Day1 Chick Development

DEVELOPMENTAL DYNAMICS 243:357–367, 2014

CRITICAL COMMENTARY

a Day-1 Chick Development

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) and eutherian (placental) embryos display significant variations. Important for understanding the evolution of early development in amniotes, the knowledge of cellular and molecular mechanisms regulating in utero 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 gene activation, 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, 2014. VC 2013 Wiley Periodicals, Inc.

namics Key words: chicken; intrauterine development; amniotes; monotremes; marsupials; eutherians; birds; reptiles; cleav- y age; blastula; gastrulation

Submitted 9 September 2013; First Decision 22 October 2013; Accepted 22 October 2013 mental D will be used here), and encompasses study the intrauterine/preimplanta- p A CASE FOR STUDYING THE FIRST DAY OF CHICK stages of rapid cell cleavages and pre- tion period of amniote development? gastrulation development. In this Answer to this question lies in under- DEVELOPMENT regard, intrauterine chick develop- standing phylogenetic diversiﬁcations

Develo The amniotes consist of two major ment is broadly equivalent to the pre- in early amniote development. It is groups of vertebrate animals, the syn- implantation development of the well-known that the yolk-less feature apsids (prototherian, metatherian, mouse embryo. During this period, a of a eutherian egg is a derived attrib- and eutherian mammals) and saurop- mouse embryo will complete the pro- ute of evolution. The marsupials have sids (reptiles and birds) (Fig. 1A). Two cess of early lineage speciﬁcation and moderately yolky eggs (Selwood, species, the mouse representing the generate three cell populations 1992; Menkhorst et al., 2009) and the mammals and the chick representing (trophoblast, epiblast, and hypoblast) monotremes have an egg organization the birds/reptiles, are currently used that make up the blastocyst (Stephen- very similar to that of the birds and as the benchmark developmental son et al., 2012; Schrode et al., 2013). reptiles (Flynn and Hill, 1938) (Fig. models for the amniotes. The list of Understanding how this period of 1B). Ancestral mammalian eggs likely chick model’s contributions to devel- development is regulated is obviously resembled a chicken egg more than opmental biology is long (Stern, important. However, intrauterine the mouse one. Early cleavage pat- 2005). Missing from this list is any stage chick embryos are generally tern of a mammalian embryo also study on developmental processes inaccessible unless the materials can varies, with the monotreme pattern taking place before oviposition. This be readily resourced from poultry again being more similar to the avian/ period of development has been varia- facilities afﬁliated with agriculture or reptilian one than to either the bly termed preovipositional, oviduc- biotechnology institutions. Why is it eutherian or marsupial one (Flynn tal, or intrauterine (the last of which desirable to use the chick model to and Hill, 1938, 1947; Eakin and

Laboratory 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: [email protected] DOI: 10.1002/dvdy.24087 Published online 29 October 2013 in Wiley Online Library (wileyonlinelibrary.com).

VC 2013 Wiley Periodicals, Inc. 358 SHENG

Fig. 1. Phylogenetic and ontogenetic relationships between the mammals and birds. A: Amniote phylogeny. The Synapsida (mammalian) group contains the monotremes, marsupials, and eutherians. The sauropsida (reptilian and avian) group contains the birds, crocodiles (omitted), turtles, liz- ards, snakes (omitted), and tuatara (omitted). B: Amniote ontogeny, showing four early stages: mature oocyte before fertilization, early cleavage, blas- namics tula, and gastrula. The monotremes, the reptiles, and the birds have similar oocyte organization, with yellow and white yolk. Marsupial eggs have y white 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. Marsupial embryos 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 marsupial embryos (blastocysts) are unilaminar, and by late blastula-stage pluriblast cells become morphologically distinguishable and will give rise to epiblast and hypoblast cells. Eutherian blastulae have mural trophectoderm cells being morphologically distinct from polar trophectoderm and inner cell mass mental D cells. The polar trophectoderm cell lineage is a eutherian invention, but is not present in all eutherian mammals. A two-layered structure (an upper p epiblast and a lower hypoblast) is conserved in reptilian/avian, monotreme, and marsupial early gastrulae. TE, trophectoderm; PrE, primitive endoderm/hypoblast; ExEm, extraembryonic. Develo Behringer, 2004) (Fig. 1B). Moreover, 1975; Eakin and Behringer, 2004), and ment may provide useful insight into at late blastula/early gastrula stages, formed but quickly lost in the rabbit, the variable and conserved features of monotreme and marsupial embryos pig, cattle, dog, cat, and many other mammalian early development. do not have their pluripotent cell pop- species (Williams and Biggers, 1990; ulation (epiblast/pluriblast) covered Flechon et al., 2004; Vejlsted et al., by a polar trophectoderm (Rauber’s) 2005; Blomberg et al., 2008; Hassoun OVERVIEW OF layer present in most eutherian mam- et al., 2009; Wolf et al., 2011). INTRAUTERINE CHICK mals and defined as part of the tro- Two conclusions can be drawn from DEVELOPMENT phectoderm layer which surrounds above-discussed phenotypic diversity and is in close association with the seen in mammalian early develop- Avian oviduct has five functional inner cell mass/epiblast (Flynn and ment. First, the “preimplantation” regions: the infundibulum, magnum, Hill, 1947; Selwood, 1992; Selwood development from cleavage to late isthmus, uterus and vagina. After and Johnson, 2006; Frankenberg blastula stage in basal extant ovulation, a mature chicken oocyte is et al., 2013) (Fig. 1B). Instead, monot- mammalian lineage (monotremes) is fertilized at the infundibulum. The remes and marsupials have a single- morphogenetically similar to the fertilized oocyte passes through the cell thick epiblast layer in direct con- intrauterine development in the magnum which secrets egg white, tact with the zona pellucida and a thin chick. Second, the “preimplantation” isthmus which makes soft shell and layer of hypoblast underneath it, simi- development in many therian mam- uterus which adds hard shell before lar to the organization known in a mals differs from that in the mouse, oviposition induced by vaginal muscle postovipositional chick embryo. Of the conventional mammalian model. contraction. The entire process takes interest, the polar trophectoderm is Given that the monotremes are not approximately 25 hr. The first cleav- not a conserved structure in the likely to become technically amenable age starts approximately 5 hr after eutherian clade. It is absent in the ten- models for embryological studies, fertilization (Olsen, 1942; Gipson, rec and elephant shrew (Wimsatt, knowledge of day-1 chick develop- 1974), and the rest of preovipositional 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 early chick 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 the developmental processes. namics y development (20 hr) takes place in epiblast and a lower discontinuous age period of intrauterine develop- the uterus. Chick development is tra- layer of hypoblast cells. The periph- ment: (1) How do open blastomeres ditionally described according to two eral cells, precursors to the area cellularize and are there maternal staging systems, the Hamburger and opaca, remain multilayered and main- determinants allocated asymmetri- Hamilton (HH) and the Eyal-Giladi tain close contact with the underlying cally during the cellularization pro- mental D and Kochav (EGK) (Eyal-Giladi and yolk cell. cess? (2) When does zygotic gene p Kochav, 1976; Kochav et al., 1980; Aside from these two publications activation start and what is the equiv- Hamburger and Hamilton, 1992; Bel- from Eyal-Giladi and colleagues (Eyal- alent stage of mid-blastula transition? lairs and Osmond, 2005; Wong et al., Giladi and Kochav, 1976; Kochav (3) What are the cellular and molecu-

Develo 2013), the latter of which is used for et al., 1980), earlier work on intrauter- lar mechanisms regulating initial sep- intrauterine embryos (Fig. 2). ine development from other authors aration of inner and outer layers and Eyal-Giladi and Kochav divided the was summarized in their first staging later increase in cell layer number? prestreak development (HH stage 1) paper (Eyal-Giladi and Kochav, 1976). Concerning the a.p. formation into 14 stages (denoted by roman Work published afterward will be dis- period of intrauterine development: numerals as EGK-I to -XIV) (Eyal- cussed in appropriate sections below. (4) When is the A-P polarity first Giladi and Kochav, 1976; Kochav established in the blastoderm? (5) et al., 1980). The intrauterine period What is the main mechanism driving covers EGK-I to -X. EGK-I to -VI are KEY ISSUES OF cell-layer reduction and what is the collectively called “cleavage” stages INTRAUTERINE CHICK earliest step in epiblast/hypoblast cell and take approximately 10 hr. EGK- DEVELOPMENT IN NEED OF lineage segregation? (6) Is the yolk VII to -X are collectively called cell a syncytium and, if so, does it FURTHER CLARIFICATION “formation of the area pellucida”(a.p. play a role in early patterning and lin- formation) stages and take another 10 Compared with other vertebrate mod- eage segregation? hr (Fig. 2). Early divisions are mero- els (zebrafish, Xenopus, and mouse), blastic. Cellularization and subgermi- very little is known about the cell and Cellularization and Maternal nal cavity formation start from molecular biology of early develop- Determinants between EGK-II to EGK-III. The blas- ment in the chick. Such knowledge toderm becomes multilayered in the would enable us to gain a glimpse of The first cell to complete the cellulari- center from EGK-III and reaches its the ancestry of and diversity in mam- zation process, i.e., with “basal” sepa- thickest (5–6 cell layer thick) at EGK- malian and amniote pregastulation ration from the yolk, does so at EGK- VI, after which the number of cell development. In the rest of this com- II to -III, when the embryo has more layers in the area pellucida decreases mentary, I will list and discuss sev- than 16 “apical-laterally” enclosed gradually to 1–2 at EGK-X, with an eral key issues in need of further cells (Eyal-Giladi and Kochav, 1976; upper continuous, single cell-layered clarification. Concerning the cleav- Bellairs et al., 1978; Kochav et al., 360 SHENG

1980; Lee et al., 2013). This process below), are fundamentally linked to hypothesis is uncertain because artifi- continues until the end of the cleav- the cellularization process. cial tilting of the blastoderm after age phase (EGK-VI), when all cells, Whether maternal determinants EGK-VI can reorient embryonic D–V including the most peripheral ones, play a role in early patterning in the axis with high efficiency (Kochav and are cellularized. Gipson (Gipson, chick embryo is still debated. Specifi- Eyal-Giladi, 1971) and multiple D–V 1974) and Bellairs et al. (1978) cation of chicken primordial germ axes can be experimentally induced in observed three types of furrow mor- cells was shown to correlate with postovipositional embryos (Lutz, phology associated with early clea- asymmetric distribution of mater- 1949; Spratt and Haas, 1960; Bertoc- vages: V- or U-shaped shallow furrow, nally deposited Vasa-positive struc- chini et al., 2004; Bertocchini and the pendulum-shaped deep furrow tures (Tsunekawa et al., 2000). Stern, 2012). An alternative explana- and slit-shaped smooth and tight fur- Embryonic dorsoventral (D–V) pat- tion, reconciling both the ooplasm- row (Fig. 3A). Both the shallow and terning, corresponding to anteropos- mediated predetermination hypothe- pendulum-shaped furrows have terior (A–P) polarization of the sis and the regulative nature of D–V numerous furrow wall protrusions, blastoderm, in quail and chick axis formation at a.p. formation and likely reflecting the process of new embryos was hypothesized to be regu- postovipositional stages, is that membrane addition similar to what is lated by asymmetric distribution of maternal asymmetry determinants known during the cellularization pro- different ooplasms at early cleavage may be localized not in dividing blas- cess in Drosophila (Lecuit and Wie- stages (Callebaut, 1987, 2005). tomeres during cellularization, but in schaus, 2000) and in meroblastic Ooplasmic materials surrounding the yolk cell cortex after cellularization, cleavages in zebrafish (Li et al., 2006) germinal vesicle, the oocyte nucleus, as reported in pregastrula zebrafish and squid (Arnold, 1969) embryos. are heterogenous. Callebaut and col- embryos (Carvalho and Heisenberg, The pendulum-shaped furrow has an leagues classified them into four 2010). Molecular nature of the puta- elaborate structure at its base called types: the a, b, g, and d ooplasms tive maternal determinants in either furrow base body, which in addition to based on histochemical staining and scenario (blastomere-inherited or yolk supplying new membrane may function radiolabeling data, and suggested cortex-inherited) is unknown. as an equivalent of midbody in driving that asymmetric inheritance of the g namics furrow deepening (Gipson, 1974) or in and d ooplasms during cleavage y controlling the transition from vertical stages provides positional cues for Zygotic Gene Activation to horizontal furrow burrowing subsequent A–P polarization. The g (ZGA) and Mid-blastula (Bellairs et al., 1978). The slit-shaped ooplasm is derived from the perinu- Transition (MBT) furrow represents cell–cell contacts clear mitochondria-rich “ticos” (thy- being established while the cellulariza- midine-incorporating cytoplasmic ZGA occurs in two waves in verte- mental D tion process is still in progress, a organelles) and the d ooplasm is the brate embryos (Tadros and Lipshitz, p phenomenon similarly observed during cytoplasmic material on the surface of 2009): an early/minor wave as the zebrafish meroblastic cleavage and the nucleus of Pander, the white yolk earliest sign of ZGA and a late/major Drosophila cellularization. substance located below the germinal wave representing a robust and large-

Develo Cytokinesis lags behind nuclear vesicle. The g and d ooplasms repre- scale increase in ZGA. In the zebra- division, but most cyokinetic events sent more “primitive” cytoplasmic fish, the minor ZGA starts at cleavage are associated with zygotic mitosis. materials than the yellow yolk which stages (64-cell stage) and the major The exceptions are the pseudo- is deposited during late stages of one at early blastula stage (Mathavan cleavage furrows (not discussed here) oocyte maturation. Although it is et al., 2005). In the mouse, the minor induced by supernumerary sperm plausible from an evo-devo perspec- ZGA starts at one-cell stage and the nuclei (Lee et al., 2013) and the edge tive that maternal determinants in an major one at two-cell stage (Wang and cells. It is unclear whether the edge avian egg are localized to these Dey, 2006); whereas in human, the cells complete the cellularization pro- ooplasms, it is unclear whether or minor ZGA starts at two-cell stage cess with no further nuclear division how asymmetric inheritance of these and the major ZGA at the four- to (thus leaving no nucleus to the ooplasms determines the A–P polar- eight-cell stage (Vassena et al., 2011; remaining yolk cell) or with an addi- ity. During EGK-I to -IV, the center of Xue et al., 2013, Yan et al., 2013). tional round of nuclear division (leav- cell proliferation, where the first two Timing of ZGA onset is influenced by ing syncytial nuclei in the yolk cell). cleavage furrows meet and where the nucleocytoplasmic ratio, maternal Cell biology of meroblastic cleavage in smallest blastomeres concentrate, is clock, transcript abortion, and epige- the chick (as outline above) and in often located off the geometric center netic modification (Tadros and Lip- other model systems (Mitchison et al., of the embryo. Dividing blastomeres shitz, 2009), the first two of which are 2012) is still very poorly understood. may therefore inherit ooplasm mate- related to oocyte organization, early Many important features of a young rials asymmetrically as they cellular- cleavage pattern and developmental chick embryo, such as the presence of ize (Fig. 3B). Like the amphibian speed. In this respect the chick a subgerminal cavity, separation of Nieuwkoop center, this ooplasm embryo resembles more the fish and the inside and outside environment, asymmetry may lead to signaling frog embryos than the mouse or polarization of early blastomeres, and asymmetry which later determines human embryos. Radioisotope label- possible asymmetric distribution of the future dorsal side at posterior end ing (Wylie, 1972) and electron micros- maternal determinants (discussed of the blastoderm. Significance of this copy (Raveh et al., 1976) studies, DAY-1 CHICK DEVELOPMENT 361 namics y mental D p Develo

Fig. 3. Cellularization, maternal determinants, and formation of cell layers. A: Model of cytokinesis. Early furrows are u/v-shaped (without imme- diate formation of cell–cell junctions) or slit-shaped (with junctional interactions). The pendulum-shaped furrows are at an intermediate stage. Fur- row 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 tight junction 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. Direct cause: 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 which with smaller surface area may be biased to become an inner cell.

using techniques which are quite in the chick starts at mid-cleavage the equivalent of mid-blastula transi- insensitive by modern-day standards, stages (EGK-II to -IV) and the major tion (MBT) in Xenopus. Cell-counting suggested that there is no prominent ZGA at late cleavage stages (EGK-IV analysis revealed that an EGK-V ribosomal RNA synthesis before EGK- to -VI). The juncture between the embryo contains approximately 2,400 III. One may predict based on these cleavage period and the a.p. formation cells and an EGK-VII embryo has lines of evidence that the minor ZGA period, EGK-VI, can thus be viewed as 36,000 cells (Park et al., 2006). 362 SHENG

Although divisions are known to be erating two outer daughters, can sec- shell is rotated along its long axis in asynchronous after the 4th cleavage ondarily give rise to inner cells the uterus (10–15 times per hr counter (Perry, 1987), these numbers translate through postdivision modifications of clock-wise if viewed from the isthmus into approximately the 11th cell cycle cell movement and cell adhesion (Par- side) with no matching rotation of the at EGK-V and the 15th cell cycle at fitt and Zernicka-Goetz, 2010). In Xen- egg yolk, resulting in the formation of EGK-VII, making it plausible that an opus, inner cell population forms coiled chalazae (two strands of dense MBT-like event occurs at EGK-VI. The through a similar parallel-to-surface egg white materials connecting the increase in cell number proceeds at a orientation of mitotic planes after 64- vitelline membrane around the yolk much slower rate afterward, reaching cell stage, while orthogonal-to-surface with the outer thick albumen under- approximately 54,000 at EGK-X (Park orientation of the mitotic plane gener- neath the soft shell) and a constant tilt et al., 2006), probably due to the ates two surface daughter cells of the blastoderm relative to its gravi- lengthening of cell cycle duration in (Chalmers et al., 2003). A third type tationally dictated position (Fig. 4A). the second half of intrauterine devel- observed in Xenopus, the oblique divi- The highest point of the blastoderm opment (Emanuelsson, 1965). sion, produces one daughter with will eventually become the posterior smaller surface area which is strongly side (the so-called von Baer’s rule). Increase in the Number of biased to undergo asymmetric divi- Results from artificial tilting of EGK- sion in the next cell cycle (Chalmers VI to -X embryos supported this Cell Layers et al., 2003). In the chick, horizontal hypothesis (Kochav and Eyal-Giladi, Blastomeres located in the middle (parallel-to-surface) orientation of the 1971; Eyal-Giladi and Fabian, 1980; complete the cellularization process cleavage plane was also hypothesized Eyal-Giladi, 1991), suggesting that at EGK-II to -III, and further cell divi- to be the cause for layer increase molecular determinants of radial- sions result in the formation of a mul- (Kochav et al., 1980), although there symmetry breaking is sensitive to tilayered organization in central is no direct evidence either in favor of gravitational cues at late cleavage and blastoderm, approximately two cells or against this model. early a.p. formation stages. These thick at EGK-III to -IV and reaching a observations do not rule out the possi- maximum of five to six cells thick at bility of radial-symmetry breaking namics EGK-VI. More peripheral regions A–P Polarity of the being determined or biased by pronu- y undergo a similar, but delayed and Blastoderm (Radial clear fusion point or maternal ooplasm less pronounced, process of layer segregation (Fig. 3B), but they do Symmetry Breaking) increase. The most peripheral part of strongly argue in favor of a regulative the blastoderm is always one-cell Although often described as such, mechanism underlying initial radial- thick. When a centrally located blas- chick embryos obtained from freshly symmetry breaking. The molecular mental D tomere separates itself from the yolk laid eggs are not radially symmetri- nature of the earliest determinants of p by forming a horizontal “basal” mem- cal. At EGK-X, morphological asym- A–P axis formation, either maternally brane, the yolk counterpart is anu- metry along the A–P axis of the biased at early cleavage stages or clear. Thus the increase in layer blastoderm is apparent with the gravitationally regulated at early a.p.

Develo number is the outcome of either rear- asymmetric distribution of hypoblast formation stages, is unknown. rangement or proliferation of already islands, and molecular asymmetry is cellularized blastomeres (Fig. 3C). also readily detectable in both the epi- Layer Reduction and Lineage Changing from a mono-layered to a blast and hypoblast cells (Peter, 1938; Segregation bi-layered structure produces an Spratt and Haas, 1960; Eyal-Giladi inside cell population sealed off from and Kochav, 1976; Sheng and Stern, From EGK-VII, the number of cell the external milieu. A similar process 1999; Bertocchini and Stern, 2012; layers in the blastoderm decreases in the mouse initiates the separation Alev et al., 2013b). At the beginning from initial ﬁve to six at EGK-VI to of cell types at the morula stage (Ste- of the a.p. formation period, EGK-VII, one to two at EGK-X. This process phenson et al., 2012; Hirate et al., initiation of layer reduction has been starts from the central-posterior 2013). Asymmetric cleavage during proposed to start from the posterior region of the blastoderm and spreads the transition from 4-cell to 16-cell side of future area pellucida (Clavert, to all areas above the subgerminal stage generates inside and outside 1960; Kochav et al., 1980; Watt et al., cavity, but not to the area opaca. Cell daughters, whereas symmetric cleav- 1993). As in most telolecithal eggs, shedding was proposed to be the age produces two outside or inside the abembryonic side of chicken yolk cause for layer reduction (Kochav cells (Sasaki, 2010). The type of divi- is heavier than the embryonic side, et al., 1980, Fabian and Eyal-Giladi, sion can be predicted based on the ori- and a developing embryo positions 1981) (Fig. 4C). Radial intercalation, entation of mitotic plane: those itself on top of the yolk if left undis- as an alternative although not mutu- parallel to the surface of the morula turbed. Vintemberger and Clavert, in ally exclusive mechanism, may also being asymmetric (by generating an a series of papers published in 1950s play a role in this process (Fig. 4C). inner, inner cell mass-biased daughter and 1960, proposed that bilateral During the process of a.p. formation, and an outer, trophectoderm-biased symmetry (i.e., A–P polarity of the the central-most, thickest area takes daughter) and those orthogonal to the blastoderm and D–V polarity of the longer time to appear translucent surface being symmetric. Symmetric embryo) is established by egg rotation than the rest of the blastoderm division of a surface blastomere, gen- in utero (Fig. 4A). The forming egg located above the subgerminal cavity. DAY-1 CHICK DEVELOPMENT 363 namics y mental D p Develo

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 salt- and-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 posterior marginal zone, located baut et al., 1998; Stern and Downs, in the turkey (Gupta and Bakst, between the Koller’s sickle (a 2012). This aspect of cell-layer reduc- 1993) and an equivalent structure, crescent-shaped lower layer cell popu- tion is not discussed in detail here. although not named, is also present lation closely associated with the Two general cell-biological features in the chick (Kochav et al., 1980). overlying epiblast) and the posterior of the area pellucida at EGK-X, how- Eventually, at EGK-X, most regions of germ wall. This part of the area pellu- ever, deserve further discussion. the area pellucida, including the area cida is often more than two cells First, the one-cell thick areas have alba, become 1–2 cell thick, whereas thick, and the dynamic nature of its only the upper, epiblast layer; while the area apaca remains multilayered morphogenesis and signaling is the two-cell thick areas contain an (Kochav et al., 1980; Andries et al., among the most prominent and upper epiblast layer and a lower loose 1983; Gupta and Bakst, 1993; Watt debated issues of early postoviposi- aggregate of primary hypoblast cells et al., 1993). A notable exception is tional development (Eyal-Giladi, (hypoblast islands). Epiblast cells in the region of the area pellucida at the 1991; Eyal-Giladi et al., 1992; Calle- one-cell thick areas are polarized with 364 SHENG

signs of basement membrane deposi- blastocyst formation and the hypo- rounding the remaining yolk, after tion, whereas epiblast cells in two-cell blast cells are generated from the the completion of cellularization at thick areas do not have a basement epiblast/pluriblast by ingression, pre- EGK-VI, is a syncytium or not. In the membrane and lag behind in the epi- sumably from both the middle and bor- zebrafish, yolk syncytium forms by thelialization process (Bortier et al., der regions of the epiblast/pluriblast fusion of the yolk cell and deep layer 1989; Harrisson et al., 1991; Lawson (Selwood, 1992; Kress and Selwood, marginal blastomeres, and plays criti- and Schoenwolf, 2001; Nakaya et al., 2006; Frankenberg et al., 2013). In cal roles in embryonic induction and 2008; Nakaya et al., 2011, 2013). Sec- eutherian mammals, hypoblast and morphogenesis (Carvalho and Heisen- ond, primary hypoblast islands have epiblast cells separate from each other berg, 2010). In monotremes, the yolk an anterior-sparse/posterior-dense from an initially mixed inner cell mass cell at late blastula is also a syncy- distribution (Kochav et al., 1980; Watt population and before the epiblast tium (Flynn and Hill, 1947). In the et al., 1993), opposite to the initiation cells adopt an epithelial structure chick, there are three possible sources of layer reduction, which starts from (Stephenson et al., 2012; Xenopoulos of zygotic nuclei which may contrib- the posterior side. The initiation of et al., 2012). It, therefore, remains pos- ute to the formation of a yolk syncy- cell shedding or intercalation and the sible that hypoblast precursor cells in tium: the most peripheral part of the speed of its continuation may, there- the chick are initially located in a salt- blastoderm, deep blastomeres in the fore, be regulated separately. and-pepper manner in the multilay- germ wall, and shed blastomeres in The epiblast was hypothesized to be ered blastoderm (including in the the area pellucida (Fig. 4D). reduced to one cell thick, and the pri- upper-most layer) and secondarily At cleavage stages, peripheral open mary hypoblast cells were derived by aggregate or ingress during the layer cells are in contact with the yolk both means of poly-ingression from the reduction/lineage sorting process. basally and peripheral-laterally. upper, epiblast layer cells (Peter, The epiblast and hypoblast layers Nuclear division in these cells gener- 1938; Fabian and Eyal-Giladi, 1981; will give rise to different embryonic ates a proximally located daughter Eyal-Giladi, 1991; Watt et al., 1993). and extraembryonic cell types later in which cellularizes like other centrally Support for this model is indirect (bot- development. Cellular changes lead- located blastomeres and a peripher- tle-shaped cells spanning both layers) ing to their formation (i.e., a.p. forma- ally located one which remains as an namics and pregastrulation development in tion and layer reduction) take place open cell. Cycles of nuclear division y the chick does not pass through any concomitant with changes in gene and cytokinesis reduce the size of the stage with a one-cell thick epiblast expression profiles. By the time a open cell successively. If the last cellu- without associated hypoblast cells. A chick embryo reaches EGK-X, at least larization step takes place before plausible interpretation is that pri- three cell populations with distinct nuclear division, no daughter nucleus mary hypoblast cells are “left-overs” molecular signatures have been is left in the yolk cell; and if it hap- mental D of the layer reduction process. An delineated: the epiblast, hypoblast, pens after nuclear division, each p incompletely epithelialized epiblast, and the area opaca cells (Fig. 4B). peripheral-most cell will leave a with no basement membrane and Each population has robust expres- daughter nucleus in the yolk cell. The labile epithelial junctions, can still sion of lineage-specific markers, many second source is the blastomeres

Develo accommodate accretion of cells from of which have been well-characterized located close to the yolk cell mem- the lower layer, resulting in bottle- in mammalian models, such as brane. From EGK-III, a subgerminal shaped cell morphology seen in sec- Nanog, PouV, Dnmt3B, and Lin28 in cavity forms in central blastoderm tions or by surface labeling. Instead, a epiblast (Lavial et al., 2007; Shin between the yolk cell membrane and well-epithelialized epiblast with form- et al., 2011; Alev et al., 2013a), Hex in cellularized central blastomeres. This ing basement membrane will resist hypoblast (Yatskievych et al., 1999), cavity expands as cellularization pro- further cellular addition, leaving non- and Eomes in area opaca cells (Per- gresses peripherally. The expansion, epithelialized lower layer cells as pri- naute et al., 2010). It is unclear how however, lags behind the cellulariza- mary hypoblast cells. The difference early these lineage specific genes ini- tion process, and the yolk cell stays in in hypoblast island density along the tiate their expression and whether close contact with peripheral deep A–P axis could, therefore, be simply their expression precedes or follows layer blastomeres even after complete due to a difference in the rate of epi- morphological changes of these three cellularization. This area of contact is thelialization of the uppermost layer cell populations (Fig. 4B). It is also maintained during early development during the process of layer reduction. worth noting that instead of defining and is called the germ wall. It is Viewed from an evolutionary per- cellular fates, specific expression of unclear whether some of the deep spective, however, the polyingression lineage markers at these early stages layer cells in the germ wall can fuse hypothesis still deserves to be tested likely marks cellular states, the labile with the yolk cell as seen in the zebra- carefully. In monotremes, the multi- and dynamic bias toward these cellu- fish. The inner margin of the germ layered blastoderm thins out into a lar fates. wall is a dynamic, shifting boundary single-layered structure, from which between the area pellucida and area the hypoblast cells are thought to Yolk Syncytium and opaca. It is also an important center come (Flynn and Hill, 1947), although for early patterning. It is unclear Blastoderm-Yolk Contact direct evidence is also lacking in this what roles the yolk cell plays in the case. In marsupials, the blastoderm is It is yet to be determined whether the maintenance of this boundary or in single-layered from the beginning of membrane enclosed structure sur- its associated molecular signaling. DAY-1 CHICK DEVELOPMENT 365

Deep cells in the area pellucida do hormonally induced retrieval method understanding of early development not contact the yolk cell membrane. (Olsen, 1942; Watt et al., 1993) and in diverse mammalian species. However, area pellucida cells that are direct sacrifice of mother hens (Ema- “shed” during the process of layer nuelsson, 1965; Gipson, 1974; Bellairs reduction drop to the floor of the sub- et al., 1978). Only eggs with soft or ACKNOWLEDGMENTS germinal cavity and associate with hard shell (from four-cell stage I thank Dr. Gary Schoenwolf for the the yolk cell tightly (Andries et al., onward) can be obtained through the invitation to write this commentary, 1983). These shed cells are also dis- first two techniques; while the third and Drs. Claudio Stern, Patrick Tam, tributed asymmetrically along the A– one can be used to obtain eggs from Jae Yong Han, members of Laboratory P axis. Whether some of them fuse fertilization to one- to two-cell divi- for Early Embryogenesis, and the with the yolk cell and whether they sion stages. Retrieved eggs can be cul- reviewers for critical comments. play any role in the function of yolk tured in vitro, with or without the syncytium are unknown. Electron shell, to postovipositional stages with REFERENCES microscopy studies of EGK-X to -XIV good survival rates (Vintemberger Alev C, Nakano M, Wu Y, Horiuchi H, chick and quail embryos (Andries and Clavert, 1960; Kochav and Eyal- Sheng G. 2013a. Manipulating the avian et al., 1983) indicate that the cortical Giladi, 1971; Perry, 1988; Naito and epiblast and epiblast-derived stem cells. regions of the yolk cell on the floor of Perry, 1989; Naito et al., 1990, 1995). Methods Mol Biol 1074:151–173. the subgerminal cavity are rich in Embryos collected from intrauterine Alev C, Wu Y, Nakaya Y, Sheng G. 2013b. Decoupling of amniote gastrulation and mitochondria, microfilaments, and eggs can be used directly for tran- streak formation reveals a morphoge- membrane invaginations and protru- scriptomics/proteomics investigations, netic unity in vertebrate mesoderm sions, suggesting that yolk cell cortex fixed for gene expression and protein induction. Development 140:2691–2696. and its associated cellular and molec- localization studies, or cultured in Andries L, Vakaet L, Vanroelen C. 1983. The subgerminal yolk surface and its ular activities are an integral part of vitro for labeling, lineage tracing, and relationship with the inner germ wall avian early development. functional analyses. Finally, an alter- edge of the stages X to XIV chick and native approach to circumvent the quail embryo. A SEM study. Anat limited access of intrauterine chick Embryol (Berl) 166:453–462. TECHNICAL LIMITATIONS Arnold JM. 1969. Cleavage furrow forma- namics embryos may be to use embryos from y AND POSSIBLE SOLUTIONS tion in a telolecithal egg (Loligo pealii). other avian species. The turkey I. Filaments in early furrow formation. The chick is a mature developmental (Gupta and Bakst, 1993) and zebra J Cell Biol 41:894–904. biology model. Tools for RNA/protein finch (unpublished data) lay their Bellairs R, Lorenz FW, Dunlap T. 1978. localization, cell labeling/tracing, eggs at much earlier stages than the Cleavage in the chick embryo. J Embryol Exp Morphol 43:55–69. imaging, overexpression/knockdown, chick. Both species have their genome

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