The Japanese Society of Developmental Biologists

Develop. Growth Differ. (2013) 55, 52–59

doi: 10.1111/dgd.12014

Review Article

A little winning streak: The reptilian-eye viewof gastrulation in birds
Federica Bertocchini, 1 * Cantas Alev, 2 Yukiko Nakaya 2 and Guojun Sheng 2 *1

Instituto de Biomedicina y Biotecnologia de Cantabria (IBBTEC), CSIC-SODERCAN-Universidad de Cantabria, Facultad
de Medicina, Avda. Cardenal Herrera Oria, s/n, 39011, Santander, Spain; and 2Lab for Early Embryogenesis, RIKEN
Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan

The primitive streak is where the mesoderm and definitive endoderm precursor cells ingress from the epiblast
during gastrulation. It is often described as an embryological feature common to all amniotes. But such a feature has not been associated with gastrulation in any reptilian species. A parsimonious model would be that theprimitive streak evolved independently in the avian and mammalian lineages. Looking beyond the primitive
streak, can one find shared features of mesoderm and endoderm formation during amniote gastrulation? Here,
we survey the literature on reptilian gastrulation and provide new data on Brachyury RNA and laminin protein
expression in gastrula-stage turtle (Pelodiscus sinensis) embryos. We propose a model to reconcile the primitive
streak-associated gastrulation in birds and the blastopore-associated gastrulation in extant reptiles.

Introduction
As a successful group with 10 000 living species, birds
occupy a unique position among the amniotes. Modern
birds evolved from theropod dinosaurs (Chiappe 2004),
and molecular phylogeny studies revealed that theirclosest living relatives are the crocodiles (Chiari et al.
2012). Birds therefore should be considered as representing the reptiles when compared to mammals. But
how representative are they of the reptiles? Extant nonavian reptiles (referred to as reptiles hereafter) consistof five major groups: the crocodiles, turtles, snakes, lizards and tuatara (Fig. 1A). Pioneers of comparativeembryology like Kupffer, Balfour, Will, Mitsukuri, Peterand Pasteels had studied reptilian embryos in great
detail (Balfour 1879; Kupffer 1882; Will 1892, 1896;
Mitsukuri 1894; Peter 1935, 1939 Pasteels 1937,
1957b). But concerning their early development, very
few papers have been published in the last two decades (Arendt & Nubler-Jung 1999; El Mouden et al.
2000; Gilland & Burke 2004; Coolen et al. 2008;
Bachvarova et al. 2009). Molecular studies on amniote*Authors to whom all correspondence should be addressed.
Email: federica.bertocchini@unican.es; sheng@cdb.riken.jp
Received 30 September 2012; revised 3 October 2012;
accepted 4 October 2012.
ª 2012 The Authors
Development, Growth & Differentiation ª 2012 JapaneseSociety of Developmental Biologists

gastrulation have been focused predominantly on two
species, the chick, representing birds, and the mouse,
representing mammals. Can we have a “reptilian-eye”
view of amniote early development? And if we consider
the chick as representing reptiles, can we reconcile thedifferences between the birds on one hand and therest of the reptiles on the other? In this article, findings
on reptilian gastrulation from old literature will be discussed together with recent molecular data. We willemphasize the lack of a phylogenetically conserved
primitive streak in reptiles. We will also highlight theirshared features (with birds) in mesoderm/endoderm
formation, and variable contributions of involution andingression to the gastrulation process. Finally, we willpropose a model to explain the evolutionary transformation from a circumblastoporal mode of mesendoderm formation in amphibians to the primitive streakassociated mode seen in birds and mammals, possiblywith an intermediate mode exemplified in reptiles.

Gastrulation and primitive streak
Gastrulation in amniotes is traditionally described as aprocess involving formation of the primitive streak. The
primitive streak manifests itself as a thick patch of cells
at the posterior edge of the area pellucida. Initially of atriangular morphology and soon taking a rod-likeshape, which elongates towards the center of theblastodisc, the primitive streak is where epiblast cells
ingress to form the endoderm and mesoderm layers

Reptilian gastrulation

(A)

(B)

53

(C)

Fig. 1. Amniote phylogenetic tree and reptilian gastrulation. (A) Phylogenetic tree of extant amniotes. Although the position of the turtles
is still controversial, we have adopted this tree based on the most recent molecular evidence (Chiari et al. 2012; Hedges 2012). (B) External morphology (dorsal view) of gastrula-stage reptilian embryos. An avian-like primitive streak is absent, and a blastopore-like structure ispresent in all species examined. In the snakes/lizards group, upper panel: lizard; lower panel: snake. (C) Sagittal sections through theblastopore of embryos slightly older than shown in (B). bp, blastopore. All images in (B) and (C) are reproduced with permission fromrespective publishers. Images for crocodile Alligator mississippiensis are from (Reese 1908), for turtle Caretta caretta from (Mitsukuri1894), for tuatara Sphenodon punctatus from (Schauinsland 1899); for lizard Lacerta agilis from (Wenckebach 1891) and Zootoca vivipara
from (Dufaure & Hubert 1961); for snake Tropidonotus natrix from (Gerhardt 1901). The same observation, that reptilian gastrulation isassociated with a blastopore instead of a primitive streak, has been reported in many other species, including the turtles [Pelodiscus sinensis (Mitsukuri & Ishikawa 1887); Clemmys japonica (Mitsukuri 1891; Will 1892); Chrysemys picta (Davenport 1896; Brachet 1914); Chelopus insculptus (Davenport 1896); Chelydra serpentina (Davenport 1896; Yntema 1968); Clemmys leprosa (Pasteels 1937); Pseudemys
virginica (Pasteels 1957b); Geoemyda trijuga and Lissemys punctata (Nayar 1959, 1966); Chelonia mydas (Miller 1985); Emys orbicularis(Mehnert 1892; Coolen et al. 2008) and Trachemys scripta (Bachvarova et al. 2009)]; the lizards [Chamaeleo vulgaris (Peter 1935); Chamaesaura anguinea, Mabuia megalura and Chamaeleo bitaeniatus (Pasteels 1957a,b); Lacerta vivipara (Hubert 1970); Agama impalearis (ElMouden et al. 2000); Platydactylus mauritanicus (Will 1890); and Lacerta muralis, Lacerta lilfordi and Lacerta viridis (Will 1896)], the snakes[Tropidonotus natrix (Ballowitz 1901); Thamnophis sirtalis (Zehr 1962)]; the tuatara [Sphenodon punctatus (Dendy 1899; Tribe & Brambell
1932)] and the crocodiles [Alligator mississippiensis (Clarke 1891); and Crocodylus niloticus (Voeltzkow 1899)].

(Stern 2004). This mode of endoderm and mesoderm
formation in amniotes is fundamentally different fromthat seen in their anamniote ancestors, that is, via
involution through the blastopore. This classical, primitive streak-centered view of amniote gastrulation isderived from analyses on eutherian mammals andbirds. The chick system, the main avian model system,has been paramount to the understanding of morphogenetic rearrangement events that accompany primitive streak formation and elongation, and of the cellularingression movement through the primitive streak thatis characteristic of amniote gastrulation. Epiblast cellintercalation in the middle posterior portion of the blastodisc results in the formation of the primitive streak(Voiculescu et al. 2007); coordinated cell ingression
through the anterior part of the early primitive streak
generates the endoderm (Kirby et al. 2003; Lawson &Schoenwolf 2003; Kimura et al. 2006); and mesoderm
precursor cells ingress through the length of theprimitive streak and interpose between the epiblastand the endoderm (Nicolet 1965; Tam & Beddington
1987; Selleck & Stern 1991; Tam et al. 1993; Psychoyos & Stern 1996; Nakaya et al. 2008; Nakaya &Sheng 2009). The original anterior-posterior positional
difference along the primitive streak translates into thefinal dorsal-ventral position in the embryo. However, in
addition to eutherian mammals and birds, the amniote
world includes two other mammalian groups, the Prototheria and Metatheria, which will not be discussed
here, and the Reptilia. So how do reptilian embryos
gastrulate?

Reptilian blastopore and primitive plate
Similar to birds, early embryogenesis in reptiles goes
through meroblastic cleavage, cellularization and blastoderm formation processes, resulting in a two-layered
structure in the area pellucida: an epiblast layer ofª 2012 The Authors
Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists

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F. Bertocchini et al.

single-cell thickness and epithelial morphology and ahypoblast layer of variable thickness and morphology(Agassiz 1857; Balfour 1879; Will 1896; Ballowitz
1901; Peter 1934; Pasteels 1937, 1957a; Dufaure &Hubert 1961; Hubert 1962; Miller 1985; El Mouden
et al. 2000). In avian embryos, initiation of gastrulation
is linked to primitive streak formation. But an avian-likeprimitive streak has not been reported in any of thereptilian species studied. What appears to be conserved among the reptiles is a blastopore-like structure
where epiblast cells are internalized to become endoderm and mesoderm cells. Representative images ofthe blastopore in each reptilian group are shown in
Figure 1B (surface views) and Figure 1C (sagittal section views), and similar observations have beenreported in many other reptilian species (Fig. 1 legend).
The blastopore is the external opening of invaginatedepiblast epithelium. The space enclosed by this invagination is the archenteron. Located posterior to theblastopore, partially constituting the posterior wall (andthe ventral-posterior wall later on) of the archenteron,is the primitive plate (also called blastoporal plate). The
primitive plate is another conserved feature in reptilian
embryos, and has been variably likened to the primitive
streak in birds, the yolk plug in amphibians, or theventral lip of the blastopore in amphibians. The reptilian blastopore itself (more precisely the anterior andlateral rims of the blastopore) has been compared tothe dorsal blastopore lip or the entire circumblastoporal lip in amphibians, or the Hensen’s node or theentire primitive streak in birds. These comparisonswere based primarily on histological analyses of fixed
reptilian embryos from above-mentioned studies(Fig. 1 legend). Before we offer our opinions on the
structural homology of these features, let us first takea look at how the endoderm and mesoderm are generated in reptiles.

Endoderm
Mechanistic understanding of mesendoderm formationin reptiles is scanty, and relies heavily on a handful ofdescriptive studies of histological sections. The few
exceptions are studies by two authors, Pasteels (1937)
and Nayar (1959, 1966), who applied dye on the epiblast of Chelonia embryos and registered the positionof the marker after a short period of time. The resultsof these histological and labeling studies pointed to amechanism based mainly on involution as the mode ofmesendoderm internalization at the site of gastrulation.
While involution of epiblast cells through the blastopore
lip is clear, ingression also appears to play a role.
These two modes combined would more satisfactorily
account for the presence of the mass of cells posi-tioned in the anterior/lateral and posterior regions of
the gastrulating reptile embryo (Coolen et al. 2008;
Bachvarova et al. 2009) (Fig. 4A). Characterization of
the lower layers in reptiles (the hypoblast or extraembryonic endoderm and the definitive endoderm) is
far from being accomplished. In chick embryos thehypoblast forms a flat layer of cells underlying the pregastrulating epiblast. The canonical view of hypoblast
formation in amniotes (mostly derived from chick studies) is based upon delamination of cells from the epiblast and their aggregation into groups called theisland of the hypoblast, followed by coalescence andspreading over the lower surface of the epiblast (Stern& Downs 2012). In reptiles, the germ cell marker Dazl
is expressed in cells embedded in the lower layer ofan early gastrulaing embryo (Bachvarova et al. 2009),
suggesting the presence of a chick-like hypoblast
layer. Histologically, the development of the lower layer
in gastrulating reptile embryos has been analyzed anddebated by a number of researchers including Will,
Kupffer, Mitsukuri, Schauinsland, Ballowitz, Peter andPasteels. Peter, for example, argued that the first
endodermal layer forms under the embryonic shield in
loco from the “deeper blastomeres” (Peter 1934), while
Pasteels proposed that in turtles and lizards the hypoblast (endophylle) forms by delamination from theupper epiblast (ectoblast) (Pasteels 1937, 1957a), likein chick. With regard to the definitive endoderm (theentoblast), Pasteels (1957a), summarizing his own
work on turtles and that of others before him, suggested that these cells involute at the blastopore lip
and account for the formation of a thin layer of endoderm cells covering the ventral surface of the epiblast.
In the midline, the axial mesendoderm called chordahypoblast is continuous with this thin layer of cells (Mitsukuri 1891; Pasteels 1957a). The hypoblast is thenpushed into extra-embryonic areas by the endoderm.
The idea of endoderm pushing the pre-existing lower
layer had been the accepted view in the amniote
experimental models, chick and mouse. However,recent experiments suggested a certain degree ofintercalation between the hypoblast and the forming
endoderm, with hypoblast cells contributing to definitive endoderm (Bertocchini & Stern 2008; Kwon et al.
2008; Burtscher & Lickert 2009). A possible partial
contribution of the hypoblast to the definitive gut epithelium in reptiles was brought up in the old literature(Mehnert 1892; Will 1892), but it is still unclear whether
and to what extent this hypoblast/endoderm mixing
occurs in reptiles. In addition to the hypoblast and the
involuting endoderm, a third population of lower layercells is recognized in the primitive plate. These endoderm cells ingress from the epiblast, and, together
with mesoderm cells (see below), form a uniform massª 2012 The Authors
Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists

Reptilian gastrulationof not yet characterized tissue. The lack of lower-layer
molecular markers, together with the paucity of information from histological sections, makes it difficult to
speculate on the positioning and movements of thedifferent dorsal and ventral components of the endoderm.

Mesoderm
Vertebrate mesoderm can be divided into four lineages: the axial, paraxial, intermediate and lateralplate. In amniotes, extraembryonic mesoderm is added
lateral to the lateral plate mesoderm (Nakazawa et al.
2006; Shin et al. 2009). These mesoderm lineages arepatterned initially along the dorso-ventral body axis ofa vertebrate embryo, so that the axial mesoderm isgenerated from the dorsal-most territory of an earlygastrula and the lateral plate from the ventral-mostFig. 2. Brachyury gene expression inlate gastrula-stage turtle (Pelodiscus
sinensis) embryos. Brachyury is usedhere as a mesendoderm precursor
marker. This gene marks the blastopore
in amphibians and the primitive streakin mammals and birds. (A) A stained
embryo (whole-mount, epiblast-sideview) used for sagittal sections. Sections through the middle and the edgeof the blastopore are shown at low
magnification, and high-magnification
panels (A1–A4) depict gradual changes
in blastopore morphology. (B) A stained
embryo (whole-mount, epiblast-sideview) used for transverse sections.
(B1)-low and (B4) low: low magnification. (B1–B4): high magnification.

55(Psychoyos & Stern 1996; Alev et al. 2010). Reptilian
embryos generate mesoderm from two populations ofcells (Coolen et al. 2008) (Figs 2, 3, 4A,B). The first
population is internalized through the blastopore lip.
The blastopore takes the shape of an anteriorwardbending narrow crescent, which changes gradually
into a posteriorward bending horseshoe shape(Fig. 4A, left). Mesoderm internalization takes placethroughout the entire width of the blastopore (Brachyury staining in Fig. 2, schematized in Fig. 4A and summarized in Fig. 4B). Similar to what is known in theanamniotes, the most axial, chordal mesoderm is internalized through the center part of the blastopore, andprogressively more lateral mesoderm types (paraxial,
intermediate and lateral plate mesoderm) are internalized through more lateral territories of the blastopore
lip (Coolen et al. 2008) (Fig. 4B). The internalized
dorsal mesoderm is initially in direct contact with the

(A)

(B)

(A1)

(B1)

(A4)

(B4)

(A1)

(B1)

(A2)

(B2)

(A3)

(B3)

(A4)

(B4)

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F. Bertocchini et al.

seen in Fig. 3), and ingression (i.e., internalized cells
lose their epithelial polarity and engage in mesenchymal like cell migration) more prominently in ventral
mesoderm (laminin breakdown in internalized cells
seen in Fig. 3). The second population of mesoderm
cells is generated from the primitive plate (Figs 2–4).
Although the boundary of the primitive plate has notbeen defined clearly in any literature and it has beenused sometimes to denote the entire area with active
gastrulation movements, we refer to the primitive plate
as an area including the ventral-posterior aspect of theinvaginated archenteron and its extension into the epiblast situated posterior to the blastopore and boundlaterally by the lateral wings of the blastopore lip (whenit has adopted a horseshoe shape) (gray area in
Fig. 4B,C). In the primitive plate, mesoderm cells areinternalized primarily through an ingression process(Fig. 3). The active zone of ingression can be visualized by the status of laminin breakdown. Cell labelingexperiments (Pasteels 1937; Nayar 1966) suggested
that epiblast cells in the primitive plate move towards
the blastopore (likely towards the active ingressionzone, which occupies a narrower territory) followed byinternalization and lateral/posterior-directed dispersal(summarized in Fig. 4B) (Pasteels 1937). Generation of
mesoderm from two spatially distinct populations of
mesodermal cells, the primitive plate derived “prostomial” mesoderm and the blastoporal invagination associated “gastral” mesoderm was already suggested
towards the end of the nineteenth century by scientists
including Wenckebach, Will and Mitsukuri (Wenckebach 1891; Will 1892; Mitsukuri 1894). The exact fateof mesoderm cells internalized through the primitiveplate is not clear. They most likely contribute to themajority of extraembryonic mesoderm and possibly
also to some parts of the lateral plate mesoderm, as
was suggested also by Mehnert, Schauinsland andPeter (Mehnert 1892; Schauinsland 1899; Peter 1934).

Fig. 3. Laminin protein localization in a gastrula-stage turtle (Pelodiscus sinensis) embryo. All sagittal sections. This embryo is ofa stage slightly younger than in Figure 2. The archenteron is not
yet connected to the subgerminal cavity. Top?Bottom panels:
Medial?Lateral sagittal sections of the blastopore. Left panels:
laminin (red). Apical surface signals are due to background staining. bp, blastopore. Arrowhead: limit of laminin expression, indicating the starting point of loss of epithelial integrity. Mesoderm
cells leaving the epiblast after losing epithelial integrity are considered to be generated by ingression. Mesoderm cells leaving theepiblast while still retaining epithelial polarity are considered to begenerated by involution. Right panels: laminin (red) + 4´6´-diamidino-2-phenylindole dihydrochloride (DAPI) (cyan) + bright field.
Scale bar, 100 lm (20 lm for each smaller unit).

Modelelongated archenteron as its superior wall and is only
later covered underneath by an inward/medialward
moving endoderm layer. The mesoderm of more ventral fate appears to be internalized by moving between
the epiblast and endoderm layers (Fig. 2). Thus the
dorso-ventral axis of mesoderm precursors in reptiles
is reflected in the medio-lateral positional difference ofthe blastopore lip through which these cells are internalized. Along the same axis, involution (i.e., internalized cells keep their epithelial polarity and movecollectively) is observed more prominently in dorsal/
axial mesoderm (laminin retention in involuted cells

So how can we compare the reptilian mode of gastrulation with what we know of in birds? It appears thatthe key difference is largely morphogenetic, whereas
the cellular sources for the endoderm and mesoderm,
the types of mesoderm generated, and the dorsoventral patterning of the mesoderm are all very similar
in birds and reptiles (Fig. 4B). The morphogenetic difference is manifested in two aspects. In the first, theblastopore formation and archenteron invagination
seen in reptiles do not take place in birds. The embryological equivalent of the blastopore in chick, the epiblast above and anterior to the Koller’s sickle, marks atopologically and functionally orthologous territory. Butª 2012 The Authors
Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists

Reptilian gastrulation

(A)

57

(B)

(C)

Fig. 4. Models for gastrulation in the reptiles. (A) Blastopore initiation, archenteron invagination, and mesoderm and endoderm formation. Left: schematic views of early-stage embryos. Not to scale (the area opaca covers a much greater area than depicted here). Right:
sagittal-section views of the boxed regions on the left. Posterior side oriented towards the right. Hypoblast/endoderm: yellow; mesoderm: red; epiblast and involuted epiblast cells: blue. Top-right panel has also the yolk syncytial cell membrane depicted. Green outlined
cells: blastopore lip at the anterior and anterior-lateral rims of the invaginated archenteron. Cells from the ventral and ventral-anteriorarchenteron will join the hypoblast layer. Definitive endoderm cells (still depicted blue in the diagram) will form from the epithelial portionof cells involuted from the blastopore lip. Mesoderm cells form from the primitive plate by ingression and from the blastopore lip by involution plus ingression or collective dissolution of epithelial characters. (B) Dorsal-ventral patterning of the mesoderm cells, which form by
involution/ingression from the blastopore (green), is reflected along the medial-lateral axis and the blastopore. The axial mesoderm isinternalized through the midline, and progressively more ventral types (paraxial, intermediate and lateral plate mesoderm) are internalized
from more lateral regions of the blastopore lip. The lateral-most regions of the blastopore lip may also generate the extraembryonicmesoderm. Mesoderm cells ingressing through the primitive plate will give rise predominantly to the extraembryonic type, but may also
contribute to the lateral plate. D, dorsal; V, ventral. (C) Model for how to reconcile the difference in mesoderm and endoderm formation
between the reptiles and the birds. Green, marginal zone or blastopore lip. Gray, primitive plate/nieuwkoop center-like structures. D, dorsal; EE, major source of the extraembryonic mesoderm; V, ventral.

instead of a blastopore, avian embryos initiate andelongate a primitive streak (Fig. 4C). This is achieved
through morphogenetic rearrangement of epiblast epithelial cells located anterior to the prospective “blastopore” prior to and during mesendoderm internalization.
In reptiles, epithelial cells in the epiblast appear also toundergo rearrangement, reflected in the dramaticallychanging morphology of the blastopore at early stagesof gastrulation (Fig. 4A,C). But this is a separate, andlikely more ancestral among the amniotes, form of
epiblast cell rearrangement. The second aspect ofmorphogenetic difference is seen in the relative contributions of involution and ingression to mesoderm

formation (Fig. 4B). Involution through the blastopore is
the main mode of mesendoderm internalization in
amphibians. Ancestral amniotes, evolving from theamphibians, may have inherited this to a large extent.
Although in birds, ingression is used as the primarymode of mesendoderm formation, it is interesting to
see that extant reptiles use a mixture of both, withinvolution being more prominent during dorsal mesendoderm formation, and ingression more prominent
during the formation of more ventral mesoderm types.
These two aspects of morphogenetic difference
between the birds and the reptiles are likely connected causally. One may speculate that an initialª 2012 The Authors
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F. Bertocchini et al.

small difference in the ancestral avian group, forinstance in the signals regulating planar cell polarity ofthe epiblast epithelium, can initiate changes leading
to both aspects of avian-specific morphogenesis. The
fact that mammalian gastrulation likewise involves aprimitive streak instead of a blastopore suggests thatsuch changes occurred at least twice independently
during amniote evolution. It is probably premature at
this moment to speculate on whether similar changes
underlie the loss of the blastopore and the gain of
the primitive streak in the avian and mammalian lineages. But the conservation in germ layer formationand patterning, as mentioned above, suggests thatsuch variations in morphogenesis do not significantly
alter the hard-wired regulatory logic of amniote gastrulation.
Finally, based on morphogenetic, cell fate and cellbiological comparisons, we propose a structural orthology between the blastopore and primitive plate on one
hand, and the primitive streak on the other (Fig. 4C).
By this we mean that the reptilian blastopore lip (dorsal
and lateral rims of the horseshoe-shaped blastopore)is evolutionarily homologous to the anterior two-thirds
or three-quarters of the avian primitive streak (including
the Hensen’s node), and both structures are homologous to the blastopore in amphibians. The blastoporalplate is evolutionarily homologous to the posterior thirdor fourth of the primitive streak, and both are dedicated mainly to generating the extraembryonic mesoderm (an amniote invention) and are probably derived
from a Nieuwkoop center-like structure in the amphibian ancestor of the amniotes (Fig. 4C).

Acknowledgments
We would like to thank Yuping Wu and Erike Sukowati(Lab for Early Embryogenesis, RIKEN Center for Developmental Biology) for help with Brachyury RNA in situ
and laminin immunofluorescence analyses, Kanako
Ota (Lab for Early Embryogenesis, RIKEN Center for
Developmental Biology) for help with obtaining copies
of publications that are not accessible electronically,
and Rosemary Bachvarova (Cornell University Medical
College) and Sylvie Mazan (Roscoff Biology Station) for
critical comments.

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ª 2012 The Authors
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ª 2012 The Authors
Development, Growth & Differentiation ª 2012 Japanese Society of Developmental Biologists