RESEARCH ARTICLE

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Characterization of the finch embryo
supports evolutionary conservation of thenaive stage of development in amniotes
Siu-Shan Mak1, Cantas Alev2†, Hiroki Nagai2†, Anna Wrabel1,2†, Yoko Matsuoka1,
Akira Honda1, Guojun Sheng2*, Raj K Ladher1,3*
1

Laboratory for Sensory Development, RIKEN Center for Developmental Biology,
Kobe, Japan; 2Laboratory for Early Embryogenesis, RIKEN Center for Developmental
Biology, Kobe, Japan; 3National Center for Biological Sciences, Bengaluru, India

Abstract Innate pluripotency of mouse embryos transits from naive to primed state as the innercell mass differentiates into epiblast. In vitro, their counterparts are embryonic (ESCs) and epiblaststem cells (EpiSCs), respectively. Activation of the FGF signaling cascade results in mouse ESCs
differentiating into mEpiSCs, indicative of its requirement in the shift between these states.
However, only mouse ESCs correspond to the naive state; ESCs from other mammals and from chickshow primed state characteristics. Thus, the significance of the naive state is unclear. In this study, we
use zebra finch as a model for comparative ESC studies. The finch blastoderm has mESC-like
properties, while chick blastoderm exhibits EpiSC features. In the absence of FGF signaling, finch
cells retained expression of pluripotent markers, which were lost in cells from chick or aged finch
epiblasts. Our data suggest that the naive state of pluripotency is evolutionarily conserved among
amniotes.
*For correspondence:
sheng@cdb.riken.jp (GS);rajladher@ncbs.res.in (RKL)†
These authors contributed
equally to this work

Competing interests: The
authors declare that nocompeting interests exist.
Funding: See page 14
Received: 24 February 2015
Accepted: 10 September 2015
Published: 11 September 2015
Reviewing editor: AlejandroSánchez Alvarado, Stowers
Institute for Medical Research,
United States
Copyright Mak et al. This
article is distributed under theterms of the Creative Commons
Attribution License, which
permits unrestricted use andredistribution provided that theoriginal author and source arecredited.

DOI: 10.7554/eLife.07178.001

Introduction
The successful isolation and in vitro culture of embryonic stem cells (ESC) from mouse embryos haveenabled technological breakthroughs and revolutionized our understanding of the molecularmechanisms regulating mammalian development (Evans, 2011). However, similar applications toother species have been lacking. One conceptual difficulty has been linking the innate pluripotency ofthe embryo with the characteristics of cultured stem cells, raising the speculation that mESCs may besolely a result of in vitro manipulations (Pauklin et al., 2011). In the mouse, ESCs are at naive state(Nichols and Smith, 2009), which recent evidence suggests is most similar to cells from embryonicday (E) 4–4.5 of mouse development (Boroviak et al., 2014). Cells taken from this stage can give rise
to derivatives of all three germ layers as well as germ cells. These cells, both in their native naive stateand in culture, express genes associated with pluripotency such as Oct3/4 (Pou5f1), Sox2, and Nanog (2).
Using defined media that includes the presence of either an inhibitor of FGF signaling or its
downstream Erk/MAP kinase transduction pathway, mouse ESCs (mESCs) can be propagated while
maintaining the expression of these pluripotency markers (Ying et al., 2008). A second pluripotent
cell type in the mouse, epiblast stem cells (mEpiSCs), is derived from embryos that are later indevelopment (E5.5) and is in what has been termed, the primed state (Brons et al., 2007). These cells
have a more limited potency and require different culture condition for in vitro propagation (Lanner
and Rossant, 2010), with a dependency on FGF-mediated ERK activation for the maintenance ofpluripotent gene expression. Pluripotent ESCs from other mammalian organisms, such as human(Thomson et al., 1998; Schatten et al., 2005), and from non-mammalian amniotes, such as chick(Pain et al., 1996), share this requirement for ERK signaling (Tesar et al., 2007). Hence, the primed

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eLife digest In animals, stem cells divide to give rise to other cells that have specialized roles inthe body. ‘Pluripotent’ stem cells—which are able to produce cells of any type—can be obtained
from young mouse embryos. Once grown in the laboratory, these cells are called naive embryonicstem cells (ESCs) and their discovery has been vitally important for understanding how mammals
develop. ESCs also have considerable medical potential because they could be used to repair or
replace tissues that have been lost to injury or disease.
A family of proteins called fibroblast growth factors (FGFs) triggers naive ESCs to mature intoanother class of stem cell that are ‘primed’ to only produce particular types of cells. Curiously, thestem cells that have been collected from other mammal embryos are already in this primed state.
Therefore, biologists wonder whether the naive state is exclusive to mice embryos, or whether it ispresent in other animals but has so far remained undetected.
The development of chick and other bird embryos shares many parallels with that of mammals.
However, embryos in chicken eggs do not contain naive ESCs. It is possible that this is due to chicken
eggs being laid when the embryos have reached a later stage in development where the naive stem
cells have already matured into the primed cells. Here, Mak et al. compared the stem cells in chickembryos to those from another bird called the zebra finch.
The experiments show that the finch embryos contain stem cells that share several features withmouse ESCs. In particular, these finch cells continue to express genes that are required for the naivestate to be maintained in the absence of FGF. On the other hand, these genes are switched off incells from chick embryos and in older zebra finch stem cells.
Mak et al.’s findings show that finch eggs are laid at an earlier stage of embryo development thanchicken eggs. The experiments also suggest that both birds and mammals have naive pluripotent
stem cells during the early stages of embryo development. In future, the zebra finch could be used asa model to study stem cells and other aspects of animal development.
DOI: 10.7554/eLife.07178.002

state of pluripotency is evolutionarily conserved in mammalian and non-mammalian amniotes.
However, the naive state has so far only been confirmed in the mouse (Ying et al., 2008) and rat(Buehr et al., 2008; Li et al., 2008; Chen et al., 2013b), raising the possibility that this state is notconserved among the amniotes. More recent reports suggested that with specific reprogramming
factors and culture conditions such a naive state may also exist for human ESCs, although the exact
nature of these naive-type human cells is under debate (Takashima et al., 2014; Theunissen et al.,2014; Ware et al., 2014). Identifying the naive state of embryogenesis in other species is therefore
central to our conceptual understanding of pluripotent stem cells.
A comparative embryology approach to address this question should include non-mammalianamniotes. These include avian species, which share key molecular and cellular features of epiblast
morphogenesis with the mammals (Sheng, 2014), yet are evolutionarily distant enough to serve as an
outgroup. As in all amniotes, fertilization of avian oocytes takes place internally and avian embryos
undergo some development prior to egg-laying (oviposition). The most widely used avian developmental
models are chicken (Gallus gallus), quail (Coturnix japonica), and zebra finch (Taeniopygia guttata).
However, chicken embryos at oviposition are already at a late blastula/early gastrulation stage (EyalGiladi and Kochav (EGK) stage X) (Eyal-Giladi and Kochav, 1976). These embryos have a columnar
epithelialized epiblast overlying scattered hypoblasts and are thus morphologically similar to E5.5 mouseembryo, later than the stage at which mESCs can be derived. The Japanese quail embryos are laid at astage later than the chicken embryos (Sellier et al., 2006), while the ovipositional stage of zebra finch
embryos is unclear. Early-staged avian embryos can generate cells that show some of the characteristics
of mammalian ES cells (Jean et al., 2015) after the introduction of reprogramming factors (Rossello
et al., 2013; Dai et al., 2014) or after the manipulation of culture conditions (Pain et al., 1996; Jeanet al., 2013). However, it was not clear how the pluripotency generated be exogenous factors related tothe pluripotent state of cells in the embryo. We decided to investigate the early development of thezebra finch (hereafter referred to as the finch) in more detail. The finches are a model system commonlyused in neurobiological studies of social behavior (Brazas and Shimizu, 2002; Svec et al., 2009),
vocalization, and learning (Jarvis, 2004; Petkov and Jarvis, 2012). These studies have led to an

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increased focus on the developmental neurobiology of zebra finch (Charvet and Striedter, 2009; Chenet al., 2013a), and in turn the embryology of the finch (Murray et al., 2013). In addition, the finch
genome has been sequenced (Warren et al., 2010) and their relatively small adult body sizes (4–7 times
smaller than adult chickens) and shorter generation time (2–3 months for finches vs 4–6 months for the
chickens) makes it feasible to breed them within a normal laboratory setting. Thus, we asked whether
finch embryos could be used for early embryogenesis and ESCs studies, complementing existing studies(Rossello et al., 2013), which together may lead to potential technological breakthroughs that facilitate
genetic and functional investigations in this model organism.
Here, we report the first molecular characterization of ovipositional finch blastoderms by quantitative
RT-PCR, immunohistochemical staining, and in situ hybridization. Our results suggested that finch
embryos are laid at stage EGK-VI to EGK-VIII, much earlier than the chicken embryos, and aremorphologically more similar to the E4–E4.5 mouse embryos from which mESCs can be derived. Cells
derived from the finch blastoderm at oviposition and cultured in the presence of a MEK inhibitor and
Leukemia inhibitory factor (LIF) retained expression of the pluripotent markers; Nanog, PouV expression,and alkaline phosphatase (AP) activity. In contrast, chicken cells taken from newly laid embryos andcultured under the same conditions did not produce Nanog, PouV, or AP-positive aggregates. Our datasuggest that birds and mammals share a common regulatory mechanism in the maintenance ofpluripotency. Finch embryos are ideally suited for the establishment and characterization of avian ESCs,
and the incorporation of recent technical improvements (Dai et al., 2014) could lead to the finch
becoming a tractable avian model for genetics and regenerative medicine.

Results
Finch oviposition is at EGK-VI prior to subgerminal cavity expansion
Avian embryos undergo varying degrees of intrauterine development prior to oviposition (egg-laying).
Chick oviposition is at the late blastula/early gastrula stage (EGK-X to EGK-XI). In other Galloanserae,
oviposition ranges from EGK-VII (Turkey [Gupta and Bakst, 1993] and Duck [Sellier et al., 2006]) to
EGK-XI (the quail [Stepinska and Olszanska, 1983]). Ratite embryos are laid at EGK-X, similar to thechick embryos (Nagai et al., 2011). The oviposition stage of Passerine (songbird) species has not been
carefully investigated, although gross morphology of newly laid embryos of the zebra finch and society
finch (Bengalese finch) suggested that they are younger than EGK-X (Yamasaki and Tonosaki, 1988;
Agate et al., 2009; Murray et al., 2013). Due to the difficulty in retrieving pre-ovipositional (<EGK-X)
chick embryos (Nagai et al., 2015), we decided to further explore the potential advantages of finch
eggs and carried out a detailed morphological and molecular characterization of ovipositional finch
embryos in order to evaluate their potential use for comparative stem cell studies as well as for genome
engineering.
Finch eggs are laid in intervals of between 20 and 29 hr, with most laid following a 24- to 25-hr
periodicity. Based on the EGK staging system for pre-oviposition chick embryos (Eyal-Giladi and
Kochav, 1976), we found that finch embryos from newly laid eggs ranged from EGK-VI to EGK-VIII,
with ∼10% of eggs at EGK-VI and the rest at EGK-VII and -VIII. To probe morphological and molecular
heterogeneity of the finch blastoderm, we first used section analysis of embryos stained for Hhex, amarker for endoderm cells (Thomas et al., 1998) (Figure 1A–C). At EGK-VI (early blastula), the embryo
was composed of similar-sized blastoderm cells organized into 7–8 layers (Figure 1A). The subgerminal(blastocoel) cavity was not as apparent as in similarly staged chick embryos (Eyal-Giladi and Kochav,
1976; Sheng, 2014). By EGK-VIII (mid-blastula stage) (Figure 1B), the subgerminal cavity and the area
pellucida became apparent and the embryo was thinner at the center of the blastoderm (4–5 cell-thick)than at EGK-VI. Cells located apically, closer to the vitelline membrane (the putative epiblast
precursors) were smaller than those located closer to the yolk side (the putative hypoblast precursors).
A low level of Hhex expression was detected in the hypoblast precursors. Post-ovipositional incubationof around 6 hr yielded finch embryos at EGK-X (Figure 1C). At this stage, the epiblast and hypoblast
were clearly separated and the epiblast showed a columnar epithelial organization. In addition, strong
Hhex expression was detected in the hypoblasts.
Epithelial organization is typically maintained by adherens junctions mediated extracellularly byhomophilic interactions of E-cadherin molecules and intracellularly by adhesion complex components
such as ß-catenin (Takeichi, 2014). To understand the transformation of the epithelial morphology
between EGK-VI to EGK-X, we asked if there were changes to E-cadherin and ß-catenin localization

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Figure 1. Morphology of the finch blastoderm at oviposition. (A–C) Finch embryos were sectioned after Hhex in situ
hybridization to reveal the morphology of the blastoderm. At EGK-VI the blastoderm is thick, with no sign ofepithelialization, and does not express Hhex (A). The blastoderm is thinner at EGK-VIII but still has not overt
epithelialisation, and express low level of Hhex transcripts in the cells of the yolk side (B). Hhex expression can bedetected clearly at EGK-X and is confined to a morphologically distinct hypoblast layer (C). (D–F) Staining of Ecadherin reveals the extent of epithelialization of the finch blastoderm. At EGK-VI, most of the blastodermal cells
appear unpolarized, however, express E-cadherin (D). At EGK-VII, while thinner, the blastoderm does not show anycolumnar cells typical of epithelial organization (E). At EGK-X, clear epithelial organization can be visualized, withpolarized E-cadherin and morphological segregation in the bright-field image (F). (G, H) ß-catenin staining confirms
the lack of epithetialisation at finch oviposition stages. The pattern at EGK-VI is similar to E-cadherin staining (G) as is
the pattern at EGK-VIII (H). In A–G, scale bar represents 100 μm. (I) Schematic of the morphology of the early, mid,and late finch blastula: The late finch blastoderm is similar to the chick embryo at oviposition. Yc—yolk cells;
bc—blastocoel; sc—subgerminal cavity; epi—epiblast; hyp—hypoblast; Op—area opaca; Pe—area pellucida.
(J) Table below showing the features of the finch at early stages.
DOI: 10.7554/eLife.07178.003

consistent with epithelialization of the early embryo. At EGK-VI, both E-cadherin (Figure 1D) andß-catenin (Figure 1G) were expressed in all cells. Staining did not appear to be enriched in any
particular region of the cell and epithelialization was not apparent. By EGK-VIII, formation of an epithelial
cell-sheet was not observed although the nuclei of the outermost, putative epiblast, cells appeared moreregularly organized (Figure 1E,H). In contrast, deep, putative hypoblast, cells appeared to down-regulate
both proteins. By EGK-X, E-cadherin was up-regulated in the lateral regions of the epiblast cells

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Developmental biology and stem cells | Genomics and evolutionary biology(Figure 1F). Their epithelial morphology was further evidenced by formation of a discrete columnar
cell-sheet. Analysis of chick embryos at oviposition revealed that it was more similar to the finch embryo
after incubation (Figure 1I), sharing features with the finch late blastula (Figure 1J).

Developmental lineages are molecularly segregated at EGK-VI prior toepithelialization of the epiblast
The reorganization of the blastoderm into an epithelialized epiblast and hypoblast raised the possibility
that epiblast formation may be concomitant with acquisition of cell fate. The early blastoderm consists ofcells fated to become the epiblast and hypoblast as well as germ cells (Eyal-Giladi et al., 1981; Hatada
and Stern, 1994; Lawson and Schoenwolf, 2003). We thus asked whether these cell identities havealready been established in the finch embryo at oviposition. As oviposition ranged from EGK-VI to
EGK-VIII, we examined these stages.
In chick, the germ cell fate is marked by immunoreactivity to the stage-specific embryonic antigen
1 (SSEA-1) and epithelial membrane antigen 1 (EMA-1) antibodies (Jung et al., 2005). In finch
embryos, immunoreactivity of these proteins were observed predominantly in the epiblast precursor(Figure 2A,B,E–H), with SSEA-1 staining being stronger than EMA-1. Few hypoblast precursor cells
were positive for these markers (Figure 2C,D,E–H). In mouse embryos, Gata4 expression marks the
specification of the hypoblast/primitive endoderm (Soudais et al., 1995) and the SoxB family
member, Sox2 expression marks the epiblast precursors (Avilion et al., 2003). In chick, a different
SoxB family member, Sox3 is expressed in epiblast precursors (Rex et al., 1997). We thus used Sox3
and Gata4 to determine the presence and distribution of these cell types in EGK-VI/VIII finch embryos.
Both immunostaining and in situ hybridization confirmed that Sox3 and Gata4 could be detected at
finch oviposition and that lineage segregation between these two cell types could be distinguished

Figure 2. Molecular characterization of the finch blastoderm at oviposition. (A–D) Whole-mount staining of EGK-VIII finch embryos of the distribution of
putative germ cell markers SSEA-1 (A, B) and epithelial membrane antigen 1 (EMA-1) (C, D) showing views from the vitelline membrane side (A, C) andyolk (lower) side (B, D). (E, F) Sections of finch embryos at oviposition showing the distribution of SSEA-1 positive cells at EGK-VI (E) and EGK-VIII (F) to
predominantly the upper side of the blastoderm. (G, H) Sections of finch embryos at oviposition showing the distribution of EMA-1 positive cells at EGK-VI(G) and EGK-VIII (H) to predominantly the upper side of the blastoderm. (I, J) Sections of finch embryos at oviposition showing the distribution of SOX3
and GATA4 protein at EGK-VI (I) and EGK-VIII (J) SOX3 and GATA4 are localized to opposite sides of the blastoderm. (K) In situ hybridization of Sox3 at
oviposition in the finch blastoderm. Section analysis, summarized in the cartoon, shows transcripts are present throughout the upper layer of theblastoderm. (L, M) In situ hybridization of Sox17 together with section analysis, summarized in the accompanying cartoon, reveals expression dispersed
throughout the blastoderm at oviposition (L), while at EGK-X expression is more predominant in the hypoblast with scattered epiblast cells (M). (N) In situ
hybridization of Gata4 at oviposition in the finch blastoderm. Section analysis, summarized in the cartoon, shows transcripts are present throughout thelower layer of the blastoderm. (O, P) In situ hybridization of Gata6 together with section analysis, summarized in the accompanying cartoon, reveals
expression more predominant in the lower layer/hypoblast at oviposition (O) and in the late blastoderm at EGK-IX/X (P).
DOI: 10.7554/eLife.07178.004

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Developmental biology and stem cells | Genomics and evolutionary biology(Figure 2I–K). Sox3 was more abundant in the cells at the vitelline membrane side (Figure 2I,K) and
Gata4 more abundant in the yolk side (Figure 2J,M). To verify the specification of the endodermal
lineage, three additional markers, Gata6, Sox17, and Hhex, were investigated. In mouse, both Gata6
and Sox17 mark all of endodermal lineages (Fujikura et al., 2002; Kanai-Azuma et al., 2002). Sox17
was expressed in the hypoblast in the finch blastoderm from oviposition, with some positive cells also
detected in the epiblasts (Figure 2L). Expression was similar after epithelization at EGK-IX/X(Figure 2M). Gata6 expression was restricted to the hypoblast lineage (Figure 2O) at EGK-VII/VIII,
which is more apparent by EGK-X (Figure 2P). Hhex expression was not detected at oviposition, but
endodermal expression could be detected from mid-blastula stage (Figure 1B,C). Gene expression at
early- and mid-blastula stages was not associated with any overt morphological segregation at either
EGK-VI or EGK-VIII. Taken together, these data suggest that the primordial germ cell, epiblast, andhypoblast precursors were already molecularly specified from early-blastula stages, prior tomorphological segregation of these cell types.

Expression of pluripotency-associated markers in finch and chickblastoderm
Our characterization suggested that the finch blastoderm at oviposition was equivalent to the blastocyst
stage mouse embryo when the epiblast precursors had not yet polarized to form an epithelium. This isalso the stage when mESCs could be derived (Boroviak et al., 2014). In mouse embryogenesis, thenaive stage is accompanied by the specific expression of a number of pluripotency-associated genes(Nichols and Smith, 2009). We thus asked whether markers of pluripotency were also expressed in the
finch blastoderm at oviposition. Nanog is a transcriptional regulator involved in cell proliferation during
early mouse development and in self-renewal of mESCs (Chambers et al., 2003; Mitsui et al., 2003).
However, upon cloning finch Nanog, it became apparent that the avian Nanog locus has undergone a
recent gene duplication event, producing Nanog and Nanog-like as suggested from chicken data
(Figure 3A) (Shin et al., 2011). Both genes showing a similar level of homology to mouse Nanog(Figure 4B). Both are expressed in the early EGK-VIII finch blastoderm, predominantly in epiblast cells,
but with scattered cells on the yolk side also being positive (Figure 3C,D). By EGK-X, Nanog-like isdifferentially enriched in the periphery of the blastoderm, in cells fated for the extra-embryonic tissue(Figure 3C). In contrast, Nanog is expressed throughout the epiblast of the EGK-X blastoderm(Figure 3D). Two additional pluripotency regulators, PouV (Pou5F1, the avian homologue of Oct3/4) and
Dnmt3b (Calloni et al., 2013), are also expressed in finch embryos at ovipoisiton in a spatial andtemporal pattern similar to that of Nanog (Figure 3E,F). By EGK-X, expression of these two markers also
became restricted to the epiblast.
To gain a broader understanding of the molecular differences between freshly laid finch and chickembryos, we used a comprehensive Q-PCR screen to compare expression levels of a number of genes
associated with early embryonic development and pluripotency. These genes have been defined fromwork in the mammalian species and adapted to avian species more recently. We first tested corepluripotency factors (Figure 4). Both finch and chick blastoderms at oviposition showed comparable
levels of expression for many of the genes tested, namely PouV, Nanog, Dnmt3b, Sall4, Cripto, Myc, andSox3. In contrast, Nanog-like, Lin28A/B and Klf2 were specifically up-regulated in the finch blastoderm.
We next looked for expression of genes associated with the naive state of pluripotency. Fbxo15 and
PRDM14, which are high in naive state ES cells, are up-regulated in the finch blastoderm. The naive state
genes Tbx3 and Tfcp2l1 show also higher levels of expression for the finch samples although not asprominent. Esrrb, an orphan nuclear receptor that has been used to reprogram fibroblasts into induced
pluripotent stem (iPS) cells (Martello et al., 2013), was not expressed in either blastoderm at oviposition.
Surprisingly, Nrob1, another gene associated with the naive state showed reciprocal expression, withhigh levels detected in the chick blastoderm. Interestingly, expression of above mentioned naive state
associated genes could be also detected in chick blastoderm samples, albeit not as robustly as in finch
samples.
To clarify this, we tested additional markers to assess whether the finch blastoderm contains
features of a full naive state and whether some of these features are lost in the transitional state of thechick blastoderm. The naive state of development includes cells that are able to give rise to the germ
cell lineage. We thus analyzed the expression of two putative markers of germ cell fated cells, Ddx4
and Dazl. Both showed higher expression in finch blastoderms at oviposition as compared to chick.

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Figure 3. Expression of pluripotent markers in the finch embryo at oviposition. (A) Comparison of the NANOG (N)and NANOG-LIKE (nl) homeodomains (HD) from chick and the finch compared to the NANOG HD from human,
mouse, rat, and zebrafish, together with percentage identity to the mouse NANOG HD. (B) Phylogenetic analysis
reveals chick and the finch NANOG and NANOG-LIKE are the result of a recent duplication in the avian lineage.
Figure 3. continued on next page

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Figure 3. Continued(C) In situ hybridization of Nanog-like at oviposition and at EGK-X in the finch blastoderm. Expression is throughout
the upper layer of the blastoderm and resolves to peripheral expression in the putative extra-embryonic region.
Diagram compiles expression revealed from sections. (D) In situ hybridization of Nanog reveals expression in thecentral part of the upper layer at finch oviposition, which expands to cover the entire epiblast at EGK-X. This isshown in sections and detailed in the diagram shown. (E) In situ hybridization of the avian Oct4 homologue PouVshows expression throughout the upper layer/epiblast of the EGK-VIII and EGK-X finch embryos. (F) Expression of
Dnmt3b shows expression throughout the upper layer of the blastula at oviposition, which resolves to central
epiblast expression at EGK-X.
DOI: 10.7554/eLife.07178.005

Cells derived from later, primed, staged blastoderm in mouse are dependent on FGF-mediated Erk
activation. We hypothesized that the chick blastoderm at oviposition would show higher levels of FGF.
Q-PCR revealed higher levels of Fgf3, Fgf4, and Fgf10 in the chick blastoderm as well as slightly
elevated levels of Fgf8 when compared with the finch blastoderm. Fgf5, which marks the mature
epiblast and is considered to be a mEpiSC marker (Lanner and Rossant, 2010), was not detected ineither chick or finch blastoderm. LIF mediates Jak-Stat3 pathway activation and has been shown to be
essential for the maintenance of ESC pluripotency in the mouse (Yoshida et al., 1994). We found that
Lif and its close homologue Il6 were both expressed in finch blastoderms but showed lower levels of
expression in the chick samples.

Expression of naive pluripotency-associated markers decreases in older
finch blastoderm
Our data suggested that the expression level of naive pluripotency markers is reduced in chickblastoderms with respect to finch blastoderms at oviposition. This could be as a result of thedifference in stage between these two embryos, but it is formally possible that this difference resultsfrom species differences. To exclude this, we performed Q-PCR on 2 stages of finch embryos; atoviposition and aged to the equivalent of the chick ovipositional stage, EGK-X (Figure 5). Markers ofgeneral pluripotency, that is, PouV, Nanog, and Dnmt3b show little change between ovipositional
finch embryos and finch embryos at EGK-X (Figure 5A). Furthermore, Nanog-like also showed only
slight differences in expression levels between the two stages of finch blastoderm (Figure 5A). In
contrast, naive pluripotency markers, the expression levels of Fbxo15, Prdm14, Tbx3, and Nrob1
showed a significant down-regulation in older finch embryos (Figure 5B).

Expression of pluripotency markers is independent of ERK activation in
isolated finch blastodermal cells
The molecular characterization described above suggested that the behavior of isolated blastoderm
cells from the chick and finch blastoderms at oviposition would be different. We hypothesized that finch
blastodermal cells may act more like mESCs, whereas chick blastodermal cells would behave moresimilarly to mEpiSCs. An important difference between mESCs and mEpiSCs is the effect of extracellular
receptor kinase (Erk) pathway inhibition. Naive mESCs culture is aided by blockage of Erk pathway in thepresence of LIF (Nichols and Smith, 2012), whereas mEpiSCs do not. In order to test this hypothesis,
individual blastoderms from finch or chick embryos at oviposition were isolated, dissociated, andcultured. Chick and finch cells that were plated and cultured without LIF and without MAP kinase
inhibition formed a monolayer of cells that eventually formed aggregates. However, these aggregatedcolonies did not show AP activity nor did they express pluripotency-associated genes (Nanog or PouV).
Inclusion of LIF in the culture media had little effect on the morphological and molecular features of
either chick or finch-dissociated blastodermal cells. Interestingly, when we added the MEK inhibitor
together with LIF in the culture media, finch blastoderm cells formed aggregates that showed strong AP
activity, as well as expressed Nanog and PouV expression (Figure 6A–C). This effect was observed
either in the presence or absence of GSK3ß inhibition. In contrast, while chick blastodermal cells
cultured with LIF and MEK inhibitor did form aggregates, these remained negative for Nanog and PouVand did not show AP activity (Figure 6D–F). We noted that some cells surrounding the chick colonies
did show weak AP activity and very weak PouV expression, but these were only visible after extended
staining incubation times, and were not found in colonies.

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Figure 4. Q-PCR characterization of finch and chick blastoderm at oviposition reveals fundamental molecular differences. Quantitative-PCR was used toassess the differences in gene expression between finch (green bars) and chick (red bars) embryos at oviposition. The markers used were associated withgeneral pluripotency (A), naive pluripotency (B), markers of primordial germ cell development (C), fibroblast growth factors (D), and leukemia inhibitory
factor (LIF) signaling (E).
DOI: 10.7554/eLife.07178.006

Q-PCR was used to compare the levels of expression of genes associated with pluripotency in the
finch blastodermal cells. Three conditions were analyzed; ovipositional finch cells with or without MEK
inhibitor, and finch blastoderms aged to the equivalent of HH2, dispersed and treated with MEK
inhibitor (Figure 6G). Maximal expression of the naive pluripotency genes, Fbxo15 and Tbx3, as well

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as the general pluripotency marker, Nanog, wasseen in ovipositional finch blastodermal cells
cultured with MEK inhibitor, with less expression
seen in cells without MEK inhibitor and in HH2
finch blastodermal cells cultured with MEK inhibitor. These data confirm that pluripotency
markers are selectively enriched in early finch
blastodermal cells cultured in the presence of
MEK inhibitor. Dnmt3b showed higher expression in ovipositional finch blastodermal cells
without MEK inhibitor. This may indicate asecond Dnmt3b-expressing cell type that is alsoenriched in these conditions. In mouse, Dnmt3b
is initially strongly expressed in the trophectoderm (Hirasawa and Sasaki, 2009), and our
initial evaluation suggests that some of theenriched cells in our cultures may be extraembryonic.

Discussion
The derivation of ES cells must reflect the innate
pluripotency of the embryo as well as the ability
to maintain this potency in vitro. Mouse datasuggest that in permissive strains, ES cells can bederived from embryos at around E4–E4.5 ofdevelopment, a stage that has been termed thenaive stage of embryogenesis (Nichols andSmith, 2009; Boroviak et al., 2014). The epiblastand hypoblast cell lineage has been specified at
this stage and is dispersed throughout the innercell mass; however, epithelialization has yet tooccur (Plusa et al., 2008; Artus et al., 2011). We
find that the finch embryo at oviposition displays
similar characteristics: although the epiblast,hypoblast, and germ line are specified, thesecells are dispersed throughout the multi-layered
blastoderm, which has yet to epithelialize. It is
likely that the sorting mechanisms that ensure
segregation of germs layers are similar to thosein the mouse, but would require further study.
Figure 5. Q-PCR characterization of finch blastoderms
However, given the accessibility of early avian
at oviposition and aged to EGK-X. Quantitative-PCR
development in the finch embryo, these andwas used to assess the differences in gene expression
other comparative studies to test hypotheses on
between finch blastoderms at laying (green bars) andthe evolution of germ layer formation in amniotes
aged to EGK-X, equivalent to chick oviposition (bluebars). The markers were used to assess generalare possible.
pluripotency (A), naive pluripotency (B). p-values <0.05
Pluripotent gene expression in blastodermal
are labeled with an asterisk.
cells derived from finch embryos is independent
DOI: 10.7554/eLife.07178.007of the Erk pathway. This is also an importantfeature of mouse ES cells. In contrast, in our
culture regimen, chick blastodermal cells do not
retain pluripotent gene expression when cultured with an ERK pathway inhibitor, a feature of mEpiSC.
Our data thus suggest that the finch blastoderm at oviposition is likely to correspond to the naivestage of embryogenesis. Furthermore, the evolutionary distance between finch and mouse suggeststhat the naive stage during embryogenesis is conserved amongst amniotes and that this stage likely
corresponds to the pre-epithelialized epiblast in amniotes (Figure 7).

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Figure 6. Finch blastodermal cell cultures retain markers of pluripotency even in the presence of MEK inhibitor.
Finch and chick embryos at oviposition were dissociated and cultured in the presence of LIF and the MEK inhibitor,
PD0325901, for 4 days. (A–C) Finch blastodermal cells form aggregates (red arrows) that show alkaline phosphatase(AP) activity (A) as well as expression of PouV (B) and Nanog (C). (D–F) Chick blastodermal cells formed aggregates(indicated as red arrowheads), but these did not show AP activity (D) nor express PouV (E) and Nanog (F) even after
extended periods of staining. (G) Q-PCR analysis of finch ovipositional blastoderms cultured in the presence of LIF
and the MEK inhibitor, PD0325901, for 4 days, as well as finch blastoderm aged to HH2/3 cultured in the presence of
LIF and PD0325901. Marker analysis was used to ascertain the pluripotent state of cells in each culture condition. Ttests were used to determine the significance of the difference in markers gene expression in finch ovipositional
blastodermal cell culture with or without MEK inhibition. p-values <0.05 are labeled with an asterisk.
DOI: 10.7554/eLife.07178.008

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Figure 7. Putative conservation of the naive stage during amniote embryogenesis. Similar to mouse pre-implantation embryos (around E4), prior to
epiblast epithelialisation, zebra finch embryos at oviposition are at early–mid-blastula stages (EGK-VI/VIII) show no overt morphological segregation
despite the expression of epiblast and primitive endoderm/hypoblast markers in distinct sub-populations of cells with the embryo. Cells from mouse andfinch at this stage do not require ERK activation for the maintenance of pluripotent marker expression in vitro. Chick embryos at oviposition are moresimilar to the mouse blastula where epithelialisation has occurred. Similarly, cells from chick and mouse embryos at this stage are unable to maintainpluripotent marker expression when ERK signaling is inhibited.
DOI: 10.7554/eLife.07178.009

The relationship between the pre-epithelialized epiblast and pluripotency is not clear. Recent datasuggest that a mesenchymal to epithelial transition is important for efficient reprogramming of somatic
cells into iPScells (Li et al., 2010; Samavarchi-Tehrani et al., 2010). Furthermore, E-cadherin, which isimportant in establishing epithelia, mediates LIF signaling during ES cell self-renewal (del Valle et al.,2013). While, this does seem contrary to the notion that ES cell derivation occurs from the preepithelialized epiblast, we found that despite a lack of epithelialization of the newly laid finch blastoderm,
E-cadherin is still highly expressed. This is similar to E-cadherin expression profile in the mouse lateblastocyst (Thomas et al., 2004). Thus, more than a mesenchymal to epithelial transition, perhaps theexpression of E-cadherin represents a metastable, transitional state during mesenchymal–epithelialtransition that is permissive for ES cell derivation. This also suggests that the ability to form epithelial-typeadhesive contacts is present but inhibited at early/mid-blastula stages. This may be due to the presenceof endogenous inhibitors of E-cadherin homophillic interactions, or alternatively it may be simply that thefull repertoire of epithelial-type adhesion molecules is not yet expressed. Consistent with this is thediffering adhesive properties of finch and chick blastodermal cells in culture. Chick blastodermal cells
were able to adhere to a wide variety of substrates; however, optimal growth of finch blastodermal cells
was only observed when type IV collagen was used as a substrate.
The question then remains: how does the pluripotent characteristics of isolated cells relate to theepiblast? One scenario could be that the epiblast consists of mixed populations of cells in varying states ofpluripotency, and that the culture conditions employed can stabilize, select for, induce, or enrich for aparticular state. Several lines of evidence suggest that this is the case. The use of MEK inhibition promotes
the production of ES cells from the epithelialized epiblast of mouse strains that are otherwise refractory for
ES cell derivation. Conversely, EpiSC cells can be isolated from the pre-epithelialized epiblast of the
majority of mouse strains that do not readily produce ES cells (Najm et al., 2011). This suggests a mixed
population of cell states, and our data support this view: finch epiblast cells at oviposition likely containsufficient numbers of naive state cells such that MEK inhibition is able to then stabilize and select for them.
The situation in avians is less clear: culture conditions and exogenously introduced genes can significantly
alter the pluripotency state of these cells. Transcriptional profiling suggests that chick ES cells, that arecultured in a complex but defined mix of growth factors and additives, are more similar to mouse ES cells
than to mouse EpiSC, but are less similar to the chicken blastodermal cells from which they are derived(Jean et al., 2015). Chick iPS cells also display characteristics of ES cells, with AP activity detectable incolonies in the presence of MEK inhibition. However, these observations do not suggest how induced

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pluripotency relates to normal early embryonic development. Our data suggest that in a minimal media,
naive pluripotency in finch blastodermal cells is insensitive to MEK inhibition, whereas chick blastodermal
cells require MEK for the expression of pluripotent markers, in these conditions. The challenge is to now
further stabilize the naive pluripotent fate of finch blastodermal cells and in combination with recent
advances in avian cell culture (Dai et al., 2014; Jean et al., 2015), develop this technology forunderstanding and manipulating avian development.

Materials and methods
Zebra finch husbandry, eggs and embryos collection
Zebra finches were maintained under a 13:11-hr light–dark cycle with constant room temperature(RT) at 23˚C. Eggs were collected every 2 hr from the onset of the light cycle and could be stored at
17–20˚C for up to 5 days without any observable drop in hatchability. Sexing of the finches was
performed by PCR amplification from genomic DNA isolated from feather pulps (Bello et al.,2001). Detailed finch husbandry protocols are available in a separate manuscript (Mak et al.,2015).
Fertile chicken eggs were purchased from Shiroyama Farm (Kanagawa, Japan). Staging of the
pre-primitive streak embryos followed a system previously described in the chick and are stated as
Eyal-Giladi and Kochav stages (EGK) -I to EGK-XIV in this manuscript (Eyal-Giladi and Kochav,1976). Hamburger and Hamilton (HH) system was used for embryos of streak stages and onward(Hamburger and Hamilton, 1951). Chicken blastoderms were collected with a paper ring. Finch
blastoderms were micro-dissected from eggs by tungsten needles owing to their small size andyolk removed as described below.

Blastodermal culture
Both chick and finch blastoderms were cut out in Ringer’s solution. At early stages, yolk is strongly
adherent to the embryo, and the bulk of this was removed by gentle aspiration with a P1000
pipetteman. This left a layer of yolk adherent to the embryo. To remove this layer, the partiallycleaned embryos were transferred to 0.45% D-Glucose (Sigma, St. Louis, MO, United States) in
HBSS+ (HBSSg) solution and left at RT for 2 hr. After incubation, this yolk layer could be lifted fromthe embryo by gentle aspiration around the periphery the embryo. Embryos were then be collected
and centrifuged at 400×g for 2 min. The pellet was resuspended in ED medium (ESGRO [EMDMillipore, Billerica, MA, United States]: DMEM in 1:1) supplemented with Glutamax (Gibco,
Waltham, MA., United States), essential amino acid (Gibco), 0.3 mM 2-mercapthoethanol, sodium
pyruvate (1:100, Gibco), and 1% fetal calf serum. The cells were plated and grown at different
culture conditions. In experiments using MEK inhibitor (PD0325901, Stemgent, Lexington, MA,
United States), GSK3ß inhibitors (CHIR99021, Stemgent), the PKC inhibitor (Gö6983 Sigma), andhuman LIF (LIF1005, EMD Millipore), half to one finch blastoderm per well of an 8-well slide chamber(177445, Nunc Nalgene, Waltham, MA, United States) was used in all experiments unless specified.
1/8 of the chick blastoderm at EGK-X was seeded in each well of an 8-well slide chamber. The
growth of avian cells on a variety of substrates was also tested. Substrates tested were 0.1% or
0.25% gelatin (G1393, Sigma) in PBS, collagen type I (C2249, Sigma), type IV (C0543, Sigma), typeVI (F1141, Sigma), recombinant E-cadherin (MAB1838, R&D Systems, Minneapolis, MN, UnitedStates), growth factor-reduced matrigel (354230, BD Biosciences, Franklin Lakes, NJ, United States),
fibronectin (F5022, Sigma) and pronectin F (Nishishita et al., 2012) (a kind gift from Shin Kawamata,Foundation of Biomedical Research and Innovation, Kobe, Japan) or laminin (354458, BD
Biosciences). Chicken cells grew much more robustly than finch cells and could grow on manydifferent surface types (0.1% or 0.25% gelatin in PBS, collagen type I, type IV, type VI, recombinantE-cadherin, matrigel, fibronectin, pronectin F, or laminin). Optimal growth for chicken blastoderm
cells was seen with type I and IV collagen, on which ES colony-like cell aggregates arose after 4-day
culture. Optimal growth for finch blastoderm cells was obtained on human placenta type IV
collagen-coated surface. We therefore used this coating for all subsequent studies. For Q-PCR
analysis, cells were dissociated from the dish using 0.25% Trypsin-EDTA, spun down, andresuspended in lysis buffer before RNA extraction using the RNeasy Micro Kit (Qiagen, Venlo,
Netherlands).

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Endogenous AP activity detection and antibody staining of embryos andcultured cells
AP activity was detected by incubating 4% PFA fixed sections of embryos/cultured cells with 4.5 μl/ml
NBT (BD Bioscience) and 3.5 μl/ml BCIP (BD Bioscience) substrates. Immunofluorescent staining was
performed using standard protocols. Briefly, sections were blocked with 2% skim milk/PBST (0.1% Triton
X-100 [Sigma] in PBS) for 1 hr at RT. The primary antibody was applied on 12-to 14-μm cryosections andthe slides were incubated overnight at 4˚C. Primary antibodies used were: SSEA-1 and EMA-1 (used in 1:200,
Developmental studies hybridoma bank), SOX3 (Wilson et al., 2001) (in 1:300, a gift of Sara Wilson,
Umeå University, Sweden), GATA6 (1:1000, R&D), E-cadherin (1:200, BD Bioscience), and ß-catenin(1:200, BD Bioscience). Slides were rinsed in PBST and appropriate secondary antibodies conjugatedwith Alexa dyes were applied. The slides were incubated at RT for 4 hr. After rinsing with PBST, thenuclei of the samples were counter-stained with DAPI (1:4000, Dojindo) and mounted in Prolong Goldanti-fade reagent (Invitrogen, Waltham MA, United States). Detection for the finch Vasa homologue
was also attempted. Six antibodies against the chick VASA homologue (CVH/DDX) were tested,
namely VN1, VN2, VC3, and VC4 (gifts from Bertrand Pain, unpublished); anti-VASA (Tsunekawa
et al., 2000) and anti-CVH (Lambeth et al., 2013). These were used on the sections of zebra finch
blastoderms and day 9 gonads, as well as the chicken day 12 gonads as a positive control. All
antibodies showed good staining in positive control chicken gonad sections; however, neither finch
blastoderm nor finch gonadal section showed immunoreactivity.

In situ hybridization of whole-mount embryos and cultured cells
In situ hybridization of embryos was performed using standard protocol (Freter et al., 2008; Alev et al.,2013). Zebra finch-specific and chicken-specific in situ riboprobe templates were generated against
cDNA libraries generated by reverse transcription PCR (SuperScript III First-strand synthesis system,
Invitrogen) from embryos of EGK-VIII, EGK-X, HH stage 4, HH8 and HH18 (Supplementary file 1A).

Quantitative RT-PCR
Total RNA was isolated from either nine stage-matched newly laid or aged blastoderms of finch or two
unincubated blastoderms of chick (EGK-X) (triplicate for each group), using QIAshredder spin columns
and RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions. First-strand cDNAs weresynthesized in parallel from equal amounts of total RNA, using oligo(dT) primers and Superscript III(Invitrogen). Real-time q-PCR was performed in quadruplet using Mesa Green qPCR Mastermix Plus for
SYBR Assay (Eurogentec, Seraing, Belgium) and ABI Prism 7900HT Fast Real-Time PCR system (Applied
Biosystems, Waltham MA, United States). Melting curve analysis was performed with SDS Software(Applied Biosystems) and gene expression calculated using mean cycle threshold (Ct) values, withnormalization to finch or chick glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. Pairwise comparisons were tested using a t-test. Primer sequences are shown in Supplementary file 1B.

Acknowledgements
We would like to thank Drs Hiroshi Nagashima, Jennifer Nichols, Hitoshi Niwa, Austin Smith, Martin
Leeb, Martin Jakt and Masatoshi Takeichi and Ms Kanako Ota for helpful discussions. We are grateful
Dr Bertrand Pain for providing various chicken-specific CVH antibodies and Dr Yukiko Nakaya for theE-cadherin staining. We appreciate the help from the Optical image analysis unit at the CDB and themembers of the Laboratory for Sensory Development. This work was supported by RIKEN CDB
intramural funding for RKL and GS as well as a JSPS fellowship for S-SM, Grant-in-aid for scientificresearch C from MEXT and director’s fund from RIKEN CDB for S-SM and RKL.

Additional information
Funding
FunderAuthorJapan Society for the Promotionof Science (JSPS)

Siu-Shan Mak

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FunderAuthor

Ministry of Education, Culture,
Sports, Science, and Technology(MEXT)

Siu-Shan Mak, Anna Wrabel, Akira
Honda

The funders had no role in study design, data collection and interpretation, or thedecision to submit the work for publication.

Author contributions
S-SM, RKL, Conception and design, Acquisition of data, Analysis and interpretation of data, Draftingor revising the article, Contributed unpublished essential data or reagents; CA, Acquisition of data,
Analysis and interpretation of data; HN, AW, Acquisition of data, Contributed unpublished essential
data or reagents; YM, AH, Acquisition of data; GS, Conception and design, Analysis andinterpretation of data, Drafting or revising the article

Additional files
Supplementary file
Supplementary file 1. (A) Details of primers that were used for generating in situ hybridization
riboprobes for the zebra finch. (B) Details of primers that were used for quantitative PCR.

·

DOI: 10.7554/eLife.07178.010

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