Characterization of the finch embryo supports evolutionary conservation of the naive stage of development in amniotes
The successful isolation and in vitro culture of embryonic stem cells ( ESC ) from mouse embryos have enabled technological breakthroughs and revolutionized our understanding of the molecular mechanisms regulating mammalian development ( Evans , 2011 ) . However , similar applications to other species have been lacking . One conceptual difficulty has been linking the innate pluripotency of the embryo with the characteristics of cultured stem cells , raising the speculation that mESCs may be solely 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 embryonic day ( 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 state and 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 in development ( 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 of pluripotent 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 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 not conserved 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-mammalian amniotes . 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 ( Eyal-Giladi 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 mouse embryo , later than the stage at which mESCs can be derived . The Japanese quail embryos are laid at a stage 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; Jean et al . , 2013 ) . However , it was not clear how the pluripotency generated be exogenous factors related to the pluripotent state of cells in the embryo . We decided to investigate the early development of the zebra finch ( hereafter referred to as the finch ) in more detail . The finches are a model system commonly used 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 increased focus on the developmental neurobiology of zebra finch ( Charvet and Striedter , 2009; Chen et 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 are morphologically 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 and cultured under the same conditions did not produce Nanog , PouV , or AP-positive aggregates . Our data suggest that birds and mammals share a common regulatory mechanism in the maintenance of pluripotency . 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 .
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 the chick 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 , a marker 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 incubation of 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 . 10 . 7554/eLife . 07178 . 003Figure 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 of epithelialization , 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 be detected clearly at EGK-X and is confined to a morphologically distinct hypoblast layer ( C ) . ( D–F ) Staining of E-cadherin 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 any columnar cells typical of epithelial organization ( E ) . At EGK-X , clear epithelial organization can be visualized , with polarized 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: http://dx . doi . org/10 . 7554/eLife . 07178 . 003 Epithelial organization is typically maintained by adherens junctions mediated extracellularly by homophilic 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 consistent with epithelialization of the early embryo . At EGK-VI , both E-cadherin ( Figure 1D ) and ß-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 more regularly 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 ( 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 ) . 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 of cells 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 have already 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 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 , and hypoblast precursors were already molecularly specified from early-blastula stages , prior to morphological segregation of these cell types . 10 . 7554/eLife . 07178 . 004Figure 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 ) and yolk ( 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 the blastoderm . ( 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 the lower 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: http://dx . doi . org/10 . 7554/eLife . 07178 . 004 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 is also the stage when mESCs could be derived ( Boroviak et al . , 2014 ) . In mouse embryogenesis , the naive 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 is differentially 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 and temporal pattern similar to that of Nanog ( Figure 3E , F ) . By EGK-X , expression of these two markers also became restricted to the epiblast . 10 . 7554/eLife . 07178 . 005Figure 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 . ( 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 the central part of the upper layer at finch oviposition , which expands to cover the entire epiblast at EGK-X . This is shown in sections and detailed in the diagram shown . ( E ) In situ hybridization of the avian Oct4 homologue PouV shows 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: http://dx . doi . org/10 . 7554/eLife . 07178 . 00510 . 7554/eLife . 07178 . 006Figure 4 . Q-PCR characterization of finch and chick blastoderm at oviposition reveals fundamental molecular differences . Quantitative-PCR was used to assess the differences in gene expression between finch ( green bars ) and chick ( red bars ) embryos at oviposition . The markers used were associated with general pluripotency ( A ) , naive pluripotency ( B ) , markers of primordial germ cell development ( C ) , fibroblast growth factors ( D ) , and leukemia inhibitory factor ( LIF ) signaling ( E ) . DOI: http://dx . doi . org/10 . 7554/eLife . 07178 . 006 To gain a broader understanding of the molecular differences between freshly laid finch and chick embryos , 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 from work in the mammalian species and adapted to avian species more recently . We first tested core pluripotency 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 , and Sox3 . 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 as prominent . 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 , with high 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 the chick 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 . 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 in either 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 . Our data suggested that the expression level of naive pluripotency markers is reduced in chick blastoderms with respect to finch blastoderms at oviposition . This could be as a result of the difference in stage between these two embryos , but it is formally possible that this difference results from species differences . To exclude this , we performed Q-PCR on 2 stages of finch embryos; at oviposition and aged to the equivalent of the chick ovipositional stage , EGK-X ( Figure 5 ) . Markers of general 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 ) . 10 . 7554/eLife . 07178 . 007Figure 5 . Q-PCR characterization of finch blastoderms at oviposition and aged to EGK-X . Quantitative-PCR was used to assess the differences in gene expression between finch blastoderms at laying ( green bars ) and aged to EGK-X , equivalent to chick oviposition ( blue bars ) . The markers were used to assess general pluripotency ( A ) , naive pluripotency ( B ) . p-values <0 . 05 are labeled with an asterisk . DOI: http://dx . doi . org/10 . 7554/eLife . 07178 . 007 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 more similarly 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 the presence 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 , and cultured . 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 aggregated colonies 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 PouV and 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 . 10 . 7554/eLife . 07178 . 008Figure 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 . T-tests 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: http://dx . doi . org/10 . 7554/eLife . 07178 . 008 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 as the general pluripotency marker , Nanog , was seen 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 a second Dnmt3b-expressing cell type that is also enriched 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 the enriched cells in our cultures may be extra-embryonic .
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 data suggest that in permissive strains , ES cells can be derived from embryos at around E4–E4 . 5 of development , a stage that has been termed the naive stage of embryogenesis ( Nichols and Smith , 2009; Boroviak et al . , 2014 ) . The epiblast and hypoblast cell lineage has been specified at this stage and is dispersed throughout the inner cell mass; however , epithelialization has yet to occur ( 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 , these cells 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 those in the mouse , but would require further study . However , given the accessibility of early avian development in the finch embryo , these and other comparative studies to test hypotheses on the evolution of germ layer formation in amniotes are possible . Pluripotent gene expression in blastodermal cells derived from finch embryos is independent of the Erk pathway . This is also an important feature 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 naive stage of embryogenesis . Furthermore , the evolutionary distance between finch and mouse suggests that the naive stage during embryogenesis is conserved amongst amniotes and that this stage likely corresponds to the pre-epithelialized epiblast in amniotes ( Figure 7 ) . 10 . 7554/eLife . 07178 . 009Figure 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 and finch at this stage do not require ERK activation for the maintenance of pluripotent marker expression in vitro . Chick embryos at oviposition are more similar to the mouse blastula where epithelialisation has occurred . Similarly , cells from chick and mouse embryos at this stage are unable to maintain pluripotent marker expression when ERK signaling is inhibited . DOI: http://dx . doi . org/10 . 7554/eLife . 07178 . 009 The relationship between the pre-epithelialized epiblast and pluripotency is not clear . Recent data suggest 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 is important 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 pre-epithelialized 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 late blastocyst ( Thomas et al . , 2004 ) . Thus , more than a mesenchymal to epithelial transition , perhaps the expression of E-cadherin represents a metastable , transitional state during mesenchymal–epithelial transition that is permissive for ES cell derivation . This also suggests that the ability to form epithelial-type adhesive contacts is present but inhibited at early/mid-blastula stages . This may be due to the presence of endogenous inhibitors of E-cadherin homophillic interactions , or alternatively it may be simply that the full repertoire of epithelial-type adhesion molecules is not yet expressed . Consistent with this is the differing 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 the epiblast ? One scenario could be that the epiblast consists of mixed populations of cells in varying states of pluripotency , and that the culture conditions employed can stabilize , select for , induce , or enrich for a particular 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 contain sufficient 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 are cultured 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 in colonies in the presence of MEK inhibition . However , these observations do not suggest how induced 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 for understanding and manipulating avian development .
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 and yolk removed as described below . 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 partially cleaned 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 from the 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 [EMD Millipore , 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 ) , and human 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 ) , type VI ( F1141 , Sigma ) , recombinant E-cadherin ( MAB1838 , R&D Systems , Minneapolis , MN , United States ) , 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 many different surface types ( 0 . 1% or 0 . 25% gelatin in PBS , collagen type I , type IV , type VI , recombinant E-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 , and resuspended in lysis buffer before RNA extraction using the RNeasy Micro Kit ( Qiagen , Venlo , Netherlands ) . 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 and the 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 conjugated with Alexa dyes were applied . The slides were incubated at RT for 4 hr . After rinsing with PBST , the nuclei of the samples were counter-stained with DAPI ( 1:4000 , Dojindo ) and mounted in Prolong Gold anti-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 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 ) . 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 were synthesized 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 , with normalization to finch or chick glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) , respectively . Pair-wise comparisons were tested using a t-test . Primer sequences are shown in Supplementary file 1B .
Innate pluripotency of mouse embryos transits from naive to primed state as the inner cell mass differentiates into epiblast . In vitro , their counterparts are embryonic ( ESCs ) and epiblast stem 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 chick show 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 .
In animals , stem cells divide to give rise to other cells that have specialized roles in the 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 embryonic stem 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 into another class of stem cell that are ‘primed’ to only produce particular types of cells . Curiously , the stem 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 is present 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 chick embryos to those from another bird called the zebra finch . The experiments show that the finch embryos contain stem cells that share several features with mouse ESCs . In particular , these finch cells continue to express genes that are required for the naive state to be maintained in the absence of FGF . On the other hand , these genes are switched off in cells 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 than chicken 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 as a model to study stem cells and other aspects of animal development .