembryonic tissues,
but when these constraints are removed byplacing the PCS or epiblast derivatives in culture, both form very similar structures (85).
This suggests that the primitive streak can be
hypothesized as a mechanical response to theboundary conditions surrounding the embryo(Fig. 4). In support of this, when ESCs areplaced in culture with trophoblast stem cells(TSCs), they form structures that resemble theearly epiblast, in which a primitive streak–likestructure can be observed to emerge at theinterface between ESCs and TSCs (88, 89). Thisis even more accentuated when the primitive
endoderm is induced in these constructions(90). The initiation of gastrulation can also be
observed in a human model of amniotic sac
formation (91), supporting the notion that theprimitive streak might be a consequence of mechanical constraints imposed on the epiblast.
Origin and function of the primitive streak:A look beyond

The PSC models of mammalian development
provide some insights into the role that gastrulation and its associated structures playin laying down the amniote body plan. In particular, they call into question whether theprimitive streak is the hallmark of gastrulation.
First, these models reveal that fate-specificschedules of gene expression and morphogenesis, including the primitive streak, are two
independent processes that are coordinatedduring embryonic development in vivo. Gastruloids, which lack a primitive streak, still
exhibit polarized gene expression and are able
to lay down a body plan, but their constituent cells are not spatially constrained at theirboundaries, as seen in embryos (Fig. 4). By contrast, when the initial aggregates are 2D andepithelial, they require higher levels of Wnt
signaling for patterning, facilitated by BMP
from the trophectoderm (88), and exhibit EMTlike movements associated with Brachyury
expression at the interface between the PSCs
and TSCs, similar to what occurs in the embryo.
This suggests that aggregates of PSCs have an
intrinsic ability to break symmetry associatedwith polarized Brachyury expression, but thesignaling threshold for these events is raised
when the cells are epithelial, and interactions

3 December 2021

with the trophectoderm bias the onset of symmetry breaking and trigger the localized emergence of the primitive streak. We surmise thathaving an epithelial substrate for the initiationof the primitive streak in embryos (40, 92) allows for controlled, precise patterning, which isabsent in gastruloids. Although PSC models(gastruloids in particular) exhibit a well-organizedoutline of the primordia for tissues and organs,this outline lacks the precision and detailed
patterning characteristic of embryos, which arenecessary for the generation of a functionalorganism. Therefore, the primitive streak may
represent a structural feature that allows alignment of the primordia and serves as a morphogenetic conduit for precision in spatial andtemporal coordination of early development.
We would like to propose that the avian
primitive streak, which has served as the reference structure for amniote gastrulation, is
not a conserved structure but rather part of acontinuum that spans species-specific structures
seen in different amniote clades. It has arisen
independently a number of times, as the common ancestor of birds and mammals likely
lacked a primitive streak (Fig. 1). One may
also view primitive streak–like structures—
including avian primitive streaks, reptilian
blastopores, and various types of mammalianprimitive streaks—in a broader sense, as amanifestation of the midline that indicatesthe emergence of anterior-posterior polarity
from the initial radial symmetry and serves asa reference point for gastrulation movement
and precise coordination of three-germ-layer
differentiation. In all amniotes, the primitive
streak–like structure begins at a precise location that is not only the posterior but also theseed of the midline of the organism. Adopting
such a view implies putting less emphasis on
the primitive streak as a distinct embryological structure and more on its roles as a conduit for symmetry breaking and coordinatedgerm-layer differentiation. Furthermore, it supports the view of gastrulation as a process that
may begin before the visualization of patterned
cell movements. Recapitulation of these functions of the primitive streak in vitro, rather
than of its morphological manifestation, will
have profound implications in developmental
and translational biology.
Our discussion supports the recent reassessment of the primitive streak as it pertains to
ethical debates surrounding rules and regulations of in vitro studies of human development(6). Currently, as a key element of ethical oversight in human embryo research, the 14-day
rule is effective in many countries, including
the US, UK, Japan, China, and Spain. This rulereflects an interdisciplinary consensus in developmental and stem cell biology and assisted
human reproduction and represents a somewhat arbitrary concept to determine the legal
rights of a human embryo (93). The scientific
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primitive streak in these 2D gastruloids (74).
However, it is not clear that these movements
are related to the presence of a primitive streakor whether they are likely to reflect the observation that Brachyury promotes cell movement (69, 75). The primitive streak in vivo isa structure with a vectorial component thatdefines the anterior-posterior axis of the embryo, which is absent in the micropattern experiments. Nevertheless, together with thefree adherent culture experiments, this workshows that a primitive streak is not necessary
to implement the schedules of gene expression
associated with the amniote body plan, as has
been suggested by perturbation experimentsof avian development in vivo, in which properly
patterned mesoderm induction could be uncoupled from primitive streak formation (76).
The coordination of signal responses andproportionate patterning of gene expression
in experimental systems in vitro highlights therole of cell collectives in cell fate decisions andprovides an opportunity to explore the role ofbiomechanical signals during gastrulation.
These arrangements provide a versatile device
to address the role that mechanochemical signaling plays in the early stages of development.
Thus, BMP and Nodal (77) as well as Wnt (78)signaling are influenced by the geometry andmechanics of the cellular environment (77).
In particular, Wnt signaling and Brachyury
expression, two key regulators of the onset
and progression of primitive streak formation,
appear to be mechanosensitive. Brachyury expression and polarized cell movements can be
induced at points of high tension in 2D culturesof human PSCs on soft substrates by Wnt/
b-catenin signaling (78). This relationship between mechanics, Wnt signaling, and Brachyury
has been described in other systems (79) andsuggests that b-catenin might act as a generalmechanotransducer during gastrulation.
When PSCs are aggregated and grown insuspension, large-scale patterns emerge thatare not predictable from adherent cultures.
Thus, under controlled culture conditions PSCs
form aggregates that, when exposed to Wnt
signaling, undergo symmetry breaking, which
is manifest in localized expression of Brachyury
at one pole of the aggregate, global shapechanges, and the expression of mesoderm- andendoderm-specific genes regionally organizedwith reference to an orthogonal coordinate
system (69, 80–86). These structures are called“gastruloids” (80), and despite the pronounced
recapitulation of fate specification and multiaxial organization, they lack a morphologicallyrecognizable primitive streak or any of theaforementioned morphological features associated with amniote gastrulation. Gastruloids
emphasize the disconnect between gene expression and morphogenesis revealed in adherent cultures. They also extend the conclusion,
derived from micropattern experiments, that

RES EARCH | R E V I E W

argument was that the primitive streak, formed
after ~14 days of human development postfertilization, represented a definitive sign ofhuman individuality—i.e., from that point on,
only one human individual would emerge,
endowed with rights and sanctity that must
be protected through ethical and legal means.
We have argued that the primitive streak isneither conserved nor necessary for germlayer formation or spatial coordinate acquisition, therefore emphasizing the arbitrarinessof the original 14-day decision and supporting
the new ISSCR guidelines. However, this lineof reasoning does not dispute the necessity ofexerting ethical oversight in human-related
developmental and stem cell biology research,nor does it preclude defining an alternative
developmental landmark as a limit for theculture of human embryos ex vivo. This should
be done, as it was by the Warnock committee,
through a consensual discussion among different stakeholders to ensure scientific andethical rigor.
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AC KNOWLED GME NTS

We thank B. Steventon, N. Moris, and C. Stern for discussions.
Funding: G.S. is supported by JST e-ASIA Joint Research Project
JPMJSC19E5, JSPS Kakenhi 18H02452 and 21H02490, andTakeda Science Foundation grants. A.M.A. is supported by an
ERC Advanced Investigator Grant (MiniEmbryoBluePrint–834580).
A.S. is supported by a grant from the National Institute for Child
Health and Human Development (1R01HD087093). Competinginterests: G.S. and A.S. declare no competing interests. A.M.E. is
an inventor on two patent applications (PCT/GB2019/052668
and PCT/GB2019/052670) filed by the University of Cambridgethat cover the generation of mouse and human gastruloids.

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The primitive streak and cellular principles of building an amniote body throughgastrulation
Guojun ShengAlfonso Martinez AriasAnn Sutherland

Science, 374 (6572), abg1727. • DOI: 10.1126/science.abg1727

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The nonconserved primitive streak
In human development, a linear structure called the primitive streak appears 14 days after fertilization. This structure
marks the transition of the embryo from having radial to bilateral symmetry. The primitive streak also gives anteriorposterior and dorsal-ventral spatial information to cells undergoing gastrulation and forming the various body cell
types. In a Review, Sheng et al. present a phylogenetic and ontogenetic overview of the primitive streak. They discussorganismal, cellular, and molecular features of the primitive streak and how it functions in amniote gastrulation.
The observation that this structure is not conserved and is not required for development in vitro has implications forembryonic stem cell–based models and considerations about human development research. —BAP