DEVELOPMENTAL DYNAMICS 243:357–367, 2014

CRITICAL COMMENTARYa

Day-1 Chick Development

Developmental Dynamics

Guojun Sheng*

The first day of chick development takes place inside the mother hen (in utero), during which the embryo
progresses from fertilization to late blastula/early gastrula formation. The salient features of developmental anatomy in this period are conserved among the sauropsids (birds and reptiles). Many of these features are also shared in prototherian (monotreme) embryos, whereas metatherian (marsupial) andeutherian (placental) embryos display significant variations. Important for understanding the evolutionof early development in amniotes, the knowledge of cellular and molecular mechanisms regulating inutero chick development may also offer valuable insight into early lineage specification in prototherians
and conserved features in mammalian early development. This commentary provides a snapshot of what
is currently known about intrauterine chick development and identifies key issues that await further
clarification, including the process of cellularization, allocation of maternal determinants, zygotic geneactivation, mid-blastula transition, cell layer increase and reduction, radial symmetry breaking, early lineage segregation, and role of yolk syncytium in early patterning. Developmental Dynamics 243:357–367,
C 2013 Wiley Periodicals, Inc.
2014. VKey words: chicken; intrauterine development; amniotes; monotremes; marsupials; eutherians; birds; reptiles; cleavage; blastula; gastrulation

Submitted 9 September 2013; First Decision 22 October 2013; Accepted 22 October 2013

A CASE FOR STUDYING THE
FIRST DAY OF CHICK
DEVELOPMENT
The amniotes consist of two majorgroups of vertebrate animals, the synapsids (prototherian, metatherian,and eutherian mammals) and sauropsids (reptiles and birds) (Fig. 1A). Two
species, the mouse representing the
mammals and the chick representing
the birds/reptiles, are currently usedas the benchmark developmental
models for the amniotes. The list ofchick model’s contributions to developmental biology is long (Stern,2005). Missing from this list is any
study on developmental processes
taking place before oviposition. This
period of development has been variably termed preovipositional, oviductal, or intrauterine (the last of which

will be used here), and encompasses
stages of rapid cell cleavages and pregastrulation development. In thisregard, intrauterine chick development is broadly equivalent to the preimplantation development of themouse embryo. During this period, amouse embryo will complete the process of early lineage specification andgenerate three cell populations(trophoblast, epiblast, and hypoblast)that make up the blastocyst (Stephenson et al., 2012; Schrode et al., 2013).
Understanding how this period ofdevelopment is regulated is obviously
important. However, intrauterine
stage chick embryos are generally
inaccessible unless the materials canbe readily resourced from poultry
facilities affiliated with agriculture orbiotechnology institutions. Why is it
desirable to use the chick model tostudy the intrauterine/preimplantation period of amniote development?
Answer to this question lies in understanding phylogenetic diversifications
in early amniote development. It is
well-known that the yolk-less featureof a eutherian egg is a derived attribute of evolution. The marsupials havemoderately yolky eggs (Selwood,
1992; Menkhorst et al., 2009) and themonotremes have an egg organization
very similar to that of the birds andreptiles (Flynn and Hill, 1938) (Fig.
1B). Ancestral mammalian eggs likely
resembled a chicken egg more thanthe mouse one. Early cleavage pattern of a mammalian embryo also
varies, with the monotreme pattern
again being more similar to the avian/
reptilian one than to either theeutherian or marsupial one (Flynn
and Hill, 1938, 1947; Eakin andLaboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan*Correspondence to: Guojun Sheng, Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology,
Kobe, Hyogo 650-0047, Japan. E-mail: sheng@cdb.riken.jp
DOI: 10.1002/dvdy.24087
Published online 29 October 2013 in Wiley Online Library (wileyonlinelibrary.com).

C 2013 Wiley Periodicals, Inc.
V

Developmental Dynamics

358 SHENG

Fig. 1. Phylogenetic and ontogenetic relationships between the mammals and birds. A: Amniote phylogeny. The Synapsida (mammalian) groupcontains the monotremes, marsupials, and eutherians. The sauropsida (reptilian and avian) group contains the birds, crocodiles (omitted), turtles, lizards, snakes (omitted), and tuatara (omitted). B: Amniote ontogeny, showing four early stages: mature oocyte before fertilization, early cleavage, blastula, and gastrula. The monotremes, the reptiles, and the birds have similar oocyte organization, with yellow and white yolk. Marsupial eggs havewhite yolk only, and the composition of marsupial white yolk differs from that in sauropsid/monotreme eggs. Eutherian eggs do not have yolk in the
traditional sense. Early cleavages are meroblastic (side and top views) and are similar in monotreme and reptilian (including bird) embryos. Marsupialembryos are holoblastic, but in many marsupial species early blastomeres extrude acellular yolk into a central cavity. Eutherian embryos are holoblastic. Monotreme, reptilian, and avian blastulae have a multilayered blastoderm positioned on top of a big yolk cell. Blastula-stage marsupialembryos (blastocysts) are unilaminar, and by late blastula-stage pluriblast cells become morphologically distinguishable and will give rise to epiblastand hypoblast cells. Eutherian blastulae have mural trophectoderm cells being morphologically distinct from polar trophectoderm and inner cell mass
cells. The polar trophectoderm cell lineage is a eutherian invention, but is not present in all eutherian mammals. A two-layered structure (an upper
epiblast and a lower hypoblast) is conserved in reptilian/avian, monotreme, and marsupial early gastrulae. TE, trophectoderm; PrE, primitive endoderm/hypoblast; ExEm, extraembryonic.

Behringer, 2004) (Fig. 1B). Moreover,at late blastula/early gastrula stages,
monotreme and marsupial embryos
do not have their pluripotent cell population (epiblast/pluriblast) coveredby a polar trophectoderm (Rauber’s)layer present in most eutherian mammals and defined as part of the trophectoderm layer which surrounds
and is in close association with theinner cell mass/epiblast (Flynn and
Hill, 1947; Selwood, 1992; Selwood
and Johnson, 2006; Frankenberg
et al., 2013) (Fig. 1B). Instead, monotremes and marsupials have a singlecell thick epiblast layer in direct contact with the zona pellucida and a thinlayer of hypoblast underneath it, similar to the organization known in a
postovipositional chick embryo. Of
interest, the polar trophectoderm is
not a conserved structure in theeutherian clade. It is absent in the tenrec and elephant shrew (Wimsatt,

1975; Eakin and Behringer, 2004), andformed but quickly lost in the rabbit,
pig, cattle, dog, cat, and many otherspecies (Williams and Biggers, 1990;
Flechon et al., 2004; Vejlsted et al.,2005; Blomberg et al., 2008; Hassoun
et al., 2009; Wolf et al., 2011).
Two conclusions can be drawn fromabove-discussed phenotypic diversity
seen in mammalian early development. First, the “preimplantation”
development from cleavage to lateblastula stage in basal extant
mammalian lineage (monotremes) ismorphogenetically similar to theintrauterine development in thechick. Second, the “preimplantation”
development in many therian mammals differs from that in the mouse,
the conventional mammalian model.
Given that the monotremes are not
likely to become technically amenablemodels for embryological studies,
knowledge of day-1 chick develop-

ment may provide useful insight into
the variable and conserved features ofmammalian early development.

OVERVIEW OFINTRAUTERINE CHICK
DEVELOPMENT
Avian oviduct has five functional
regions: the infundibulum, magnum,
isthmus, uterus and vagina. Afterovulation, a mature chicken oocyte is
fertilized at the infundibulum. The
fertilized oocyte passes through themagnum which secrets egg white,isthmus which makes soft shell anduterus which adds hard shell before
oviposition induced by vaginal muscle
contraction. The entire process takes
approximately 25 hr. The first cleavage starts approximately 5 hr after
fertilization (Olsen, 1942; Gipson,1974), and the rest of preovipositional

Developmental Dynamics

DAY-1 CHICK DEVELOPMENT 359

Fig. 2. Intrauterine chick development. Two staging systems, the EGK and HH, are currently used to delineate the developmental stage of earlychick embryos. The EGK stages are used for intrauterine and prestreak stages. The intrauterine period, covering approximately 25 hr in total, is
divided into 10 stages: EGK-I to EGK-X. From fertilization to first cell division: 0–5 hr. EGK-I to -VI: cleavage stages (5–15 hr). EGK-VII to EGK-X:
area pellucida formation period (15–25 hr). a.p., area pellucida; a.o., area opaca; epi, epiblast; hypo, hypoblast. See the text for detail of thedevelopmental processes.

development (20 hr) takes place inthe uterus. Chick development is traditionally described according to two
staging systems, the Hamburger and
Hamilton (HH) and the Eyal-Giladi
and Kochav (EGK) (Eyal-Giladi and
Kochav, 1976; Kochav et al., 1980;
Hamburger and Hamilton, 1992; Bellairs and Osmond, 2005; Wong et al.,2013), the latter of which is used for
intrauterine embryos (Fig. 2).
Eyal-Giladi and Kochav divided theprestreak development (HH stage 1)
into 14 stages (denoted by romannumerals as EGK-I to -XIV) (EyalGiladi and Kochav, 1976; Kochav
et al., 1980). The intrauterine period
covers EGK-I to -X. EGK-I to -VI arecollectively called “cleavage” stages
and take approximately 10 hr. EGKVII to -X are collectively called“formation of the area pellucida” (a.p.
formation) stages and take another 10
hr (Fig. 2). Early divisions are meroblastic. Cellularization and subgerminal cavity formation start frombetween EGK-II to EGK-III. The blastoderm becomes multilayered in thecenter from EGK-III and reaches its
thickest (5–6 cell layer thick) at EGKVI, after which the number of cell
layers in the area pellucida decreases
gradually to 1–2 at EGK-X, with an
upper continuous, single cell-layered

epiblast and a lower discontinuous
layer of hypoblast cells. The peripheral cells, precursors to the area
opaca, remain multilayered and maintain close contact with the underlying
yolk cell.
Aside from these two publications
from Eyal-Giladi and colleagues (EyalGiladi and Kochav, 1976; Kochav
et al., 1980), earlier work on intrauterine development from other authors
was summarized in their first staging
paper (Eyal-Giladi and Kochav, 1976).
Work published afterward will be discussed in appropriate sections below.

KEY ISSUES OF
INTRAUTERINE CHICK
DEVELOPMENT IN NEED OF
FURTHER CLARIFICATION
Compared with other vertebrate models (zebrafish, Xenopus, and mouse),very little is known about the cell andmolecular biology of early development in the chick. Such knowledge
would enable us to gain a glimpse of
the ancestry of and diversity in mammalian and amniote pregastulation
development. In the rest of this commentary, I will list and discuss several key issues in need of further
clarification. Concerning the cleav-age period of intrauterine development: (1) How do open blastomeres
cellularize and are there maternal
determinants allocated asymmetrically during the cellularization process? (2) When does zygotic geneactivation start and what is the equivalent stage of mid-blastula transition?
(3) What are the cellular and molecular mechanisms regulating initial separation of inner and outer layers andlater increase in cell layer number?
Concerning the a.p. formation
period of intrauterine development:(4) When is the A-P polarity firstestablished in the blastoderm? (5)What is the main mechanism driving
cell-layer reduction and what is theearliest step in epiblast/hypoblast celllineage segregation? (6) Is the yolkcell a syncytium and, if so, does it
play a role in early patterning and lineage segregation?

Cellularization and Maternal
Determinants
The first cell to complete the cellularization process, i.e., with “basal” separation from the yolk, does so at EGKII to -III, when the embryo has morethan 16 “apical-laterally” enclosed
cells (Eyal-Giladi and Kochav, 1976;
Bellairs et al., 1978; Kochav et al.,Developmental Dynamics

360 SHENG

1980; Lee et al., 2013). This processcontinues until the end of the cleavage phase (EGK-VI), when all cells,
including the most peripheral ones,
are cellularized. Gipson (Gipson,1974) and Bellairs et al. (1978)
observed three types of furrow morphology associated with early cleavages: V- or U-shaped shallow furrow,the pendulum-shaped deep furrow
and slit-shaped smooth and tight furrow (Fig. 3A). Both the shallow andpendulum-shaped
furrowshave
numerous furrow wall protrusions,likely reflecting the process of new
membrane addition similar to what is
known during the cellularization process in Drosophila (Lecuit and Wieschaus, 2000) and in meroblastic
cleavages in zebrafish (Li et al., 2006)
and squid (Arnold, 1969) embryos.
The pendulum-shaped furrow has anelaborate structure at its base called
furrow base body, which in addition to
supplying new membrane may function
as an equivalent of midbody in driving
furrow deepening (Gipson, 1974) or incontrolling the transition from verticaltohorizontal
furrowburrowing(Bellairs et al., 1978). The slit-shaped
furrow represents cell–cell contacts
being established while the cellularization process is still in progress, aphenomenon similarly observed during
zebrafish meroblastic cleavage andDrosophila cellularization.
Cytokinesis lags behind nuclear
division, but most cyokinetic events
are associated with zygotic mitosis.
The exceptions are the pseudocleavage furrows (not discussed here)
induced by supernumerary sperm
nuclei (Lee et al., 2013) and the edge
cells. It is unclear whether the edge
cells complete the cellularization process with no further nuclear division(thus leaving no nucleus to theremaining yolk cell) or with an additional round of nuclear division (leaving syncytial nuclei in the yolk cell).
Cell biology of meroblastic cleavage inthe chick (as outline above) and inother model systems (Mitchison et al.,2012) is still very poorly understood.
Many important features of a young
chick embryo, such as the presence ofa subgerminal cavity, separation of
the inside and outside environment,
polarization of early blastomeres, andpossible asymmetric distribution ofmaternal determinants (discussed

below), are fundamentally linked tothe cellularization process.
Whether maternal determinants
play a role in early patterning in thechick embryo is still debated. Specification of chicken primordial germ
cells was shown to correlate withasymmetric distribution of maternally deposited Vasa-positive structures (Tsunekawa et al., 2000).
Embryonic dorsoventral (D–V) patterning, corresponding to anteroposterior (A–P) polarization of theblastoderm, in quail and chickembryos was hypothesized to be regulated by asymmetric distribution ofdifferent ooplasms at early cleavage
stages (Callebaut, 1987, 2005).
Ooplasmic materials surrounding thegerminal vesicle, the oocyte nucleus,
are heterogenous. Callebaut and colleagues classified them into fourtypes: the a, b, g, and d ooplasms
based on histochemical staining andradiolabeling data, and suggested
that asymmetric inheritance of the gand d ooplasms during cleavage
stages provides positional cues for
subsequent A–P polarization. The gooplasm is derived from the perinuclear mitochondria-rich “ticos” (thymidine-incorporating
cytoplasmic
organelles) and the d ooplasm is thecytoplasmic material on the surface ofthe nucleus of Pander, the white yolk
substance located below the germinal
vesicle. The g and d ooplasms represent more “primitive” cytoplasmic
materials than the yellow yolk which
is deposited during late stages ofoocyte maturation. Although it isplausible from an evo-devo perspective that maternal determinants in an
avian egg are localized to theseooplasms, it is unclear whether or
how asymmetric inheritance of theseooplasms determines the A–P polarity. During EGK-I to -IV, the center ofcell proliferation, where the first two
cleavage furrows meet and where thesmallest blastomeres concentrate, isoften located off the geometric centerof the embryo. Dividing blastomeres
may therefore inherit ooplasm materials asymmetrically as they cellularize (Fig. 3B). Like the amphibian
Nieuwkoop center, this ooplasm
asymmetry may lead to signaling
asymmetry which later determines
the future dorsal side at posterior endof the blastoderm. Significance of thishypothesis is uncertain because artificial tilting of the blastoderm after
EGK-VI can reorient embryonic D–Vaxis with high efficiency (Kochav andEyal-Giladi, 1971) and multiple D–V
axes can be experimentally induced in
postovipositional
embryos(Lutz,
1949; Spratt and Haas, 1960; Bertocchini et al., 2004; Bertocchini and
Stern, 2012). An alternative explanation, reconciling both the ooplasmmediated predetermination hypothesis and the regulative nature of D–Vaxis formation at a.p. formation andpostovipositional stages, is thatmaternal asymmetry determinants
may be localized not in dividing blastomeres during cellularization, but inyolk cell cortex after cellularization,
as reported in pregastrula zebrafish
embryos (Carvalho and Heisenberg,2010). Molecular nature of the putative maternal determinants in either
scenario (blastomere-inherited or yolk
cortex-inherited) is unknown.

Zygotic Gene Activation(ZGA) and Mid-blastulaTransition (MBT)ZGA occurs in two waves in vertebrate embryos (Tadros and Lipshitz,2009): an early/minor wave as theearliest sign of ZGA and a late/major
wave representing a robust and largescale increase in ZGA. In the zebrafish, the minor ZGA starts at cleavage
stages (64-cell stage) and the major
one at early blastula stage (Mathavan
et al., 2005). In the mouse, the minor
ZGA starts at one-cell stage and the
major one at two-cell stage (Wang andDey, 2006); whereas in human, theminor ZGA starts at two-cell stage
and the major ZGA at the four- toeight-cell stage (Vassena et al., 2011;
Xue et al., 2013, Yan et al., 2013).
Timing of ZGA onset is influenced by
nucleocytoplasmic ratio, maternal
clock, transcript abortion, and epigenetic modification (Tadros and Lipshitz, 2009), the first two of which are
related to oocyte organization, earlycleavage pattern and developmental
speed. In this respect the chickembryo resembles more the fish andfrog embryos than the mouse or
human embryos. Radioisotope labeling (Wylie, 1972) and electron microscopy (Raveh et al., 1976) studies,Developmental Dynamics

DAY-1 CHICK DEVELOPMENT 361

Fig. 3. Cellularization, maternal determinants, and formation of cell layers. A: Model of cytokinesis. Early furrows are u/v-shaped (without immediate formation of cell–cell junctions) or slit-shaped (with junctional interactions). The pendulum-shaped furrows are at an intermediate stage. Furrow base body, not drawn here, is located at the base of the pendulum and functions to drive vertical deepening or horizontal burrowing of the
furrow. Completion of the cellularization process requires a supply of new plasma membrane, which can be provided either apically (before tightjunction formation) or basolaterally (after tight junction formation). B: Ooplasms are deposited in the oocyte in a radially symmetric manner. In one
scenario, dividing blastomeres inherit these ooplasms symmetrically (top). However, early cleavages are known to be radially asymmetric, possibly
due to off-center pronuclear fusion, gravitational effects on yolk materials, or stochastic variation in peripheral extension of meroblastic cleavage
furrows. This results in an asymmetric inheritance of maternal determinants (bottom). The side of the embryo inheriting more g and d ooplasmic
materials (surrounding and within in the nucleus of Pander, respectively) (Callebaut, 2008) will become or be biased to become posterior. Arrows:
Hypothetical axis from yolk center to blastoderm center. C: Two possible causes for the increase in cell layer number from one to two. Directcause: Horizontal (parallel to blastoderm surface) cleavage plane leads to the formation of outer and inner daughters. Indirect cause: Vertical(orthogonal to blastoderm surface) cleavage plane (not shown) or oblique cleavage plane (shown) generates two surface daughters, one of whichwith smaller surface area may be biased to become an inner cell.

using techniques which are quite
insensitive by modern-day standards,
suggested that there is no prominentribosomal RNA synthesis before EGKIII. One may predict based on theselines of evidence that the minor ZGA

in the chick starts at mid-cleavage
stages (EGK-II to -IV) and the major
ZGA at late cleavage stages (EGK-IV
to -VI). The juncture between thecleavage period and the a.p. formation
period, EGK-VI, can thus be viewed as

the equivalent of mid-blastula transition (MBT) in Xenopus. Cell-counting
analysis revealed that an EGK-V
embryo contains approximately 2,400
cells and an EGK-VII embryo has
36,000 cells (Park et al., 2006).

362 SHENG

Although divisions are known to be
asynchronous after the 4th cleavage(Perry, 1987), these numbers translate
into approximately the 11th cell cycle
at EGK-V and the 15th cell cycle at
EGK-VII, making it plausible that an
MBT-like event occurs at EGK-VI. The
increase in cell number proceeds at amuch slower rate afterward, reachingapproximately 54,000 at EGK-X (Parket al., 2006), probably due to thelengthening of cell cycle duration in
the second half of intrauterine development (Emanuelsson, 1965).

Developmental Dynamics

Increase in the Number ofCell Layers
Blastomeres located in the middle
complete the cellularization process
at EGK-II to -III, and further cell divisions result in the formation of a multilayered organization in central
blastoderm, approximately two cells
thick at EGK-III to -IV and reaching amaximum of five to six cells thick at
EGK-VI. More peripheral regions
undergo a similar, but delayed andless pronounced, process of layer
increase. The most peripheral part ofthe blastoderm is always one-cell
thick. When a centrally located blastomere separates itself from the yolk
by forming a horizontal “basal” membrane, the yolk counterpart is anuclear. Thus the increase in layer
number is the outcome of either rearrangement or proliferation of alreadycellularized blastomeres (Fig. 3C).
Changing from a mono-layered to abi-layered structure produces aninside cell population sealed off fromthe external milieu. A similar process
in the mouse initiates the separationof cell types at the morula stage (Stephenson et al., 2012; Hirate et al.,2013). Asymmetric cleavage during
the transition from 4-cell to 16-cellstage generates inside and outsidedaughters, whereas symmetric cleavage produces two outside or inside
cells (Sasaki, 2010). The type of division can be predicted based on the orientation of mitotic plane: thoseparallel to the surface of the morula
being asymmetric (by generating an
inner, inner cell mass-biased daughter
and an outer, trophectoderm-biased
daughter) and those orthogonal to thesurface being symmetric. Symmetric
division of a surface blastomere, gen-erating two outer daughters, can secondarily give rise to inner cells
through postdivision modifications ofcell movement and cell adhesion (Parfitt and Zernicka-Goetz, 2010). In Xenopus, inner cell population forms
through a similar parallel-to-surface
orientation of mitotic planes after 64cell stage, while orthogonal-to-surface
orientation of the mitotic plane generates two surface daughter cells(Chalmers et al., 2003). A third typeobserved in Xenopus, the oblique division, produces one daughter withsmaller surface area which is strongly
biased to undergo asymmetric division in the next cell cycle (Chalmers
et al., 2003). In the chick, horizontal(parallel-to-surface) orientation of thecleavage plane was also hypothesized
to be the cause for layer increase(Kochav et al., 1980), although there
is no direct evidence either in favor of
or against this model.

A–P Polarity of theBlastoderm (Radial
Symmetry Breaking)Although often described as such,chick embryos obtained from freshlylaid eggs are not radially symmetrical. At EGK-X, morphological asymmetry along the A–P axis of theblastoderm is apparent with theasymmetric distribution of hypoblast
islands, and molecular asymmetry is
also readily detectable in both the epiblast and hypoblast cells (Peter, 1938;
Spratt and Haas, 1960; Eyal-Giladi
and Kochav, 1976; Sheng and Stern,
1999; Bertocchini and Stern, 2012;
Alev et al., 2013b). At the beginningof the a.p. formation period, EGK-VII,
initiation of layer reduction has been
proposed to start from the posteriorside of future area pellucida (Clavert,
1960; Kochav et al., 1980; Watt et al.,1993). As in most telolecithal eggs,
the abembryonic side of chicken yolk
is heavier than the embryonic side,
and a developing embryo positions
itself on top of the yolk if left undisturbed. Vintemberger and Clavert, ina series of papers published in 1950s
and 1960, proposed that bilateralsymmetry (i.e., A–P polarity of theblastoderm and D–V polarity of theembryo) is established by egg rotationin utero (Fig. 4A). The forming egg

shell is rotated along its long axis in
the uterus (10–15 times per hr counterclock-wise if viewed from the isthmusside) with no matching rotation of theegg yolk, resulting in the formation ofcoiled chalazae (two strands of dense
egg white materials connecting thevitelline membrane around the yolk
with the outer thick albumen underneath the soft shell) and a constant tiltof the blastoderm relative to its gravitationally dictated position (Fig. 4A).
The highest point of the blastoderm
will eventually become the posteriorside (the so-called von Baer’s rule).
Results from artificial tilting of EGKVI to -X embryos supported thishypothesis (Kochav and Eyal-Giladi,
1971; Eyal-Giladi and Fabian, 1980;
Eyal-Giladi, 1991), suggesting thatmolecular determinants of radialsymmetry breaking is sensitive togravitational cues at late cleavage andearly a.p. formation stages. Theseobservations do not rule out the possibility of radial-symmetry breaking
being determined or biased by pronuclear fusion point or maternal ooplasm
segregation (Fig. 3B), but they dostrongly argue in favor of a regulative
mechanism underlying initial radialsymmetry breaking. The molecularnature of the earliest determinants of
A–P axis formation, either maternallybiased at early cleavage stages orgravitationally regulated at early a.p.
formation stages, is unknown.

Layer Reduction and Lineage
Segregation
From EGK-VII, the number of cell
layers in the blastoderm decreases
from initial five to six at EGK-VI to
one to two at EGK-X. This process
starts from the central-posterior
region of the blastoderm and spreads
to all areas above the subgerminal
cavity, but not to the area opaca. Cell
shedding was proposed to be thecause for layer reduction (Kochav
et al., 1980, Fabian and Eyal-Giladi,1981) (Fig. 4C). Radial intercalation,as an alternative although not mutually exclusive mechanism, may also
play a role in this process (Fig. 4C).
During the process of a.p. formation,
the central-most, thickest area takes
longer time to appear translucent
than the rest of the blastoderm
located above the subgerminal cavity.

Developmental Dynamics

DAY-1 CHICK DEVELOPMENT 363

Fig. 4. A–P polarization, lineage segregation, layer reduction, and yolk syncytium formation. A: A–P polarity. Despite possible biases imposed at
an earlier stage (see Fig. 3B), anterior–posterior polarity of a chick blastoderm first becomes visible at EGK-VII. This is a regulative process. Egg
shell is rotated counter clock-wise in utero (if viewed from the isthmus side), resulting in a tilt of the blastoderm (middle) away from its preferred
position (left). Gravitational forces reorient the yolk and yolk cortical materials, and side of the embryo in closer contact with the nucleus of Pander
ooplasms (top side) will become posterior. B: Two possible mechanisms for the segregation of the epiblast, hypoblast and area opaca cell lineages. In one scenario, lineage specification starts when the blastoderm is multilayered. The epiblast and hypoblast lineages are mixed in a saltand-pepper manner. Sorting takes place during layer reduction, eventually resulting in bilaminar organization with an upper epiblast and a lower
hypoblast layer. In the second scenario, lineage specification follows layer reduction. C: Two possible mechanisms for layer reduction: cell shedding and radial intercalation. D: Three possible sources of syncytial nuclei. Black arrow: shed cells on the floor of subgerminal cavity. Green arrow:germ wall deep layer cells. Red arrow: edge cell.

This central area is called area alba
in the turkey (Gupta and Bakst,
1993) and an equivalent structure,
although not named, is also present
in the chick (Kochav et al., 1980).
Eventually, at EGK-X, most regions of
the area pellucida, including the area
alba, become 1–2 cell thick, whereas
the area apaca remains multilayered(Kochav et al., 1980; Andries et al.,
1983; Gupta and Bakst, 1993; Wattet al., 1993). A notable exception is
the region of the area pellucida at theposterior marginal zone, locatedbetween the Koller’s sickle (acrescent-shaped lower layer cell population closely associated with theoverlying epiblast) and the posterior
germ wall. This part of the area pellucida is often more than two cells
thick, and the dynamic nature of its
morphogenesis and signaling isamong the most prominent anddebated issues of early postovipositional
development(Eyal-Giladi,
1991; Eyal-Giladi et al., 1992; Calle-baut et al., 1998; Stern and Downs,
2012). This aspect of cell-layer reduction is not discussed in detail here.
Two general cell-biological featuresof the area pellucida at EGK-X, however, deserve further discussion.
First, the one-cell thick areas have
only the upper, epiblast layer; while
the two-cell thick areas contain an
upper epiblast layer and a lower loose
aggregate of primary hypoblast cells(hypoblast islands). Epiblast cells in
one-cell thick areas are polarized with

Developmental Dynamics

364 SHENG

signs of basement membrane deposition, whereas epiblast cells in two-cell
thick areas do not have a basement
membrane and lag behind in the epithelialization process (Bortier et al.,
1989; Harrisson et al., 1991; Lawson
and Schoenwolf, 2001; Nakaya et al.,2008; Nakaya et al., 2011, 2013). Second, primary hypoblast islands haveananterior-sparse/posterior-dense
distribution (Kochav et al., 1980; Wattet al., 1993), opposite to the initiationof layer reduction, which starts fromthe posterior side. The initiation ofcell shedding or intercalation and the
speed of its continuation may, therefore, be regulated separately.
The epiblast was hypothesized to be
reduced to one cell thick, and the primary hypoblast cells were derived bymeans of poly-ingression from theupper, epiblast layer cells (Peter,
1938; Fabian and Eyal-Giladi, 1981;
Eyal-Giladi, 1991; Watt et al., 1993).
Support for this model is indirect (bottle-shaped cells spanning both layers)and pregastrulation development in
the chick does not pass through any
stage with a one-cell thick epiblast
without associated hypoblast cells. A
plausible interpretation is that primary hypoblast cells are “left-overs”
of the layer reduction process. An
incompletely epithelialized epiblast,with no basement membrane andlabile epithelial junctions, can still
accommodate accretion of cells fromthe lower layer, resulting in bottleshaped cell morphology seen in sections or by surface labeling. Instead, awell-epithelialized epiblast with forming basement membrane will resist
further cellular addition, leaving nonepithelialized lower layer cells as primary hypoblast cells. The difference
in hypoblast island density along theA–P axis could, therefore, be simply
due to a difference in the rate of epithelialization of the uppermost layer
during the process of layer reduction.
Viewed from an evolutionary perspective, however, the polyingression
hypothesis still deserves to be tested
carefully. In monotremes, the multilayered blastoderm thins out into asingle-layered structure, from which
the hypoblast cells are thought tocome (Flynn and Hill, 1947), although
direct evidence is also lacking in thiscase. In marsupials, the blastoderm issingle-layered from the beginning of

blastocyst formation and the hypoblast cells are generated from theepiblast/pluriblast by ingression, presumably from both the middle and border regions of the epiblast/pluriblast(Selwood, 1992; Kress and Selwood,
2006; Frankenberg et al., 2013). In
eutherian mammals, hypoblast andepiblast cells separate from each otherfrom an initially mixed inner cell mass
population and before the epiblast
cells adopt an epithelial structure(Stephenson et al., 2012; Xenopoulos
et al., 2012). It, therefore, remains possible that hypoblast precursor cells in
the chick are initially located in a saltand-pepper manner in the multilayered blastoderm (including in theupper-most layer) and secondarily
aggregate or ingress during the layer
reduction/lineage sorting process.
The epiblast and hypoblast layers
will give rise to different embryonicand extraembryonic cell types later indevelopment. Cellular changes leading to their formation (i.e., a.p. formation and layer reduction) take placeconcomitant with changes in gene
expression profiles. By the time achick embryo reaches EGK-X, at least
three cell populations with distinct
molecular signatures have beendelineated: the epiblast, hypoblast,
and the area opaca cells (Fig. 4B).
Each population has robust expression of lineage-specific markers, manyof which have been well-characterized
in mammalian models, such as
Nanog, PouV, Dnmt3B, and Lin28 inepiblast (Lavial et al., 2007; Shinet al., 2011; Alev et al., 2013a), Hex inhypoblast (Yatskievych et al., 1999),
and Eomes in area opaca cells (Pernaute et al., 2010). It is unclear how
early these lineage specific genes initiate their expression and whether
their expression precedes or follows
morphological changes of these threecell populations (Fig. 4B). It is also
worth noting that instead of defining
cellular fates, specific expression of
lineage markers at these early stages
likely marks cellular states, the labileand dynamic bias toward these cellular fates.

Yolk Syncytium andBlastoderm-Yolk Contact
It is yet to be determined whether themembrane enclosed structure sur-rounding the remaining yolk, after
the completion of cellularization at
EGK-VI, is a syncytium or not. In thezebrafish, yolk syncytium forms byfusion of the yolk cell and deep layer
marginal blastomeres, and plays critical roles in embryonic induction andmorphogenesis (Carvalho and Heisenberg, 2010). In monotremes, the yolkcell at late blastula is also a syncytium (Flynn and Hill, 1947). In thechick, there are three possible sourcesof zygotic nuclei which may contribute to the formation of a yolk syncytium: the most peripheral part of theblastoderm, deep blastomeres in thegerm wall, and shed blastomeres in
the area pellucida (Fig. 4D).
At cleavage stages, peripheral open
cells are in contact with the yolk bothbasallyandperipheral-laterally.
Nuclear division in these cells generates a proximally located daughter
which cellularizes like other centrally
located blastomeres and a peripherally located one which remains as anopen cell. Cycles of nuclear divisionand cytokinesis reduce the size of theopen cell successively. If the last cellularization step takes place before
nuclear division, no daughter nucleus
is left in the yolk cell; and if it happens after nuclear division, each
peripheral-most cell will leave adaughter nucleus in the yolk cell. The
second source is the blastomeres
located close to the yolk cell membrane. From EGK-III, a subgerminal
cavity forms in central blastoderm
between the yolk cell membrane andcellularized central blastomeres. Thiscavity expands as cellularization progresses peripherally. The expansion,
however, lags behind the cellularization process, and the yolk cell stays inclose contact with peripheral deep
layer blastomeres even after complete
cellularization. This area of contact ismaintained during early development
and is called the germ wall. It is
unclear whether some of the deep
layer cells in the germ wall can fusewith the yolk cell as seen in the zebrafish. The inner margin of the germwall is a dynamic, shifting boundary
between the area pellucida and area
opaca. It is also an important centerfor early patterning. It is unclear
what roles the yolk cell plays in themaintenance of this boundary or in
its associated molecular signaling.

Developmental Dynamics

DAY-1 CHICK DEVELOPMENT 365

Deep cells in the area pellucida do
not contact the yolk cell membrane.
However, area pellucida cells that are“shed” during the process of layer
reduction drop to the floor of the subgerminal cavity and associate withthe yolk cell tightly (Andries et al.,1983). These shed cells are also distributed asymmetrically along the A–
P axis. Whether some of them fusewith the yolk cell and whether theyplay any role in the function of yolk
syncytium are unknown. Electron
microscopy studies of EGK-X to -XIV
chick and quail embryos (Andries
et al., 1983) indicate that the corticalregions of the yolk cell on the floor of
the subgerminal cavity are rich inmitochondria, microfilaments, andmembrane invaginations and protrusions, suggesting that yolk cell cortex
and its associated cellular and molecular activities are an integral part ofavian early development.

TECHNICAL LIMITATIONSAND POSSIBLE SOLUTIONS
The chick is a mature developmental
biology model. Tools for RNA/protein
localization, cell labeling/tracing,
imaging, overexpression/knockdown,
and transcriptomics/proteomics analyses are all available. Recent progressesinavian
transgenesis(McGrew et al., 2004; van de Lavoir
et al., 2006; Park and Han, 2012;
Nishijima and Iijima, 2013) make itpossible to generate chicken lines
with ubiquitous or promoter-specificgreen fluorescent protein expression,
allowing sophisticated imaging andmolecular analyses in the future. The
only major limitation in studying
intrauterine development is theaccess to these embryos. Large poultry facilities are common in mostcountries, but basic developmental
biologists often do not have access tothese facilities. Some universities still
maintain poultry farms affiliated with
their agriculture or biotechnology
departments. Scientists working inthese departments are best suited forbridging this gap.
Three methods have been used toretrieve intrauterine eggs for earlystage embryos. They are the physically induced retrieval method (Olsen,
1942; Eyal-Giladi and Kochav, 1976;
Park et al., 2006; Lee et al., 2013),

hormonally induced retrieval method(Olsen, 1942; Watt et al., 1993) anddirect sacrifice of mother hens (Emanuelsson, 1965; Gipson, 1974; Bellairs
et al., 1978). Only eggs with soft or
hard shell (from four-cell stage
onward) can be obtained through thefirst two techniques; while the third
one can be used to obtain eggs fromfertilization to one- to two-cell division stages. Retrieved eggs can be cultured in vitro, with or without theshell, to postovipositional stages withgood survival rates (Vintemberger
and Clavert, 1960; Kochav and EyalGiladi, 1971; Perry, 1988; Naito andPerry, 1989; Naito et al., 1990, 1995).
Embryos collected from intrauterine
eggs can be used directly for transcriptomics/proteomics investigations,
fixed for gene expression and protein
localization studies, or cultured invitro for labeling, lineage tracing, andfunctional analyses. Finally, an alternative approach to circumvent thelimited access of intrauterine chickembryos may be to use embryos fromother avian species. The turkey(Gupta and Bakst, 1993) and zebra
finch (unpublished data) lay theireggs at much earlier stages than thechick. Both species have their genome
sequenced (Dalloul et al., 2010;
Warren et al., 2010), and the early
development, from cleavage to hypoblast formation, of the turkey embryo
has also been described (Gupta and
Bakst, 1993).

CONCLUSIONS
Intrauterine chick development isdivided into 10 EGK stages (EGK-I to-X). EGK-I to -VI are collectively
called cleavage stages, during which
the cell number increases from 1 toapproximately 10,000 and the embryo
attains the equivalent of mid-blastula
stage. The area pellucida forms
between EGK-VII to -X, during which
the embryo establishes bilateral symmetry and specifies three basic celllineages, the epiblast, hypoblast, andarea opaca. Intrauterine developmentin the chick is similar to the“preimplantation” (cleavage to lateblastula) development in the monotremes, and the knowledge gleaned
from studies of day-1 chick embryos
will enable a better mechanistic

understanding of early development
in diverse mammalian species.

ACKNOWLEDGMENTS
I thank Dr. Gary Schoenwolf for theinvitation to write this commentary,and Drs. Claudio Stern, Patrick Tam,Jae Yong Han, members of Laboratory
for Early Embryogenesis, and thereviewers for critical comments.

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