Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction

Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction View ORCID Profile Aurélien Villedieu , Olinda Alegria-Prévot , Carole Phan , View ORCID Profile Yu Ieda , View ORCID Profile Francis Corson , View ORCID Profile Jérôme Gros doi: https://doi.org/10.1101/2025.05.15.654239 Aurélien Villedieu 1 Institut Pasteur, Université de Paris, CNRS UMR3738, Developmental and Stem Cell Biology Department , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Aurélien Villedieu Olinda Alegria-Prévot 1 Institut Pasteur, Université de Paris, CNRS UMR3738, Developmental and Stem Cell Biology Department , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Carole Phan 1 Institut Pasteur, Université de Paris, CNRS UMR3738, Developmental and Stem Cell Biology Department , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yu Ieda 1 Institut Pasteur, Université de Paris, CNRS UMR3738, Developmental and Stem Cell Biology Department , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yu Ieda Francis Corson 2 Laboratoire de Physique de l’Ecole Normale Supérieure, CNRS, ENS, Université PSL, Sorbonne Université, Université de Paris , 75005 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francis Corson Jérôme Gros 1 Institut Pasteur, Université de Paris, CNRS UMR3738, Developmental and Stem Cell Biology Department , F-75015 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jérôme Gros For correspondence: jgros{at}pasteur.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract In birds and mammals, the formation of the primitive streak, the hallmark of the primary axis and site of gastrulation, is thought to occur when an anterior displacement of the hypoblast (visceral endoderm in mice) lifts its inhibition on the posterior epiblast, enabling the activation of NODAL signaling. Although the anterior movement of the murine visceral endoderm is well documented, the dynamics of the avian hypoblast remain poorly understood. Here, using live imaging and quantitative image analysis, we find that the hypoblast is mechanically coupled to the epiblast and does not migrate away from its posterior end. Instead, the hypoblast moves and deforms passively, in response to the forces transmitted from the epiblast that shape the primitive streak, after its induction. Furthermore, we show that the posterior hypoblast does not exert an inhibitory effect on the epiblast but instead expresses NODAL , which activates primitive streak formation. NODAL concomitantly regulates gene expression in the hypoblast, patterning it along the anteroposterior axis. Our results thus redefine how the primary axis is established in avians, demonstrating that the displacement of the hypoblast and its concomitant anteroposterior patterning are consequences — rather than drivers — of primitive streak induction, downstream of NODAL signaling. Essential summary NODAL asymmetric expression in the hypoblast activates primitive streak formation in the avian embryo. Introduction In amniotes, the basal side of the epiblast is covered by a layer of extra-embryonic endodermal cells called the hypoblast ( Figure 1A , top). The hypoblast plays a critical role in inducing the primitive streak, which defines the embryonic primary axis. In mice and rabbits, the hypoblast (called visceral endoderm [VE] in mice) secretes inhibitors of NODAL and WNT signaling pathways (e.g. CER1, LEFTY1 and DKK1) that inhibit primitive streak formation in the epiblast 1 , 2 . In mice, this inhibition is initially located in the distal part of the VE and is then relocated to the anterior side of the embryo owing to an active migration of the VE 3 . This anterior movement of the VE, whose direction has been proposed to be predetermined by early molecular asymmetries in the VE 4 , 5 , 6 , ensures the definitive positioning of the primitive streak at the posterior end of the epiblast and prevents the formation of additional primitive streak 1 , 7 . Download figure Open in new tab Figure 1: Counter-rotating flows shape the hypoblast. (A) Schematic of the ex ovo culture system used for high-resolution live imaging of the hypoblast. (B) Quail blastoderm viewed from the hypoblast side at 2h post-laying, and (B’) associated schematic showing the different hypoblast populations. The transition region joining the hypoblast islands (in orange) and the posterior hypoblast (in blue) is represented in green. (C) First image of a time-lapse imaging of the hypoblast in a memGFP transgenic embryo (2h post-laying). Arrows show the averaged hypoblast flows between 2h and 12h. The yellow square marks the location shown in closeup in Figure 1F . (D) Cumulated deformation of an initially squared grid (computed from PIV-calculated velocity fields) between 2h-12h, color-coded for area changes and overlaid on the last timeframe of the time-lapse experiment (12h) (D’) The grid is colored according to populations pictured in Figure 1B’ . Asterisk: region masked by debris, in which PIV calculation is not reliable. (E-E’) Averaged velocity field within the hypoblast between 7-9h and between 10-12h (average of 9 embryos) and corresponding decomposition into rotational and divergent components of the flow. (F) High magnification time series of the yellow region shown in (C) at 2h, 6h and 12h. Yellow contours: region tracked by PIV. Scale bars: 1 mm (C-E’), 100 µm (D). Scale vectors: 100 µm/h. In avian embryos, the site of primitive streak formation has also been proposed to result from interactions with the hypoblast, although its role has long been debated 8 , 9 , 10 , 11 , 12 , 13 , 14 . The hypoblast had initially been proposed to act as an inducer of primitive streak formation 8 , 11 , 15 , but this view was later challenged. Based on transmitted light time-lapse videomicroscopy and graft experiments, it has been proposed that the hypoblast is displaced anteriorly by the emergence of another extra-embryonic endoderm population, the junctional endoblast (also named secondary hypoblast), at the posterior end of the blastoderm 16 , 17 , 18 , 19 (stages EGK XIII-XIV). The existence of this endoblast population and its role in displacing the hypoblast found support through the observation that the expression of genes in the hypoblast (e.g. FOXA2, HHEX, CRESCENT , etc.) including NODAL and WNT inhibitors (e.g. CER1 and DKK1 ) becomes progressively relocated to the anterior end of the hypoblast, as the primitive streak appears in the posterior epiblast 13 , 20 , 21 , 22 , 23 . From these observations, it was concluded that the emergence of the endoblast, by displacing the primary hypoblast anteriorly, relieves the posterior epiblast from inhibitors secreted by the hypoblast, entailing the formation of the primitive streak 13 , 16 . An inhibitory role of the hypoblast in primitive streak formation is supported by experiments involving its surgical ablation and reported to induce supernumerary axes — albeit at low frequencies 13 . Thus, as in mice, an anterior movement of the hypoblast relative to the epiblast would be a critical step in instructing primitive streak formation and positioning 1 , 24 , 25 . However, this model lacks critical experimental support in avians. While original studies have described an overall anterior motion of the hypoblast 17 , 19 , 20 , 26 , 27 , these are based on poorly resolved transmitted light imaging or experiments in which group of cells are sparsely labelled and followed using vital dye or carbon particles. Furthermore, despite the central role of a hypoblast motion relative to the epiblast in the proposed “relief-of-inhibition” model for primitive streak induction, such relative motion has not been directly observed. Indeed, counter-rotating tissue flows accompanying primitive streak formation occur simultaneously in the epiblast 14 , 28 , 29 , and how they compare with the tissue flows of the hypoblast is not known. Finally, it is unclear whether the anterior relocation of gene expression within the hypoblast results from a physical displacement or from genetic regulation within the hypoblast itself. In this study, we characterize the dynamics of the avian hypoblast and show that it is passively displaced by the forces driving primitive streak formation in the epiblast. Thus, the posterior hypoblast remains in constant contact with the presumptive primitive streak territory of the epiblast, as gastrulation movements take place. These findings which are inconsistent with a relief of inhibition prompted us to re-evaluate the inhibitory role of the hypoblast in controlling primitive streak formation. At odds with earlier proposal, we find that NODAL expression initiates in the posterior hypoblast and then propagates to the epiblast, where it induces the formation of the primitive streak while patterning the hypoblast along the anteroposterior axis. Collectively, our findings, which clarify the dynamics and role of the hypoblast, redefine the cellular and molecular mechanisms underlying primary axis induction in avians. Results Counter-rotating flows shape the hypoblast To capture the dynamics of the hypoblast, we first developed a culture system for high-resolution imaging of transgenic quail embryos expressing fluorescent reporters ( Figure 1A ). In this set-up, the Early Chick (EC) culture system 30 is inverted onto a glass-bottom dish, and silicone oil is placed between the hypoblast and coverslip (see Methods). Using this system, hypoblast development could be imaged from 2h to 12h after egg-laying (corresponding to EGK XI-XIV 31 ) in 9 embryos expressing a membrane-bound green fluorescent protein 29 (memGFP; Figure 1B-F , Movie 1). These movies provide a cell-resolved description of the developmental stages defined macroscopically by Eyal-Giladi and Kochav 31 . In line with their observations, we observe that at the laying stage (stage EGK XI), the avian hypoblast consists of isolated non-epithelialized islands in its center, joined by a denser, opaque posterior hypoblast crescent known as Koller’s or Rauber’s sickle 15 , surrounded by a thicker peripheral ring of another extra-embryonic tissue called the germ wall 16 , 31 ( Figure 1B-B’ , Supplementary Figure 1A ). This organization is confirmed by immunofluorescence against FOXA2 (also called HNF-3β), which is expressed throughout the hypoblast at this stage 22 ( Supplementary Figure 1B ). At around 5h, the hypoblast, which initially comprised rounded cells, start to fuse to form a cohesive epithelial tissue (Stage EGK XIII 31 ; Figure 1F , Movie 1). The cohesiveness of the hypoblast from 7h can also be observed when an attempt is made to separate the epiblast from the hypoblast by microsurgery: while it is difficult to remove hypoblast islands at 2-5h, it becomes easy to dissect the hypoblast in one piece after 7h ( Supplementary Figure 1C ). The motion of the hypoblast can be quantified over time using Particle Image Velocimetry (PIV; see Methods; Figure 1C-F , Movie 1). Between 2-6h, the initially rounded cells of the hypoblast epithelialize in situ without major tissue movement, as revealed by following PIV-tracked regions ( Figure 1F and Movie 1). From 5h onwards, the hypoblast shows large-scale tissue flows. Surprisingly, we find that the hypoblast does not display a strict anterior migration but exhibits counter-rotating flows ( Figure 1C, 1E-E’ ), very similar to the ones that accompany primitive streak formation in the epiblast 29 , 32 . As a result, the shape of the primitive streak can be recognized in the posterior hypoblast, which converges along the medio-lateral axis and extends along the anteroposterior axis ( Figure 1D-F ). However, several differences remain compared to the morphogenesis of the primitive streak in the posterior epiblast. First, the posterior hypoblast expands as it converges and elongates ( Figure 1D-E’ ), in contrast to the extensive tissue contraction that characterizes the crescent-shaped presumptive primitive streak territory in the epiblast 29 , 32 . Second, cells of the posterior hypoblast, facing the primitive streak, elongate along the anteroposterior axis ( Figure 1F , Movie 1), in contrast to the mediolateral elongation of cells in the epiblast evidencing an actively driven convergent extension of the primitive streak. Decomposing the flows into rotational (incompressible) and divergent (area changes) components 29 reveals the persistence of counter-rotating flows throughout the 10 hours movie and identifies an area expansion flow that is maximal from 10 to 12h ( Figure 1E-E’ ). To ensure that the PIV calculation of the hypoblast flows is not mistaken by integrating fluorescence from epiblast cells, the hypoblast of wild type (WT) embryo was surgically replaced by a hypoblast expressing a H2b-GFP reporter gene 33 at 7h ( Supplementary Figure 2A-B ). The resulting chimeras, in which only the hypoblast cells express H2b-GFP, confirmed the presence of the above-mentioned counter-rotating flows and tissue deformation ( Supplementary Figure 2C-D , Movie 2). The epiblast entrains and deforms the hypoblast during primitive streak formation To characterize the motion of the hypoblast relative to the epiblast, we photoconverted small regions of transgenic embryos expressing the green-to-red photoconvertible mEOS2 protein 34 at 2h. Because of the low Z-resolution of confocal excitation, both epiblast and hypoblast cells were photoconverted in these regions, which were then imaged by orientating the blastoderm on its hypoblast or epiblast side (see Methods). Despite extensive tissue movement, 6 hours later, photoconverted cells in the hypoblast and epiblast remained in close association, although cells from hypoblast regions were more dispersed than their epiblast counterparts ( Figure 2A , Movie 3). Thus, the hypoblast and epiblast display qualitatively similar counter-rotating flows, and the two layers show little relative displacement. To be more quantitative, we grafted early hypoblast islands expressing tdTomato:Myosin 29 onto a memGFP-expressing host at 2h, prior to hypoblast epithelialization, so that grafted cells could be incorporated during the epithelialization of the hypoblast, and we simultaneously live imaged the epiblast and hypoblast dynamics thanks to the differential expression of tdTomato:Myosin and memGFP ( Figure 2B , Movie 4). Quantification of the epiblast and hypoblast flows show that they are highly correlated, confirming results obtained with mEOS2 photoconversion ( Figure 2C , Supplementary Figure 3A-C ). Subtracting the velocity fields of the hypoblast from those of the epiblast ( Figure 2C’ , Supplementary Figure 3D ) reveals that the relative displacement between these tissue layers is small compared to the overall displacement of the epiblast and hypoblast (compare Figures 2C and 2C’ ) and corresponds mostly to an area expansion flow ( Figure 2D-D’ ). Altogether, these experiments suggested that the rotational component of the flow observed in the hypoblast might result from mechanical coupling with the epiblast, in which active stresses at the embryo margin produce the large-scale rotational tissue flows associated with primitive streak formation 29 . To test this hypothesis, a porous filter was inserted between the hypoblast and the epiblast at 7h ( Figure 2E ). If diffusible molecules can be exchanged through this filter, mechanical contact between the two layers is however prevented 35 . In this condition, tissue flows occurred normally in the epiblast, resulting in primitive streak formation as in control embryos in which the hypoblast was dissected and put back in place without inserting a filter ( Figure 2F-F’ ); the hypoblast however did not exhibit counter-rotating flows, unlike control embryos, but only exhibited a small area expansion (n=7/7 with filter, n=4/4 controls, Figure 2G-G’ , Movie 5). These results demonstrate that mechanical coupling between the epiblast and hypoblast is required for the rotational tissue flows to occur in the hypoblast, whereas they proceed autonomously in the epiblast. They further show that these flows arise from the passive transmission of active forces generated in the epiblast while only an area expansion is intrinsic to the hypoblast. Consistent with this, we find that the hypoblast shows no detectable levels of junctional phosphorylated Myosin II (which is a read out of active force generation), in contrast to the epiblast, where supracellular cables of phosphorylated Myosin II could be readily identified at the margin of the same embryo, as previously published 29 ( Supplementary Figure 2E ). Altogether, these results show that the tissue flows observed in the hypoblast can be understood as the sum of a passive rotational flow entrained by the active forces generated in the epiblast owing to mechanical coupling and an active but modest expansion flow intrinsic to the hypoblast layer ( Figure 2H ). Download figure Open in new tab Figure 2: The epiblast entrains and deforms the hypoblast during primitive streak formation. (A) mEOS2 transgenic embryo immediately after (left) and 6h after (right) the photoconversion of 16 square regions. Photoconverted cells of the hypoblast and of the epiblast are shown in magenta and green, respectively (in n=7/7 embryos, photoconverted hypoblast and epiblast regions show little relative displacement 6h after photoconversion). (B) memGFP transgenic embryo (green) grafted with hypoblast cells expressing tdTomato:Myosin (magenta) at 2h and 12h. (C) Averaged epiblast and hypoblast flows between 4h-12h (from n=10 grafted embryos). (C’) Averaged difference between hypoblast and epiblast flows shown in (C). (D) Cumulative deformations of the hypoblast (grid in magenta) between 4h-12h. 5 initially horizontal stripes of the grid are shown in plain magenta and corresponding stripes in the epiblast are shown in plain green, to visualize the relative deformation between the two tissue layers. (D’) Grid representing the averaged deformation of the hypoblast after the deformation of the epiblast has been subtracted. (E) Sketch illustrating the intercalation of a porous filter between hypoblast and epiblast at 7h post-laying. (F, G) Grid showing the cumulative deformation between 7h-12h of the epiblast (F) and the hypoblast (G) with (bottom) or without (top) filter intercalation. A circular region (yellow circle) in the center of the hypoblast at 7h is tracked by PIV in (G). Color code represents area changes. (F’, G’) Quantification of the medio-lateral convergence of the primitive streak in the epiblast (F’) and aspect ratio of PIV-tracked regions in the hypoblast (G’) at 12h with or without intercalation of a porous filter (F’, n=6 embryos with or without filter, n . s .: non-significant, Welch test p-value = 0.73; G’, n=4 and 7, **: p-value<0.001, Welch test p-value=0.005). (H) Diagram of the hypoblast motion as the sum of a passive reaction to the contractile forces generated in the epiblast and a small active deformation (red: active flows, blue: passive flows, light blue arrow: propagation by mechanical coupling). Scale bars: 1 mm. Scale vectors: 100 µm/h. NODAL expression initiates in the posterior hypoblast and propagates in correlation with the progressive patterning of the hypoblast Our results show that there is little relative displacement between the hypoblast and the epiblast at the onset of gastrulation. Thus, the hypoblast remains in constant contact with the primitive streak during its formation, an observation which is inconsistent with an inhibitory function of the hypoblast on primitive streak induction. We thus decided to reanalyze the expression of NODAL and GDF1 , two key inducers of the primitive streak 16 , 36 , using Hairpin Chain Reaction-based RNA fluorescent in situ hybridization (HCR-RNA-FISH). At odds with earlier reports 23 , 37 , 38 , we found that NODAL is first expressed in the posterior hypoblast (corresponding to the Koller’s sickle) at 2h (EGK XI), before the epiblast expresses NODAL or GDF1 , in both quail and chicken embryos ( Figure 3A-B , Supplementary Figure 4 ). To characterize the temporal evolution of NODAL expression, we labeled quail embryos between 2 hours (EGK XI) and 8 hours (EGK XIII), timing them precisely based on the progression of their gastrulation movements (see Methods). By spatially aligning and averaging fluorescent signals of 6-11 embryos per timing (see Methods), we generated archetypal maps of NODAL RNA levels over time ( Figure 3B ). These maps reveal that NODAL expression in the hypoblast increased over time and then appeared in the posterior epiblast by 4h, reaching expression levels comparable to those of the hypoblast by 6h. Thus, a wave of NODAL expression, initiating from the posterior hypoblast, propagates across the epiblast and hypoblast, forming an anteroposterior gradient in both tissue layers. GDF1 expression, on the other hand, was detected later than NODAL , at around 5h (EGK XII) and specifically in the epiblast ( Supplementary Figure 4 ). As observed for NODAL, GDF1 expression increases over time in the epiblast and then appears in the hypoblast, but in a more anterior pattern than NODAL . These results suggest that GDF1 is downstream of NODAL during primitive streak induction, although it remains possible that the HCR-RNA-FISH probes used here do not allow detection of very low expression levels. Download figure Open in new tab Figure 3: NODAL expression initiates in the posterior hypoblast and propagates in correlation with the progressive patterning of the hypoblast. (A) NODAL RNA levels at 2h post-laying. Left: hypoblast view, right: cryosection of the posterior zone (location of the section is marked by a yellow line). NODAL is expressed specifically in the hypoblast in n=3/3 sectioned embryos. (B) Archetypal map of NODAL RNA levels in hypoblast and epiblast at 2h (average of 7 embryos), 4h (average of 6 embryos), 6h (average of 11 embryos) and 8h (average of 7 embryos, see Methods). (C) Immunofluorescence for FOXA2 and FISH of NODAL RNA at 2h, 8h and 12h. (D) Average anteroposterior profiles of FOXA2 and NODAL RNA levels at 2h (average of 7 embryos +/-std), 8h (average of 7 embryos +/-std) and 12h (average of 4 embryos +/-std). (E) FISH of CER1 and NODAL RNA at 4h, 6h, 8h and 12h. Scale bars: 100 µm (A right), 1 mm (A left, C). We then investigated how this NODAL expression gradient relates to the antero-posterior patterning of the hypoblast. We first characterized the pattern of FOXA2 in relation to NODAL expression. As previously published 21 , 22 , we observed an initially homogeneous localization of FOXA2 in the hypoblast at 2h, that over the course of 12h progressively took the shape of a gradient, decaying from anterior to posterior (EGK XIV, Figure 3C left). Notably, this anteroposterior gradient of FOXA2 is anticorrelated with the gradient of NODAL expression ( Figure 3C-D ). As mentioned above, this anterior relocation of FOXA2 expression (along with other genes) has been attributed to the displacement of the hypoblast by the invading endoblast 13 , 16 , 21 . However, backtracking the NODAL -high/FOXA2-low domain from 12h to 2h, using hypoblast tissue flows from the same embryo, maps this region to the transition between the posterior hypoblast and the central hypoblast islands, where FOXA2 is initially high and NODAL expression emerges ( Supplementary Figure 5 ). We also analyzed the pattern of CER1 expression in relation to the one of NODAL . Surprisingly, we observe very low, if any, CER1 expression early on and found that CER1 starts to be reliably detected in the anterior hypoblast from 6h, in a pattern which is, like FOXA2, opposite to NODAL expression ( Figure 3E ). Altogether these analyses argue that the anterior wave of FOXA2 and CER1 expression results from a genetic regulation in the hypoblast that is concomitant to the propagation of the wave of NODAL expression, rather than from the emergence of a new cell population (endoblast) displacing the hypoblast. NODAL signaling in the posterior hypoblast induces the primitive streak NODAL activity is well known to induce ectopic primitive streak formation 13 , 39 , strongly suggesting that the posterior hypoblast is indeed an inducer rather than an inhibitor of primary axis formation. To test this, we surgically removed hypoblast cells (both the posterior hypoblast and hypoblast islands) of quail embryos at 2h ( Supplementary Figure 6A ). In 7/9 cases, this prevented primitive streak formation and NODAL expression, while the epiblast expanded extensively, presumably as the result of tension-induced stretching arising during epiboly 40 , which is unaffected by hypoblast surgical ablation ( Figure 4A , Movie 7). In the remaining 2/9 cases, a reduced primitive streak formed, probably due to incomplete ablation of the posterior hypoblast ( Supplementary Figure 6B ). Similar results were obtained when the hypoblast was removed in chicken embryos incubated for 2h ( Supplementary Figure 6C ). Interestingly, an intentional incomplete removal of the hypoblast, leaving two pieces of posterior hypoblast on each side of the ablated zone ( Supplementary Figure 6A ) resulted in the formation of two primitive streaks facing the untouched hypoblast in 7/11 cases ( Figure 4A , Movie 7), or a single primitive streak but shifted from its original site in the remaining 4/11 cases ( Supplementary Figure 6B ). These results not only show that, in the absence of the hypoblast, the primitive streak is not induced, but they also provide a possible explanation for the previously proposed inhibitory role of the hypoblast 13 . That proposal was based on supernumerary axes induction upon surgical ablation of the hypoblast — an outcome that could also be explained by incomplete ablation. Notably, the ablation of the hypoblast at later stages (7h) had no noticeable effect on primitive streak formation and NODAL expression, whether it was performed in quail or in chicken embryos ( Supplementary Figure 6D ), suggesting that the posterior hypoblast is required for primitive streak induction but dispensable for its maintenance. Finally, to confirm that the posterior hypoblast is an inducer of primitive streak formation, we grafted a posterior hypoblast anteriorly, which resulted in the induction of an ectopic primary axis in 6/6 cases ( Supplementary Figure 6A , Figure 4A ). This ectopic graft induced a decrease in Download figure Open in new tab Figure 4: NODAL signaling in the posterior hypoblast induces the primitive streak. (A) Top: Diagram illustrating the performed microsurgeries. From left to right: no ablation (control), hypoblast ablation, hypoblast partial ablation, anterior graft of posterior hypoblast. Middle: epiblast deformation maps between 2h-12h for each microsurgery. Bottom: NODAL RNA expression in the epiblast at 12h, for each microsurgery. White arrows show primitive streaks (marked by tissue contraction and NODAL expression). (B) Top: Schematic illustrating an anterior graft of a posterior hypoblast (left) and the deposition of an activin-coated bead anteriorly (right). Middle: NODAL RNA expression in the hypoblast at 12h. Bottom: Immunofluorescence for FOXA2 in the hypoblast at 12h. Dotted lines indicate the position of the graft, red dot indicates the position of the grafted bead, white arrows indicate the induction of NODAL expression and low FOXA2 levels in the hypoblast. (C) Immunofluorescence for FOXA2 and FISH for CER1 and NODAL RNA in hypoblast of control (DMSO) and SB505124-treated embryos at 10h. (C’) Averaged FOXA2 levels (+/-std) along the anteroposterior axis (marked by the dotted white lines in c) in the hypoblast of control or SB505124-treated embryos (n=4 control, n=9 SB505124-treated, black line indicates A-P positions for which Welch test p-values < 0.01). (D) Graph summarizing the inductive effect of the posterior hypoblast on the surrounding epiblast and hypoblast (light pink: induction of NODAL expression, light green: downregulation of FOXA2, dashed blue lines: induction of CER1 expression). Scale bars: 1 mm. FOXA2 levels and an ectopic NODAL activation in the hypoblast of the host embryo ( Figure 4B left panels, n=6/6). Similar results on primitive streak formation and NODAL /FOXA2 levels were obtained by depositing a bead coated with Activin A, a known surrogate of NODAL activity 41 , on the hypoblast ( Figure 4B right panels, n=7/7). On the contrary, inhibition of NODAL receptor by systemic SB505124 treatment prevented primitive streak formation as well as FOXA2 downregulation and CER1 expression in the hypoblast ( Figure 4C-C’ ). Taken together, these results demonstrate that NODAL signaling mediates the inducing activity of the posterior hypoblast and that it concomitantly patterns the hypoblast along its antero-posterior axis by shaping FOXA2 and CER1 expression domains ( Figure 4D ). The anterior hypoblast is unable to inhibit primitive streak formation The anterior hypoblast has been proposed to inhibit primitive streak formation through the activity of CER1 13 . Although it has been shown that ectopic expression of CER1 displaces the site of primitive streak formation 13 , whether the anterior hypoblast inhibits primary axis formation has not been directly tested. However, as explained above, we could only detect a clear CER1 expression after primitive streak formation has initiated, and only in the anterior hypoblast ( Figure 3E ). To test whether undetectable levels of CER1 expression or other inhibitors possibly expressed in the hypoblast can inhibit primitive streak formation, we grafted fluorescent anterior hypoblast cells from a 2h-donor onto the posterior pole of a WT 2h-host and monitored effect on primitive streak formation and NODAL expression in the host after 10h ( Figure 5A ). Not only did we observe that grafts do not inhibit NODAL expression in the posterior epiblast, but on the contrary, we found that NODAL expression and FOXA2 downregulation were induced in the grafted hypoblast ( Figure 5A , Supplementary Figure 7A-B ). We thus conclude that the anterior hypoblast is unable to inhibit the NODAL -mediated induction of primitive streak at 2h. To check whether this inhibition could be effective later on, when the anterior hypoblast starts to express CER1 ( Figure 3E ), we performed similar grafts of 7h-anterior hypoblast onto the posterior side of a 7h- or a 2h-host ( Figure 5B and Supplementary Figure 7C ). These homochronic and hetero-chronic grafts show that although the 7h-anterior hypoblast does express CER1 , it is unable to inhibit NODAL expression, but instead becomes patterned into a posterior hypoblast, as revealed by NODAL and FOXA2 expression levels. Altogether, these results show that NODAL signaling in the posterior blastoderm overcomes inhibitory signals produced by the anterior hypoblast. To test whether NODAL induction in these grafts arises from interaction with the epiblast or hypoblast host, a posterior 7h-epiblast fragment (without hypoblast) expressing NODAL was grafted onto the anterior pole of a 2h-host, where the hypoblast expresses FOXA2 but not NODAL . The grafted epiblast induced the formation of an ectopic primitive streak, but importantly induced NODAL expression and FOXA2 downregulation in the host hypoblast ( Figure 5C , Supplementary Figure 5D ), unlike controls, in which an anterior epiblast fragment was grafted anteriorly ( Supplementary Figure 7E ). Altogether, these results reveal a positive feedback loop from the epiblast to the hypoblast, maintaining NODAL activity in the posterior blastoderm — an effect that, on its own, is sufficient to pattern the hypoblast along its anteroposterior axis ( Figure 5D ). Download figure Open in new tab Figure 5: The anterior hypoblast is unable to inhibit primitive streak formation. (A) Top: Schematic illustrating anterior graft of transgenic hypoblasts in the posterior of a WT host at 2h. Bottom: Epiblast (left) and closeup hypoblast images (right) at 12h of the obtained chimera. Note the unaffected NODAL expression in the epiblast and the induction of NODAL expression in the grafted cells. (B) Schematic illustrating the grafting of anterior hypoblasts onto the posterior side of a non-fluorescent host at 7h. Epiblast (left) and closeup hypoblast images (right) at 12h of the obtained chimera. Note the absence of effect of the grafts on NODAL expression in the epiblast and the induction of NODAL expression in the grafted cells. (C) Top: Schematic illustrating the grafting of a WT 7h-posterior epiblast onto the anterior side of the epiblast of a memGFP transgenic 2h-host. Middle: Epiblast view of the obtained chimera right after grafting (left) and 10h after graft (right). Dotted light blue contour: grafted tissue. Bottom: Hypoblast view 10h after graft of the obtained chimera stained for NODAL RNA and closeup on the anterior side. Note the induction of NODAL expression in the hypoblast neighboring the grafted epiblast (n=5/5 chimeras). (D) Schematic diagram illustrating the positive feedback loop on NODAL expression between the hypoblast and the epiblast after initial induction of NODAL expression by the posterior hypoblast. Scale bars: 500 µm (A left, B left), 50 µm (A right, B right), 1 mm (C). Discussion In this study, we show that the original radial symmetry of the blastoderm is broken in the posterior hypoblast, through the localized activation of NODAL expression. The propagation of NODAL expression induces the formation of the primitive streak in the epiblast and patterns the hypoblast along the anteroposterior axis, repressing FOXA2 posteriorly and activating CER1 anteriorly. In the epiblast, NODAL induces the large-scale tissue flows leading to primitive streak formation, which concomitantly propagate to the hypoblast through mechanical coupling. Thus, the motion of the hypoblast and its patterning are a consequence and not a cause of primitive streak induction, at odds with the current model in avians and its generalization to amniotes. In avians, the anterior motion of the hypoblast was proposed to be driven by the intercalation of the endoblast in the posterior, that would displace the hypoblast anteriorly 16 , 17 , 19 , 27 , 42 . Our data, capturing for the first time the dynamics of the hypoblast at both cell and tissue scales, demonstrate that the hypoblast forms by island fusion and epithelialization without an anterior displacement of the hypoblast. Although we cannot rule-out the contribution of radial intercalation during the epithelization process (between 2-7h, cells rearrange extensively), we do not observe in our dynamic imaging any displacement of the hypoblast relative to the epiblast preceding primitive streak induction. Instead, we show that the hypoblast passively deforms through the transmission of the forces generated in the epiblast that shape the primitive streak 14 , 29 , 32 while the hypoblast itself only shows an intrinsic but modest area expansion. Interestingly, it was previously reported that the extracellular matrix at the interface between the epiblast and hypoblast also exhibits counter-rotating flows 43 , as observed in the epiblast and the hypoblast. It is thus possible that the extracellular matrix may act as a link that transmits active forces from the epiblast to the hypoblast. Because the hypoblast is entrained and deformed by the epiblast, it remains in constant contact with it during primitive streak induction and formation. Thus, there cannot be a relief of inhibition and indeed an inhibitory function of the hypoblast at all during this process. Consistent with this, we find that expression of NODAL , a critical factor in primitive streak formation that was thought to be downstream of GDF1 16 , 36 , initiates in the posterior hypoblast and subsequently propagates and increases in both the epiblast and hypoblast. This propagation of NODAL expression is reminiscent of that observed in patterned human embryonic stem cell colonies, in which NODAL expression propagates through a short-range relay mechanism 44 . Furthermore, our analysis of the temporal evolution of NODAL and GDF1 expression indeed suggests that GDF1 is downstream of NODAL during primitive streak induction. Such a view is consistent with the recent finding that PITX2 , a known target of NODAL in other developmental contexts 45 , regulate GDF1 expression during primitive streak induction 46 and also with the finding that, in other species, GDF1 is a cofactor of NODAL that might be inactive on its own 47 , 48 . Importantly, surgical ablation of the hypoblast performed in this study prevented primitive streak induction, while anterior grafts of posterior hypoblast induced ectopic primitive streak, confirming the inducing activity of the hypoblast. It is worth noting that similar experiments have been published, but their results were however not mutually consistent. On the one hand, the inhibitory role of the hypoblast was concluded from surgical ablation of the hypoblast, since this procedure occasionally led to the induction of multiple axes 13 . Here we show that supernumerary axes form when isolated pieces of posterior hypoblast are intentionally left behind during surgical ablation. As expected for a local induction process, each of these isolated hypoblast regions induces a primitive streak in the overlying epiblast, resulting in the formation of multiple axes. We note that the complete surgical ablation of the hypoblast is particularly difficult to achieve at these early stages, when the hypoblast is composed of non-epithelialized islands. We therefore controlled that most if not all hypoblast cells were removed following surgical ablation using FOXA2 immunofluorescence and NODAL HCR-RNA-FISH ( Supplementary Figure 6A ). On the other hand, the inductive power of the posterior blastoderm has been identified by various series of grafts, but whether the inductive region was located in the epiblast or in the hypoblast was unclear 11 , 20 , 42 , 49 . Callebaut et al. performed grafts of posterior hypoblast (i.e. Koller’s/Rauber’s sickle) onto explants of central epiblast, which resulted in ectopic primitive streak induction 50 . Here, we obtain similar results and further show that NODAL activity mediates the inducing activity of the posterior hypoblast. Finally, although it is possible that NODAL inhibitors expressed in the anterior hypoblast might act to restrict the activity of NODAL and prevent the formation of ectopic primitive streak later on, as observed in mouse 1 , such inhibition of the hypoblast is unlikely to play a role in positioning the endogenous primitive streak, since CER1 is not expressed early on and the anterior hypoblast is unable to inhibit endogenous NODAL activity when grafted posteriorly. The progressive anterior relocation of a series genes including NODAL inhibitors and FOXA2 in the hypoblast 20 , 21 , 22 , 23 has been attributed to the displacement of the hypoblast 13 , 21 . We show that the regionalization of FOXA2 and CER1 , and presumably other genes (e.g. DKK1, CRESCENT , etc.) is not established by the emergence and invasion of a new endoblast population as previously proposed, but by a genetic regulation induced by NODAL activity within the hypoblast. Indeed, we find that CER1 , which was proposed to initially inhibit NODAL signaling 13 , is expressed at undetectable or very low levels in the hypoblast at earliest stages. Transplantation experiments further show that the anterior hypoblast is unable to inhibit primary axis formation in the posterior, but instead becomes itself patterned into a posterior identity, as revealed by NODAL induction and FOXA2 downregulation. Furthermore, transplanting posterior epiblast fragments from 7h-donners, in which NODAL expression has been initiated, in anterior epiblasts shows that the anterior hypoblast of the host downregulates FOXA2 , an effect which is mimicked by the deposition of Activin A beads. These results place the patterning of the hypoblast concomitant to, if not downstream of, primitive streak formation. More generally, our results show that primitive streak initiation is positioned by the localized activity of an inducer (NODAL) and not by a relief of inhibition. This result, which redefines how primary axis formation is regulated, has several important implications. First, as early avian development is regulative, and multiple primitive streaks can be induced along the embryo margin upon mechanical perturbations of the epiblast 51 , 52 , it could be conceived that a primitive streak can spontaneously self-organize at any location along the margin, but that this potential is initially and homogenously inhibited 38 , 51 , 53 . However, experiments in which the hypoblast is surgically removed show that this is not the case: in the absence of an inducing trigger (NODAL from the hypoblast) the epiblast cannot self-organize any (single or multiple) primitive streak at its margin. This further suggests that the self-organizing property of the embryo margin 52 requires at least permissive, if not instructive signals from the hypoblast. Second, our study traces back the first molecular event breaking the original radial symmetry of the blastoderm to the localized expression of NODAL in the hypoblast, begging the question of the mechanisms responsible for this restricted expression. Embryonic polarity has been reported to be fixed prior to oviposition, during intrauterine development, under the influence of gravity 54 . How gravity eventually results in NODAL expression specifically in the posterior hypoblast remains to be examined. Finally, in mice, several studies have shown that molecular asymmetries in the VE precede its motion and it has been proposed that they predetermine its direction 4,5,6,56,57 . Our results in avian embryos are consistent with the view that changes in gene expression breaks the radial symmetry of the embryo prior to hypoblast/VE directional motion. The recent identification of counter-rotating flows associated with the anterior movement of the VE in mice 55 is intriguing and — in the light of our findings — should encourage the characterization of the tissues flows occurring in the epiblast and their possible role on the anterior motion of the VE. Author contributions The project was conceptualized by A.V. and J.G. A.V. and J.G. designed experiments. A.V. and Y.I. performed experiments. A.V and F.C. designed quantitative image analyses approaches. A.V. and J.G. analyzed the data. A.V. and J.G. wrote the manuscript and generated the figures with inputs from F.C. O.A-P. and C.P. generated the tdTomato:Myosin transgenic quail line and managed transgenic egg supply. Competing interest statement The authors declare no competing interests. Materials and Methods Animals All experimental methods and animal husbandry for transgenic quails were performed in accordance with the guidelines of the European Union 2010/63/UE, approved by the Institut Pasteur ethics committee authorization #dha210003, and under the GMO agreement 2432. Imaging Hypoblast imaging Transgenic quail eggs were collected after oviposition and placed in a 38.5°C incubator for 2h to facilitate embryo collection without damaging the hypoblast. Embryos were then collected (stage EGK XI) using a paper filter ring and cultured on a semi-solid nutritive medium made of thin chicken albumen, agarose (0.2%), glucose and NaCl, as described previously 27 . The lid of a 35mm Petri dish was filled with a more solid nutritive medium (with 0.4% agarose), and an embryo was transferred on it, with its epiblast surface and vitelline membrane facing the semi-solid nutritive medium. The lid with the embryo was then inverted onto a glass-bottom dish (Mattek) containing silicone oil (Sigma-Aldrich 378321). This way, the embryo is in contact with the silicone oil on its hypoblast side, enabling confocal live imaging through the coverslip. Embryos were imaged at 38.5 °C using a Zeiss LSM 900 or LSM 980 microscope and 20× or 10× objectives. The time interval between two consecutive frames was ranging from 6-20 min. mEOS2 photoconversion and imaging Transgenic mEOS2 quail eggs were collected 2h after oviposition and placed on a glass-bottom dish filled with semi-solid nutritive medium. The dish was placed upside down, without lid on a Zeiss LSM 980 microscope with a focus on the hypoblast. 150×150µm square regions were photoconverted at different locations of the hypoblast using a 10× objective. Both epiblast and hypoblast cells were photoconverted in these regions. A photoconversion calibration curve was calculated before each imaging session, and the amount of blue light giving 95% of maximal photoconversion was chosen for subsequent photoconversion. Embryos were imaged (both red and green emission) from their hypoblast and then epiblast sides just after photoconversion and 6h later, using a 10× objective. Fluorescent signal from the epiblast and hypoblast were projected and digitally aligned with each other using the transmitted light image of the embryo, which was collected during the imaging of the embryos. Simultaneous imaging of hypoblast and epiblast using a chimera Hypoblast cells were collected from a 2h embryo expressing tdTomato:Myosin and grafted onto the hypoblast of a host expressing memGFP. The resulting chimera was placed on a glass-bottom dish (Mattek) and was imaged from its epiblast side, using a Zeiss LSM 980 or LSM 900 microscope and 5× objective, as described previously 26 . The pinhole of the microscope was open wide to collect the red fluorescent signal from grafted cells through the epiblast. The time interval between two consecutive frames was 6-7 min. Microsurgery For all microsurgeries, embryos were collected at 2h after oviposition, and, if needed, incubated for 5h at 38.5°C to reach 7h after oviposition. Before each surgery, the embryo was carefully immersed drop by drop in Hank’s Balanced Salt Solution (HBSS) using a 10 µl pipette. An eyebrow hair mounted on a Pasteur pipette was used to peel off the desired hypoblast tissue (2h hypoblast islands, 2h-posterior hypoblast, 7h-full or anterior hypoblast) or to cut an approximately 500×300µm region of epiblast. Tissues from the donner were transferred in HBSS onto a host using a tapered Pasteur pipette whose tip was cut-off. Each explant was slightly incised on one side before transfer, to ensure proper orientation during the graft. Filter intercalation The hypoblast of a 7h-embryo was first removed and temporarily transferred onto nutritive semi-solid medium. A polycarbonate filter (Millipore RTTP01300) immersed in HBSS, was placed on the epiblast. The hypoblast was then transferred onto the filter using a 10 µl pipette whose tip was cut-off. An eyebrow hair was used to orient and position the hypoblast. Pharmacological treatments SB505124 treatment 100µM SB505124 (Sigma-Aldrich S4696, 1% DMSO) and 1% DMSO (control) were added to the culture medium. Activin-coated bead Heparin-agarose beads (Sigma-Aldrich H6508) were soaked for 2 days at 4°C in a solution containing Fast Green (1:2 dilution, to help visualize the beads) and Activin A (ProteinTech HZ-1138, 50ng/µl). They were then quickly rinsed in HBSS and grafted onto a 2h embryo using a Pasteur pipette. Tissue flow analysis Tissue flows were calculated using Particle Image Velocimetry (PIV) and decomposed into a divergent and a rotational component, as previously described 26 . Spatial alignment of the hypoblast films was performed using the two centers of the counter-rotating flows at 8h as landmarks. Regions masked by debris appearing towards the end of the movies have been filtered out and were not taken into consideration during the analysis. Calculated vector fields were averaged across animals to obtain an average map; regions covered by less than 3 animals were not displayed. To compare epiblast and hypoblast flows, epiblast flows were analyzed by PIV as previously described 26 , while grafted hypoblast islands were manually tracked. The average hypoblast velocity field was calculated by averaging the velocities calculated over all the animals (providing coverage of the entire hypoblast). The trajectory of each manually tracked hypoblast island was corrected for epiblast movement at each time interval to calculate the movement of the hypoblast relative to the epiblast. Immunostaining, RNA-FISH and related image analysis Immunostaining Embryos were fixed overnight at 4°C in 4% formaldehyde solution. They were then incubated 3×10 min in PBT (1×PBS with 1% Triton 100X and 1% SDS) and overnight in PBT containing rabbit anti-FOXA2 (Proteintech, Cat #22574-1-AP, 1/1000 dilution), or in Can Get Signal Solution B (Toyobo) containing rabbit phospho-Myosin light chain 2 (Cell Signaling, Cat#3674, 1/200 dilution) and mouse anti-ZO1 (Invitrogen, Cat#33-9100, 1/400 dilution). Embryos were rinsed 3×10 min in PBT, incubated for 2h at room temperature with PBT containing secondary antibodies (Invitrogen, donkey anti-rabbit-647 and donkey anti-mouse-555, 1/2000 dilution), for 10 min in PBT containing Hoechst (1/2000 dilution) and for 3×10 min in PBT. They were mounted between two glass coverslips so that both sides (epiblast and hypoblast) can be imaged. HCR-RNA-FISH Embryos were fixed overnight at 4°C in 4% formaldehyde solution and were then labelled using probes specific for quail NODAL, GDF1 and CER1 and following the manufacturer’s protocol (Molecular Instruments). The NODAL probe was revealed using an amplifier B5-647 or B5-546, the GDF1 probe using an amplifier B1-546, and the CER1 probe using B3-647. Immunostainings against FOXA2 were performed after HCR-RNA-FISH labelling. Cryosection Embryos were embedded in 7.5% gelatin/15% sucrose and sectioned sagittally using a Leica CM3050S cryostat. Sections were degelatinized in PBS at 37°C for 30 min. Imaging and extraction of epiblast and hypoblast signals HCR-RNA-FISH- and immunolabelled embryos were imaged with a Zeiss LSM 980 or LSM 900 microscope using a 20× or 10× objective (3µm z-resolution) on each side (epiblast and hypoblast sides). Hoechst nuclear signal was used to detect the most superficial plane corresponding to epiblast or hypoblast (depending on embryos’ orientation) and to project fluorescent signals, using custom Matlab code. The projection of the epiblast and hypoblast were then aligned with each other using the contours of the embryo. Spatio-temporal alignment and calculation of archetype maps Temporal alignment: Embryos used for establishing the evolution of the NODAL expression pattern at 4h, 6h and 8h were timed based on the progression of their gastrulation movements, as previously published 49 . Embryos were imaged and their tissue flows calculated on the fly, allowing to fix them at the onset of primitive streak contraction (4h), at 25% medio-lateral primitive streak contraction (6h) or at 50% contraction (8h). Images of the HCR labelling were aligned with the last frame of the live movie using blastoderm contours. Spatial alignment: the contour of blastoderms was determined at 2h (either directly on the 2h fixed sample, or on the live movie aligned with the staining). A circle was fitted onto blastoderm contours, and its diameter was reduced by 10%. This reduced circle and the position of the posterior pole were used to align all the images ( Figure 3B ). Signal normalization: NODAL HCR-RNA-FISH signal, which takes the form of a cloud of points each corresponding to the detection of single RNA molecules, was binarized by adjusting a threshold value for each image so as sparse dots were well segmented. NODAL expression levels in the epiblast and hypoblast were then averaged for all animals of the same timing. Download figure Open in new tab Supplementary Figure 1: Complementary information on hypoblast imaging and hypoblast populations identification. (A) Left: Still image from Movie 1 showing the hypoblast view of an embryo expressing the fluorescent reporter memGFP at 2h. Middle: Optical cross-section along the dotted yellow line. Right: Closeup of the region indicated by the white rectangle. The image is color coded for the Z axis (blue: surface of the deep layers, magenta/orange: superficial layer), highlighting hypoblast islands discontinuity. Orange, green and blue bars, on the right, show the location of hypoblast islands, transition zone and posterior hypoblast respectively. (B) Left: FOXA2 immunofluorescence at 2h. Right: Closeup of the region indicated by the white rectangle. Orange, green, and blue bars, on the right, show the location of hypoblast islands, transition zone and posterior hypoblast, respectively. (C) FOXA2 immunofluorescence after hypoblast removal at 7h (left: embryo without hypoblast, right: removed hypoblast, n=2 embryos). Scale bars: 1 mm. Download figure Open in new tab Supplementary Figure 2: Confirmation of tissue flow monitoring with a chimera. (A) Schematic illustrating a chimera obtained by replacing the hypoblast of a 7h wild-type host embryo (gray) with a hypoblast from a 7h embryo expressing H2b-GFP (green). (B) Still image of Movie 2 of the grafted hypoblast at 7.5h (30 min after graft) and 12h. (C) Cumulated deformation of an initially square grid between 7.5h-12h using PIV. Color code indicates area changes. n=4/4 imaged chimera show similar cumulated deformation. (D) Velocity field in the grafted hypoblast at 8h and 12h. n=4/4 imaged chimeras show similar motion fields. Scale bars: 1 mm Scale vector: 100 µm/h. Download figure Open in new tab Supplementary Figure 3: Measure of epiblast and hypoblast flows and phospho-Myosin localization. (A) Tracking of tdTomato:Myosin grafted hypoblast cells imaged through the epiblast (158 manual tracking) for a representative chimera. Color code indicates timing (from 4h to 12h). (B) Tracks in the corresponding epiblast, obtained by PIV using memGFP signal. Initial points of epiblast tracks match those chosen for hypoblast tracking. (C) Averaged velocity vectors between 4h-12h in epiblast and hypoblast for the chimera displayed in (A-B). (D) Tracks of the differential motion between the hypoblast and epiblast for the chimera displayed in (A-C). (E) Immunofluorescence for phospho-Myosin light chain 2 and ZO1 in the hypoblast and the epiblast at 8h post-laying (absence of cortical phospho-Myosin light chain 2 in hypoblast is observed in n=3/3 labeled embryos). Scale bars: 1 mm (A-D), 50 µm (E). Scale vectors: 100 µm/h Download figure Open in new tab Supplementary Figure 4: Comparison of NODAL and GDF1 expression patterns using HCR-RNA-FISH. (A) NODAL (in magenta) and GDF1 expression (in yellow) at 2h, 5h, 8h and 12h in posterior epiblast and posterior hypoblast of quail embryos. Note the absence of specific signal in epiblast and hypoblast for GDF1 at 2h (observed in n=9/9 embryos). Note the appearance of NODAL and GDF1 expression in the posterior epiblast at 5h (observed in n=4/4 embryos); and the difference in GDF1 and NODAL expression patterns at 8h and 12h (observed in n=5/5 embryos at 8h and n=9/9 embryos at 12h). (B) NODAL (in magenta) and GDF1 expression (in yellow) at 3h and 6h post-laying in posterior epiblast and posterior hypoblast of chicken embryos. Note the absence of specific signal in epiblast and hypoblast for GDF1 at 3h (observed in n=3/3 embryos). Note the appearance of NODAL and GDF1 expression in the posterior epiblast at 6h (observed in n=3/3 embryos). Scale bars: 1 mm. Download figure Open in new tab Supplementary Figure 5: Backtracking the hypoblast population expressing NODAL at 12h. Backtracking the NODAL -high FOXA2-low region of the hypoblast using hypoblast tissue flows from 12h to 2h. In all of the 4 embryos analyzed, this region is backtracked to the transition zone between hypoblast islands and the posterior hypoblast where FOXA2 is high and NODAL expression emerges (see Supplementary Figures 1 and 4). Scale bars: 1 mm. Download figure Open in new tab Supplementary Figure 6: Complementary information on hypoblast microsurgeries related to Figure 4 . (A) From left to right: schematics illustrating different microsurgeries (from left to right): 2h-hypoblast ablation, partial posterior ablation of 2h hypoblast and 2h-posterior hypoblast extraction, used for grafting on anterior position. Bottom: Verification of NODAL expression and FOXA2 levels at 2h for the control and right after surgery for the different microsurgeries. Dashed lines represent ablated region. Note that the precise contours of the posterior hypoblast being unclear under transmitted light, partial and centered posterior ablation can result in one domain of NODAL expression (n=4/11) instead of two (n=7/11), as intended by the surgery. (B) Infrequent phenotypes observed after 2h-hypoblast ablation (left, n=2/9) or partial posterior 2h-hypoblast ablation (right, n=4/11), complementary to the ones shown in Figure 4 . Top: schematics illustrating the different microsurgeries. Middle: epiblast cumulative deformation between 2h-12h (color code represents area changes). Bottom: NODAL expression in the epiblast at 12h (white line: embryo contours). White arrows show primitive streaks. (C) Top: Transmitted light image in control and 18h after surgery of chicken embryos whose hypoblast was ablated at 2h of incubation. Bottom: NODAL expression in the epiblast at 20h (white line: embryo contours). White arrows show primitive streaks. Absence of primitive streak after hypoblast ablation at 2h was observed in 5/6 cases, development of a miniature embryo was observed in 1/6 cases. (D) Left: schematic illustrating hypoblast ablation at 7h. Center: Epiblast deformation between 7h-15h in embryos whose hypoblast was ablated at 7h. A single primitive streak was observed in n=25/25 animals. Right: transmitted light image 24h after surgery of quail or chicken embryos whose hypoblast was ablated at 7h, single primitive streak was observed in n=63/63 ablated quail embryos and n=20/20 chicken embryos. Scale bar: 1 mm. Download figure Open in new tab Supplementary Figure 7: Complementary information on the role of anterior hypoblast and anterior epiblast on primary axis formation, related to Figure 5 . (A) FOXA2 immunofluorescence at 12h, following an anterior 2h-hypoblast graft on a 2h-host. Top: Schematic illustrating the grafting of fluorescent anterior hypoblast cells onto the posterior side of a non-fluorescent host at 2h. Bottom: Hypoblast of the obtained chimera at 12h stained for FOXA2 (grafted cells are in green). Anterior and posterior closeups (yellow squares in top panels), showing high FOXA2 levels in cells grafted anteriorly, and low FOXA2 levels in cells grafted posteriorly. (B) Averaged (+/-std) FOXA2 levels in hypoblast from graft and host (n=5 chimeras) in chimeras obtained by grafting 2h-anterior hypoblast graft onto a 2h-host. (C) Top: Schematic illustrating the heterochronic grafting of anterior 7h-hypoblast onto the posterior side of a non-fluorescent 2h-host. Epiblast (bottom left) and closeup hypoblast images (bottom right) at 12h of the obtained chimera. Note the absence of effect of the grafts on NODAL expression in the epiblast and the induction of NODAL expression in the grafted cells. (D) Top: Schematic illustrating the grafting of a non-fluorescent 7h-posterior epiblast fragment onto the anterior epiblast of a fluorescent 2h-host. Middle: Epiblast view of the obtained chimera right after (left) and 10h after the graft (right). Dotted light blue contour: grafted tissue. Bottom: Hypoblast view 10h after the graft stained for FOXA2 and NODAL RNA and closeup on the anterior side. Note the downregulation of FOXA2 and induction of NODAL expression in hypoblast host cells neighboring the epiblast graft (n=5/5 chimeras). (E) Top: Schematic illustrating the grafting of a non-fluorescent 7h-anterior epiblast fragment onto the anterior epiblast of a fluorescent 2h-host. Middle: Epiblast view of the obtained chimera right after (left) and 10h after graft (right). Dotted light blue contour: grafted tissue. Bottom: Hypoblast view 10h after graft of the obtained chimera stained for FOXA2 and NODAL RNA and closeup on the anterior side. Note the absence of FOXA2 downregulation and induction of NODAL expression in hypoblast host cells neighboring the epiblast graft (n=5/5 chimeras). Scale bars: 1 mm (A top, C top, D top and middle, E top and middle), 50 µm (A closeups, B right, D closeup, E closeup) Movie1: Quantitative description of hypoblast dynamics by videomicroscopy (1) Live imaging of the hypoblast of an embryo expressing the fluorescent reporter memGFP from to 2h to 12h post-laying. Z is color-coded as in Supplementary Figure 1 . (2) PIV-calculated flows. 2 regions tracked using PIV. (3) PIV-calculated flows and deformation corresponding to Figure 1D-D’ . (4) Average flows and average decomposition into rotational and divergent components. Movie2: Confirmation of hypoblast dynamics with a chimera Live imaging of the hypoblast of an H2b-GFP transgenic embryo grafted onto a WT host, and the associated PIV-calculated flows and deformations. Movie3: Study of hypoblast and epiblast relative movements using photoconversion Photoconversion of square boxes in the blastoderm, imaging of hypoblast and epiblast sides after photoconversion and after 6h. The epiblast of the embryo was live imaged between 2h and 8h to ensure that proper development occurred. Movie4: Simultaneous imaging of hypoblast and epiblast movements using a 2-color chimera (1) Simultaneous live imaging of the epiblast of a transgenic memGFP host embryo grafted with hypoblast cells expressing tdTomato:Myosin. PIV-calculated deformation of the epiblast and manual tracking of hypoblast cells are displayed. (2) Quantitative comparison of epiblast and hypoblast motions. Movie5: Effect of filter intercalation on hypoblast dynamics Live imaging of a transgenic H2b-GFP hypoblast grafted on a host embryo with or without filter intercalation, and the associated PIV-calculated deformation grids and tracked circular regions. Movie6: Effect of full or partial hypoblast ablation on epiblast dynamics Live imaging of the epiblast of a control embryo and embryos in which the hypoblast has been fully or partially removed, and the associated PIV-calculated deformation grids. Transmitted light allows to visualize regions lacking hypoblast as a result of the ablations. Acknowledgements We are grateful to Arthur Michaut, Alexander Chamolly and Carolina Parada for their advice on experiments and data analysis. Work in J.G. lab is funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement n° 866186 to J.G), the Agence Nationale de la Recherche (LabEx Revive), the Centre National de la Recherche Scientifique (CNRS) and Institut Pasteur. Y.I was supported by a stipend from the Pasteur - Paris University (PPU) International PhD program and by 4th-year PhD fellowship from the Fondation pour la Recherche Médicale (FRM). For the purpose of open access, the authors have applied a CC-BY public copyright license to any Author Manuscript version arising from this submission. References 1. ↵ Perea-Gomez , A. et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks . Dev. Cell 3 , 745 – 756 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 2. ↵ Idkowiak , J. , Weisheit , G. , Plitzner , J. & Viebahn , C. Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo . Dev. Genes Evol . 214 , 591 – 605 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 3. ↵ Srinivas , S. , Rodriguez , T. , Clements , M. , Smith , J. C. & Beddington , R. S. P. Active cell migration drives the unilateral movements of the anterior visceral endoderm . Development 131 , 1157 – 1164 ( 2004 ). OpenUrl Abstract / FREE Full Text 4. ↵ Yamamoto , M. et al. Nodal antagonists regulate formation of the anteroposterior axis of the mouse embryo . Nature 428 , 387 – 392 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 5. ↵ Takaoka , K. , Yamamoto , M. & Hamada , H. Origin of body axes in the mouse embryo . Curr. Opin. Genet. Dev . 17 , 344 – 350 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 6. ↵ Morris , S. A. et al. Dynamics of anterior-posterior axis formation in the developing mouse embryo . Nat. Commun . 3 , 1 – 10 ( 2012 ). OpenUrl CrossRef PubMed 7. ↵ Kimura , C. et al. Visceral endoderm mediates forebrain development by suppressing posteriorizing signals . Dev. Biol . 225 , 304 – 321 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 8. ↵ Waddington , C. H. Experiments on the Development of Chick and Duck Embryos, Cultivated in vitro . Philos. Trans. R. Soc. London 221 , 179 – 230 ( 1932 ). OpenUrl CrossRef 9. ↵ Azar , Y. & Eyal-Giladi , H. Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments . J. Embryol. Exp. Morphol . Vol. 61 , 133 – 144 ( 1981 ). OpenUrl PubMed Web of Science 10. ↵ Mitrani , E. & Eyal-Giladi , H. Hypoblastic cells can form a disk inducing an embryonic axis in chick epiblast . Nature 289 , 800 – 802 ( 1981 ). OpenUrl CrossRef PubMed 11. ↵ Khaner , O. Axis Determination in the Avian Embryo . Curr. Top. Dev. Biol . 28 , 155 – 180 ( 1993 ). OpenUrl CrossRef PubMed 12. ↵ Khaner , O. The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast . Proc. Natl. Acad. Sci. U. S. A . 92 , 10733 – 10737 ( 1995 ). OpenUrl Abstract / FREE Full Text 13. ↵ Bertocchini , F. & Stern , C. D. The hypoblast of the chick embryo positions the primitive streak by antagonizing nodal signaling . Dev. Cell 3 , 735 – 744 ( 2002 ). OpenUrl CrossRef PubMed 14. ↵ Voiculescu , O. , Bertocchini , F. , Wolpert , L. , Keller , R. E. & Stern , C. D. The amniote primitive streak is defined by epithelial cell intercalation before gastrulation . Nature 449 , 1049 – 1052 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 15. ↵ Callebaut , M. , Van Nueten , E. , Bortier , H. & Harrisson , F. Positional information by Rauber’s sickle and a new look at the mechanisms of primitive streak initiation in avian blastoderms . J. Morphol . 255 , 315 – 327 ( 2003 ). OpenUrl CrossRef PubMed 16. ↵ Raffaelli , A. & Stern , C. D. 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Morphogenetic movements in the lower surface of the unincubated and early chick blastoderm . J. Exp. Zool . 144 , 139 – 157 ( 1960 ). OpenUrl CrossRef 27. ↵ Stern , C. D. The marginal zone and its contribution to the hypoblast and primitive streak of the chick embryo . Development 109 , 667 – 682 ( 1990 ). OpenUrl Abstract / FREE Full Text 28. ↵ Gräper , L. Die Primitiventwicklung des Hühnchens nach stereokinematographischen Untersuchungen, kontrolliert durch vitale Farbmarkierung und verglichen mit der Entwicklung anderer Wirbeltiere . Dev. Genes Evol . 116 , 382 – 429 ( 1929 ). OpenUrl 29. ↵ Saadaoui , M. , Rocancourt , D. , Roussel , J. , Corson , F. & Gros , J. A tensile ring drives tissue flows to shape the gastrulating amniote embryo . Science (80- .). 367 , 453 – 458 ( 2020 ). OpenUrl Abstract / FREE Full Text 30. ↵ Chapman , S. C. , Collignon , J. , Schoenwolf , G. C. & Lumsden , A. Improved method for chick whole-embryo culture using a filter paper carrier . Dev. Dyn . 220 , 284 – 289 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 31. ↵ Kochav , S. , Ginsburg , M. & Eyal-Giladi , H. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick: I.General Morphology . Dev. Biol . 79 , 296 – 308 ( 1980 ). OpenUrl CrossRef PubMed Web of Science 32. ↵ Rozbicki , E. et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation . Nat. Cell Biol . 17 , 397 – 408 ( 2015 ). OpenUrl CrossRef PubMed 33. ↵ Serralbo , O. et al. Transgenesis and web resources in Quail . Elife 9 , 1 – 22 ( 2020 ). OpenUrl CrossRef PubMed 34. ↵ Moreau , C. et al. Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position . Curr. Biol . 29 , 35 – 50 .e4 ( 2019 ). OpenUrl CrossRef PubMed 35. ↵ Eyal-Giladi , H. & Wolk , M. The inducing capacities of the primary hypoblast as revealed by transfilter induction studies . Wilhelm Roux. Arch. Entwickl. Mech. Org . 165 , 226 – 241 ( 1970 ). OpenUrl CrossRef PubMed 36. ↵ Shah , S. B. et al. Misexpression of chick Vg1 in the marginal zone induces primitive streak formation . Development 124 , 5127 – 5138 ( 1997 ). OpenUrl Abstract 37. ↵ Skromne , I. & Stern , C. D. A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo . Mech. Dev . 114 , 115 – 118 ( 2002 ). OpenUrl CrossRef PubMed 38. ↵ Bertocchini , F. , Skromne , I. , Wolpert , L. & Stern , C. D. Determination of embryonic polarity in a regulative system: Evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo . Development 131 , 3381 – 3390 ( 2004 ). OpenUrl Abstract / FREE Full Text 39. ↵ Schier , A. F. & Shen , M. M. Nodal signalling in vertebrate development . Nature 403 , 385 – 389 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 40. ↵ Michaut , A. , Chamolly , A. , Villedieu , A. , Corson , F. & Gros , J. A tension-induced morphological transition shapes the avian extra-embryonic territory . Curr. Biol . 35 , 1 – 12 ( 2025 ). OpenUrl CrossRef PubMed 41. ↵ Lee , H. C. , Hastings , C. & Stern , C. D. The extra-embryonic area opaca plays a role in positioning the primitive streak of the early chick embryo . Development 149 , ( 2022 ). 42. ↵ Azar , Y. & Eyal-Giladi , H. Marginal zone cells: the primitive streak-inducing component of the primary hypoblast in the chick . J. Embryol. Exp. Morphol . Vol 52 , 79 – 88 ( 1979 ). OpenUrl PubMed Web of Science 43. ↵ Zamir , E. A. , Rongish , B. J. & Little , C. D. The ECM moves during primitive streak formation - Computation of ECM versus cellular motion . PLoS Biol . 6 , 2163 – 2171 ( 2008 ). OpenUrl CrossRef 44. ↵ Liu , L. et al. Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids . Nat. Commun . 13 , 1 – 12 ( 2022 ). OpenUrl CrossRef PubMed 45. ↵ Shiratori , H. et al. Two-step regulation of left-right asymmetric expression of Pitx2: Initiation by nodal signaling and maintenance by Nkx2 . Mol. Cell 7 , 137 – 149 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 46. ↵ Torlopp , A. et al. The transcription factor Pitx2 positions the embryonic axis and regulates twinning . Elife 3 , e03743 ( 2014 ). OpenUrl CrossRef PubMed 47. ↵ Montague , T. G. & Schier , A. F. Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm . Elife 6 , 1 – 24 ( 2017 ). OpenUrl CrossRef PubMed 48. ↵ Tanaka , C. , Sakuma , R. , Nakamura , T. , Hamada , H. & Saijoh , Y. Long-range action of Nodal requires interaction with GDF1 . Genes Dev . 21 , 3272 – 3282 ( 2007 ). OpenUrl Abstract / FREE Full Text 49. ↵ Khaner , O. & Eyal-Giladi , H. The embryo-forming potency of the posterior marginal zone in stages X through XII of the chick . Dev. Biol . 115 , 275 – 281 ( 1986 ). OpenUrl CrossRef PubMed 50. ↵ Callebaut , M. , Nueten, E. Van , Nassauw, L. Van , Bortier , H. & Harrisson , F. Only the endophyll-Rauber’ s sickle complex and not cells derived from the caudal marginal zone induce a primitive streak in the upper layer of avian blastoderms . Reprod. Nutr. Dev . 38 , 449 – 463 ( 1998 ). OpenUrl CrossRef PubMed 51. ↵ Spratt , N. T. & Haas , H. Integrative mechanisms in development of the early chick blastoderm. I. Regulative potentialitiy of separated parts . J. Exp. Zool 97 – 137 ( 1960 ). 52. ↵ Caldarelli , P. et al. Self-organized tissue mechanics underlie embryonic regulation . Nature 633 , ( 2024 ). 53. ↵ Lee , H. C. et al. Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity . Nat. Commun . 15 , ( 2024 ). 54. ↵ Kochav , S. & Eyal-Giladi , H. Bilateral symmetry in chick embryo determination by gravity . Science (80- .). 171 , 1027 – 1029 ( 1971 ). OpenUrl Abstract / FREE Full Text 55. ↵ Stower , M. et al. Single-cell phenomics reveals behavioural and mechanical heterogeneities underpinning collective migration during mouse anterior patterning . bioRxiv ( 2023 ). 56. Torres-Padilla , M. E. et al. The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation . Dev. Biol . 309 , 97 – 112 ( 2007 ). OpenUrl CrossRef PubMed 57. Rivera-Pérez , J. A. & Magnuson , T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3 . Dev. Biol . 288 , 363 – 371 ( 2005 ) OpenUrl CrossRef PubMed Web of Science 1. Perea-Gomez , A. et al. Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks . Dev. Cell 3 , 745 – 756 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 2. Idkowiak , J. , Weisheit , G. , Plitzner , J. & Viebahn , C. Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo . Dev. Genes Evol . 214 , 591 – 605 ( 2004 ). OpenUrl CrossRef PubMed Web of Science 3. Srinivas , S. , Rodriguez , T. , Clements , M. , Smith , J. C. & Beddington , R. S. P. Active cell migration drives the unilateral movements of the anterior visceral endoderm . Development 131 , 1157 – 1164 ( 2004 ). OpenUrl Abstract / FREE Full Text 4. Kimura , C. et al. Visceral endoderm mediates forebrain development by suppressing posteriorizing signals . Dev. Biol . 225 , 304 – 321 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 5. Waddington , C. H. Experiments on the Development of Chick and Duck Embryos, Cultivated in vitro . Philos. Trans. R. Soc. London 221 , 179 – 230 ( 1932 ). OpenUrl CrossRef 6. Azar , Y. & Eyal-Giladi , H. Interaction of epiblast and hypoblast in the formation of the primitive streak and the embryonic axis in chick, as revealed by hypoblast-rotation experiments . J. Embryol. Exp. Morphol . Vol. 61 , 133 – 144 ( 1981 ). OpenUrl PubMed Web of Science 7. Mitrani , E. & Eyal-Giladi , H. Hypoblastic cells can form a disk inducing an embryonic axis in chick epiblast . Nature 289 , 800 – 802 ( 1981 ). OpenUrl CrossRef PubMed 8. Khaner , O. Axis Determination in the Avian Embryo . Curr. Top. Dev. Biol . 28 , 155 – 180 ( 1993 ). OpenUrl CrossRef PubMed 9. Khaner , O. The rotated hypoblast of the chicken embryo does not initiate an ectopic axis in the epiblast . Proc. Natl. Acad. Sci. U. S. A . 92 , 10733 – 10737 ( 1995 ). OpenUrl Abstract / FREE Full Text 10. Bertocchini , F. & Stern , C. D. The hypoblast of the chick embryo positions the primitive streak by antagonizing nodal signaling . Dev. Cell 3 , 735 – 744 ( 2002 ). OpenUrl CrossRef PubMed 11. Voiculescu , O. , Bertocchini , F. , Wolpert , L. , Keller , R. E. & Stern , C. D. The amniote primitive streak is defined by epithelial cell intercalation before gastrulation . Nature 449 , 1049 – 1052 ( 2007 ). OpenUrl CrossRef PubMed Web of Science 12. Callebaut , M. , Van Nueten , E. , Bortier , H. & Harrisson , F. Positional information by Rauber’s sickle and a new look at the mechanisms of primitive streak initiation in avian blastoderms . J. Morphol . 255 , 315 – 327 ( 2003 ). OpenUrl CrossRef PubMed 13. Raffaelli , A. & Stern , C. D. Signaling events regulating embryonic polarity and formation of the primitive streak in the chick embryo . Current Topics in Developmental Biology vol. 136 ( Elsevier Inc ., 2020 ). 14. Vakaet , L. Cinephotomicrographic investigations of gastrulation in the chick blastoderm . Arch. Biol. (Liege) . 81 , 387 – 426 ( 1970 ). OpenUrl PubMed 15. Stern , C. D. & Canning , D. R. Origin of cells giving rise to mesoderm and endoderm in chick embryo . Nature 343 , 273 – 275 ( 1990 ). OpenUrl CrossRef PubMed Web of Science 16. Eyal-Giladi , H. , Debby , A. & Harel , N. The posterior section of the chick’s area pellucida and its involvement in hypoblast and primitive streak formation . Development 116 , 819 – 830 ( 1992 ). OpenUrl Abstract 17. Bachvarova , R. F. , Skromne , I. & Stern , C. D. Induction of primitive streak and Hensen’s node by the posterior marginal zone in the early chick embryo . Development 125 , 3521 – 3534 ( 1998 ). OpenUrl Abstract 18. Foley , A. C. , Skromne , I. & Stern , C. D. Reconciling different models of forebrain induction and patterning : a dual role for the hypoblast . Development 3854 , 3839 – 3854 ( 2000 ). OpenUrl 19. Chapman , S. C. , Schubert , F. R. , Schoenwolf , G. C. & Lumsden , A. Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos . Dev. Biol . 245 , 187 – 199 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 20. Lee , H. C. et al. Molecular anatomy of the pre-primitive-streak chick embryo . Open Biol . 10 , ( 2020 ). 21. Rivera-Pérez , J. A. , Mager , J. & Magnuson , T. Dynamic morphogenetic events characterize the mouse visceral endoderm . Dev. Biol . 261 , 470 – 487 ( 2003 ). OpenUrl CrossRef PubMed 22. Stern , C. D. & Downs , K. M. The hypoblast (visceral endoderm): An evo-devo perspective . Development 139 , 1059 – 1069 ( 2012 ). OpenUrl Abstract / FREE Full Text 23. Spratt , N. T. & Haas , H. Morphogenetic movements in the lower surface of the unincubated and early chick blastoderm . J. Exp. Zool . 144 , 139 – 157 ( 1960 ). OpenUrl CrossRef 24. Stern , C. D. The marginal zone and its contribution to the hypoblast and primitive streak of the chick embryo . Development 109 , 667 – 682 ( 1990 ). OpenUrl Abstract / FREE Full Text 25. Gräper , L. Die Primitiventwicklung des Hühnchens nach stereokinematographischen Untersuchungen, kontrolliert durch vitale Farbmarkierung und verglichen mit der Entwicklung anderer Wirbeltiere . Dev. Genes Evol . 116 , 382 – 429 ( 1929 ). OpenUrl 26. Saadaoui , M. , Rocancourt , D. , Roussel , J. , Corson , F. & Gros , J. A tensile ring drives tissue flows to shape the gastrulating amniote embryo . Science . 367 , 453 – 458 ( 2020 ). OpenUrl Abstract / FREE Full Text 27. Chapman , S. C. , Collignon , J. , Schoenwolf , G. C. & Lumsden , A. Improved method for chick whole-embryo culture using a filter paper carrier . Dev. Dyn . 220 , 284 – 289 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 28. Kochav , S. , Ginsburg , M. & Eyal-Giladi , H. From cleavage to primitive streak formation: A complementary normal table and a new look at the first stages of the development of the chick: I.General Morphology . Dev. Biol . 79 , 296 – 308 ( 1980 ). OpenUrl CrossRef PubMed Web of Science 29. Rozbicki , E. et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation . Nat. Cell Biol . 17 , 397 – 408 ( 2015 ). OpenUrl CrossRef PubMed 30. Serralbo , O. et al. Transgenesis and web resources in Quail . Elife 9 , 1 – 22 ( 2020 ). OpenUrl CrossRef PubMed 31. Moreau , C. et al. Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position . Curr. Biol . 29 , 35 – 50 .e4 ( 2019 ). OpenUrl CrossRef PubMed 32. Eyal-Giladi , H. & Wolk , M. The inducing capacities of the primary hypoblast as revealed by transfilter induction studies . Wilhelm Roux. Arch. Entwickl. Mech. Org . 165 , 226 – 241 ( 1970 ). OpenUrl CrossRef PubMed 33. Shah , S. B. et al. Misexpression of chick Vg1 in the marginal zone induces primitive streak formation . Development 124 , 5127 – 5138 ( 1997 ). OpenUrl Abstract 34. Skromne , I. & Stern , C. D. A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo . Mech. Dev . 114 , 115 – 118 ( 2002 ). OpenUrl CrossRef PubMed 35. Bertocchini , F. , Skromne , I. , Wolpert , L. & Stern , C. D. Determination of embryonic polarity in a regulative system: Evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo . Development 131 , 3381 – 3390 ( 2004 ). OpenUrl Abstract / FREE Full Text 36. Schier , A. F. & Shen , M. M. Nodal signalling in vertebrate development . Nature 403 , 385 – 389 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 37. Michaut , A. , Chamolly , A. , Villedieu , A. , Corson , F. & Gros , J. A tension-induced morphological transition shapes the avian extra-embryonic territory . Curr. Biol . 35 , 1 – 12 ( 2025 ). OpenUrl CrossRef PubMed 38. Lee , H. C. , Hastings , C. & Stern , C. D. The extra-embryonic area opaca plays a role in positioning the primitive streak of the early chick embryo . Development 149 , ( 2022 ). 39. Azar , Y. & Eyal-Giladi , H. Marginal zone cells: the primitive streak-inducing component of the primary hypoblast in the chick . J. Embryol. Exp. Morphol . Vol 52 , 79 – 88 ( 1979 ). OpenUrl PubMed Web of Science 40. Zamir , E. A. , Rongish , B. J. & Little , C. D. The ECM moves during primitive streak formation - Computation of ECM versus cellular motion . PLoS Biol . 6 , 2163 – 2171 ( 2008 ). OpenUrl CrossRef 41. Liu , L. et al. Nodal is a short-range morphogen with activity that spreads through a relay mechanism in human gastruloids . Nat. Commun . 13 , 1 – 12 ( 2022 ). OpenUrl CrossRef PubMed 42. Shiratori , H. et al. Two-step regulation of left-right asymmetric expression of Pitx2: Initiation by nodal signaling and maintenance by Nkx2 . Mol. Cell 7 , 137 – 149 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 43. Torlopp , A. et al. The transcription factor Pitx2 positions the embryonic axis and regulates twinning . Elife 3 , e03743 ( 2014 ). OpenUrl CrossRef PubMed 44. Montague , T. G. & Schier , A. F. Vg1-Nodal heterodimers are the endogenous inducers of mesendoderm . Elife 6 , 1 – 24 ( 2017 ). OpenUrl CrossRef PubMed 45. Tanaka , C. , Sakuma , R. , Nakamura , T. , Hamada , H. & Saijoh , Y. Long-range action of Nodal requires interaction with GDF1 . Genes Dev . 21 , 3272 – 3282 ( 2007 ). OpenUrl Abstract / FREE Full Text 46. Khaner , O. & Eyal-Giladi , H. The embryo-forming potency of the posterior marginal zone in stages X through XII of the chick . Dev. Biol . 115 , 275 – 281 ( 1986 ). OpenUrl CrossRef PubMed 47. Callebaut , M. Nueten , E. Van Nassauw , L. Van , Bortier , H. & Harrisson , F. Only the endophyll-Rauber’ s sickle complex and not cells derived from the caudal marginal zone induce a primitive streak in the upper layer of avian blastoderms . Reprod. Nutr. Dev . 38 , 449 – 463 ( 1998 ). OpenUrl CrossRef PubMed 48. Spratt , N. T. & Haas , H. Integrative mechanisms in development of the early chick blastoderm. I. Regulative potentialitiy of separated parts . J. Exp. Zool 97 – 137 ( 1960 ). 49. Caldarelli , P. et al. Self-organized tissue mechanics underlie embryonic regulation . Nature 633 , ( 2024 ). 50. Lee , H. C. et al. Regulation of long-range BMP gradients and embryonic polarity by propagation of local calcium-firing activity . Nat. Commun . 15 , ( 2024 ). 51. Kochav , S. & Eyal-Giladi , H. Bilateral symmetry in chick embryo determination by gravity . Science . 171 , 1027 – 1029 ( 1971 ). OpenUrl Abstract / FREE Full Text 52. Stower , M. et al. Single-cell phenomics reveals behavioural and mechanical heterogeneities underpinning collective migration during mouse anterior patterning . bioRxiv ( 2023 ). 53. Torres-Padilla , M. E. et al. The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation . Dev. Biol . 309 , 97 – 112 ( 2007 ). OpenUrl CrossRef PubMed 54. Rivera-Pérez , J. A. & Magnuson , T. Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3 . Dev. Biol . 288 , 363 – 371 ( 2005 ). OpenUrl CrossRef PubMed Web of Science View the discussion thread. Back to top Previous Next Posted May 17, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction Aurélien Villedieu , Olinda Alegria-Prévot , Carole Phan , Yu Ieda , Francis Corson , Jérôme Gros bioRxiv 2025.05.15.654239; doi: https://doi.org/10.1101/2025.05.15.654239 Share This Article: Copy Citation Tools Live imaging and functional characterization of the avian hypoblast redefine the mechanisms of primitive streak induction Aurélien Villedieu , Olinda Alegria-Prévot , Carole Phan , Yu Ieda , Francis Corson , Jérôme Gros bioRxiv 2025.05.15.654239; doi: https://doi.org/10.1101/2025.05.15.654239 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15171) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)