Formation of the vertebrate embryo: Moving beyond the Spemann organizer.

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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

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Formation of the vertebrate embryo: Moving beyond the Spemann organizer

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Bernard Thisse, Christine Thisse ∗ Department of Cell Biology, University of Virginia, Charlottesville, VA, USA

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a b s t r a c t

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Article history: Available online xxx

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Keywords: Organizer Morphogen Gradient BMP Nodal

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Contents

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During the course of their classic experiments, Hilde Mangold and Hans Spemann discovered that the dorsal blastopore lip of an amphibian gastrula was able to induce formation of a complete embryonic axis when transplanted into the ventral side of a host gastrula embryo. Since then, the inducing activity of the dorsal lip has been known as the Spemann or dorsal organizer. During the past 25 years, studies performed in a variety of species have led to the identification of molecular factors associated with the properties of this tissue. However, none of them is, by itself, able to induce formation of the main body axis from a population of naive pluripotent embryonic cells. Recently, experiments performed using the zebrafish (Danio rerio) revealed that the organizing activities present in the embryo are not restricted to the Spemann organizer but are distributed along the entire blastula/gastrula margin. These organizing activities result from the interaction between two opposing gradients of morphogens, BMP and Nodal, that are the primary signals that trigger the cascade of developmental events leading to the organization of the embryo. These studies mark the end of the era during which developmental biologists saw the Spemann organizer as the core element for the organization of the vertebrate embryonic axis and, instead, provides opportunities for the experimental control of morphogenesis starting with a population of embryonic pluripotent cells that will be instructed using those two morphogen gradients. © 2015 Published by Elsevier Ltd.

The Spemann organizer and its molecular nature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .00 More than just the Spemann organizer: the entire blastula/gastrula margin has organizing properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Molecular nature of the organizing activities of the blastula/gastrula margin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Organization of a complete embryonic axis with opposing gradients of BMP and Nodal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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1. The Spemann organizer and its molecular nature In the early 1920s, in Freiburg University (Germany), a PhD student, Hilde Mangold, under the direction of her advisor Hans Spemann, grafted the dorsal blastopore lip, the region where gastrulation starts, from a weakly pigmented salamander gastrula to

∗ Corresponding author at: Department of Cell Biology, University of Virginia, School of Medicine, 1340 Jefferson Park Avenue, Charlottesville, VA 22903, USA. Tel.: +1 434 243 6613. E-mail address: [email protected] (C. Thisse).

the ventral side of a more highly pigmented species. This allowed her to distinguish the cells contributed to the graft from those of the host embryo. Mangold found that the transplanted dorsal tissue gave rise mostly to notochord, while the neighboring cells from the host were induced to form a Siamese twin containing dorsal tissues such as somites and central nervous system. The most remarkable finding was that the neural folds were built from recipient cells and not from donor cells. The transplant had altered the fate of the overlying cells, which normally would have make skin (epidermis) and produced, instead, a second head [1]. Spemann and Mangold used the term “induction” for the ability of one group of cells to influence the fate of another and called this dorsal lip region ‘the organizer’, known subsequently as the ‘Spemann organizer’. Hans

http://dx.doi.org/10.1016/j.semcdb.2015.05.007 1084-9521/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Thisse B, Thisse C. Formation of the vertebrate embryo: Moving beyond the Spemann organizer. Semin Cell Dev Biol (2015), http://dx.doi.org/10.1016/j.semcdb.2015.05.007

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ARTICLE IN PRESS B. Thisse, C. Thisse / Seminars in Cell & Developmental Biology xxx (2015) xxx–xxx

Spemann was awarded the Nobel Prize for Medicine or Physiology in 1935 for this discovery. During the past 25 years, a number of studies, performed in a variety of species have identified the molecular activities associated with the properties of the Spemann organizer and identified their functions both in amphibian (reviewed in [2,3]) and in fish (reviewed in [4]) embryos. A summary of these results is presented in Fig. 1 for the zebrafish embryo. The function of the Spemann organizer involves a number of factors acting mostly as transcriptional repressors or as antagonists of growth factors involved in complex regulatory pathways. The general view is that two signaling centers, a ventral center and a dorsal center (the Spemann organizer) are active in the embryo and that they antagonize each other. Ventrally, two sets of morphogens, the bone morphogenetic proteins (BMPs) and Wnts induce ventral and posterior identities into the embryo. Dorsally, secreted antagonists and transcriptional repressors prevent the activity and the expression of the ventral morphogens, protecting the dorsal side of the embryo from their ventralizing and posteriorizing activities. In turn, activity of Chordin (Chd), the main BMP antagonist, is negatively regulated by ventrally expressed factors. In addition, transcriptional repressors induced by Wnt and BMP prevent expression of dorsal genes (such as chd, goosecoid and dharma) in lateral and ventral domains and the FGF signaling pathway, negatively regulated by feedback inhibitors, progressively restrict bmp gene transcription to the ventral half of the embryo. Finally, transcriptional repressors expressed in the dorsal center prevent the expression of the ventral genes in that territory: Dharma represses bmp2b as well as vent and vox while Goosecoid and FoxA3 repress transcription of wnt8a. Altogether,

the interplay between these various regulatory proteins establishes a stable ventral to dorsal gradient of BMP activity and prevents expression of Wnt in the dorsal most part of the blastula/gastrula margin allowing for the formation of dorsal and anterior tissues. 2. More than just the Spemann organizer: the entire blastula/gastrula margin has organizing properties The Spemann organizer is a source of antiventralizing factors. Therefore, in grafting experiments, it can function only in territories that express the ventral morphogens, BMP and Wnt, where it represses both their expression and their activity, establishing or reshaping gradients of these ventral signals. Because the Spemann organizer acts as a modifier of the activity of existing morphogen gradients, it does not carry organizing function(s) by itself. Attraction of cells in an embryonic axis, regulation of their proliferation and control of their identity to build tissues and organs with precise 3-dimentional shapes requires more than the antagonistic and repressing activities of the Spemann organizer. Therefore, the signals directly responsible for these organizing functions were not identified in studies that have characterized the molecular nature of the classic Spemann organizer. A search for such organizing activities has been performed using the zebrafish model [5,6]. Because of the particular type of segmentation of this species (meroblastic, discoidal) the embryo develops on top of the yolk, which is progressively covered by embryonic cells during the blastula and gastrula stages. At the onset of gastrulation, the blastopore lip (called the “blastoderm margin” or simply “margin”) is hugely expanded compared to its amphibian

Fig. 1. Major interactions between the dorsal center (Spemann organizer) and the ventral center in early zebrafish gastrula. A schematic of a zebrafish embryo at the onset of gastrulation is shown in the center. Factors on the right act in the dorsal center to control the activity of the ventralizing factors: the bone morphogenetic proteins (BMP2b, BMP7, BMP4) [17,34–37] and of the posteriorizing [38] and ventralizing [39] factor: Wnt8a [40], in the dorsalmost domain of the embryo. The action of BMPs and Wnt is antagonized by dorsally secreted factors (Chordin [8], Noggin1 [7] and Follistatin like 1b [9]). Activity of Chordin, the main BMP antagonist, is negatively regulated by ventrally expressed modulators that include tolloid [41], BMP1 [42,43], twisted gastrulation – Tsg [44,45] and crossveinless 2 – Cvl2 [46]. The FGF signaling pathway progressively restricts the expression of bmp genes to ventral territory [47] by terminating the positive autoregulatory loop required to maintain transcription of bmp coding genes (by promoting the degradation of Smad1/5 that positively control bmp gene transcription [48] and our unpublished observation). The activity of FGF [47,49] that signal through the FGF receptor 1, Ras, Raf, ERK1/2 signaling pathway, is attenuated by the feedback antagonists Spry2, Spry4 and SEF (Interleukin 17 Receptor D) [50–52]. Wnt8a activity is negatively regulated by dorsally secreted antagonists (Frzb and Dickkopf1 [53]) while Wnt8a expression is repressed dorsally by two transcription repressors goosecoid and FoxA3 that prevent Wnt8a from posteriorizing the anterior neural plate [13]. Wnt8a and BMP induce expression of the transcription repressors, Vent, Vox and Ved that prevent the expression in lateral and ventral position of the dorsal gene chordin, dharma, and goosecoid [54–58]. In turn, dharma prevents transcription at the dorsal margin of vent, ved and vox [58]. It also represses transcription of bmp2b at the early blastula stage in that dorsal domain [59]. Action of the ventral morphogens is indicated with green arrows; negative interactions through protein–protein interactions are indicated in red, transcriptional repressions are indicated in dark blue; transcriptional activations are indicated by open light blue arrows.


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counterpart, encircling the yolk (700 ␮m in diameter) at its equator. It is therefore much easier to isolate portions of the margin of a fish gastrula than it is in the case of the blastopore lip of the amphibian and to use them in grafting experiments designed to look for their potential organizing activities. Contrary to the classical Spemann–Mangold experiments in which cells were grafted in a ventral marginal position (a territory with high Wnt and BMP activity), the analysis in zebrafish has been performed by grafting portions of the margin into the animal pole of an early blastula host. This territory appears to be the most neutral of the embryo and contains only naive pluripotent cells at early blastula stages. When the dorsal margin of an early gastrula (also called the embryonic shield), the zebrafish equivalent to the dorsal blastopore lip of an amphibian, is grafted into the animal pole of a blastula stage host (Fig. 2A), it does not induce formation of a complete embryonic axis and has only very limited organizing activity. The only structures that develop in response to signals from this graft are axial mesendoderm derivatives: prechordal plate, notochord and floor plate (Fig. 2B). These tissues are those that are normally formed by cells of the dorsal margin and the grafts do not recruit significant numbers of animal pole cells to form additional ectopic structures. Surprisingly, a graft from the ventral margin of a donor embryo (at blastula or gastrula stage) put into the animal pole of a blastula host results in the organization of a well-differentiated tail (Fig. 2C). This tail contains epidermis, neural tube, somites and blood but lacks axial mesoderm as well as tissues (adaxial cells and ventral spinal cord), which are induced by factors secreted by notochord and floor plate. This secondary tail is composed of cells from the

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graft and cells from the host animal pole that have been recruited and organized by the graft. The piece of ventral margin grafted into the animal pole shares all of the properties associated with an organizer and has therefore been defined as the tail organizer [5]. Because the dorsal margin gives rise to axial mesendoderm while the ventral margin carries tail organizing properties, it has been hypothesized that other territories may be organized by the lateral margin. As predicted, pieces of early gastrula lateral margin grafted at the animal pole of a host blastula induce organization of posterior head and trunk tissues ([6]; Fig. 2D). Therefore these studies revealed that the lateral and ventral blastula/gastrula margin carries multiple organizing activities: the lateral margin organizes the posterior part of the head and the trunk while the ventral margin organizes the tail. In contrast, cells of the dorsal margin differentiate into axial tissues but have little organizing activity when placed in a neutral territory. Altogether, analyzes performed in zebrafish established that instead of being restricted to the dorsal most territory (the Spemann/dorsal organizer), the organizing properties present in the embryo are distributed all along the blastula/gastrula margin (Fig. 2E), which acts as a continuous organizer [6]. Gene expression studies have revealed that the pieces of margin that, when grafted into the animal pole of a blastula, organize ectopic tail, trunk or posterior head do not contain any of the molecular activities such as BMP antagonists (Noggin1 – Nog1 [7], Chordin – Chd [8], Follistatin like 1b – Fstl1b [9]), the Wnt antagonist Sfrp3/Frzb [10] or transcription factors (such as Dharma [11], Goosecoid [12], or FoxA3 [13]) identified as mediators of the function of the Spemann organizer. It is therefore clear that the

Fig. 2. The entire blastula/gastrula margin acts as an organizer. (A) Grafts taken from various portions of the early gastrula margin (green: ventral margin; orange: lateral margin; red: dorsal margin) of a donor embryo at early gastrula (shield stage), when transplanted into the animal pole of a blastula (sphere stage) embryo give rise (B) to formation of ectopic axial mesendoderm for grafts from the dorsal margin (the Spemann organizer), (C) to formation of a tail that contains (right panel) cells from the graft (labeled in green) as well as cells recruited from the host (unlabeled) for grafts from the ventral margin and (D) to formation of posterior head and trunk for grafts from the lateral margin (ant. trunk: anterior trunk; ov: otic vesicle). These experiments demonstrate that each portion of the embryonic margin (ventral, lateral, dorsal) has organizing properties. Therefore, while in the classical view the embryo is organized by the dorsal marginal tissue (Spemann organizer, (B)), (E) the organizing activities in the embryo are in fact distributed all along the blastula/gastrula margin, which acts as a global organizer.


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molecular process(es) that result in the organization of a tail, trunk and posterior head are independent from those described for the function of the classic Spemann organizer. 3. Molecular nature of the organizing activities of the blastula/gastrula margin The blastula/early gastrula margin is the embryonic territory where most of the major signaling pathways involved in early embryonic development: Nodal, FGF, non-canonical Wnt, retinoic acid and the zygotic canonical Wnt/␤-catenin are active. This is also true for BMP signaling but in this case this pathway is active in the whole ventral half of the embryo from the margin to the animal pole [14]. To identify which of these signals or combination of signals may be responsible for the organizing properties of blastula/gastrula marginal cells, expression of the different ligands activating these signaling pathways has been induced at the animal pole of embryos by injection of in vitro synthesized mRNAs coding for these factors into a single animal pole blastomere at the 128-cell stage (Fig. 3A). This injected blastomere gives rise to a clone of cells

that secrete ligand(s) translated from the injected mRNA(s). This results in the formation of a gradient of activity for these factors, which is maximal in the center of the secreting clone and that decreases progressively as one moves further away from the secreting cells. Injection of mRNA coding for BMPs (BMP2b, BMP7 or BMP4) has almost no effect on the embryo morphology ([6,15]; Fig. 3C). Because bmp gene expression, which starts soon after the midblastula transition, is very strong at the animal pole until the end of the blastula stage [16–18], adding more BMP transcripts in this region of the embryo at the blastula stage has little or no effect. In contrast, injection of mRNA coding for Nodal (either Ndr1/Squint or Ndr2/Cyclops) induces animal pole cells to form an ectopic notochord ([19]; Fig. 3D). This local gain of function mimics the effect of grafting dorsal marginal cells of an early gastrula at the animal pole of a host blastula (Fig. 2B) strongly supporting that Nodal induces the properties associated with the Spemann organizer. In agreement with this hypothesis, localized gain of function of Nodal at the ventral blastula/gastrula margin induces formation of a complete secondary axis [6,20]. This reproduces exactly the results of Spemann–Mangold grafting experiments performed in fish [21].

Fig. 3. The organizing properties of the blastula/gastrula margin depend on the ratio of Nodal to BMP activity. (A) Injection of a mix of BMP, Nodal and GFP mRNAs into one animal pole blastomere at the 128-cell stage gives rise to a clone of cells secreting BMP and Nodal at the animal pole of the embryo (illustrated here at late blastula stage). (B) Schematic presenting the territories where BMP (green, ventral half of the embryo) and Nodal signaling (red, margin) pathways are active at the onset of gastrulation. The curve at the bottom illustrates schematically the activity of these signals along the blastula/gastrula margin for BMP (green curve), high in the ventral domain and progressively decreasing to be null at the dorsal margin while Nodal activity is present all along the margin with more Nodal activity in the dorsal most territories. Therefore the BMP/Nodal ratio of activity is high ventrally and decreases progressively toward dorsal marginal position. (C) Injection of BMP mRNA and GFP mRNA at the animal pole does not result in formation of ectopic tissue (the BMP expressing cells populate preferentially the ventral epidermis). (D) Injection of Nodal mRNA alone induces formation of an ectopic notochord, growing in the head. This reproduces precisely the properties of grafts of the dorsal margin (the Spemann organizer) at the animal pole of a blastula embryo. (E) When the ratio between BMP mRNA/Nodal mRNA injected is high, this results in the organization of an ectopic tail at the animal pole, from animal pole cells. This tail contains both cells derived from the injected blastomere as well as cells from the animal pole (labeled in green) that have been recruited in the ectopic structure. Injection of this RNA mix with a high BMP/Nodal ratio perfectly reproduces the organizing activity of the ventral embryonic margin (Fig. 2C). (F) Injection of a mixture of BMP mRNA and Nodal mRNA with medium value for the BMP/Nodal ratio results in organization of trunk structures while (G) injection of a low ratio of BMP mRNA and Nodal mRNA results in organization of an ectopic posterior head (ot: otic vesicle). These medium and low ratios of BMP/Nodal signaling reproduce the organizing activity identified at the lateral margin (Fig. 2D).


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Stimulation of animal pole cells by the Wnt/␤-catenin signaling pathway through injection of Wnt8a mRNA leads to outcomes sim204 ilar to those observed upon Nodal injection [5]. However, at the 205 128-cell stage, expression of a canonical Wnt ligand, such as Wnt8a, 206 causes the activation of the maternal ␤-catenin pathway. There207 fore stimulation of the animal pole cells by a Wnt ligand does not 208 reproduce the physiological function of the zygotic Wnt/␤-catenin 209 pathway at the late blastula/early gastrula stage. Instead, activa210 tion of the maternal ␤-catenin pathway results in transcription of 211 nodal genes and therefore it is likely that the effects observed for 212 the ectopic expression of Wnt8a at the animal pole are, in fact, 213 mediated by Nodal. 214 Territories in which BMP and Nodal are active overlap exten215 sively: BMP activity is present in the ventral half of the embryo at 216 the late blastula/early gastrula stage [14] while Nodal signaling is 217 observed all along the margin with increased activity on the dorsal 218 most territory (as revealed with the expression of Antivin/Lefty1, 219 Q3 a feedback antagonist and direct target of Nodal [22,23]). By 220 analyzing the relative level of activity for these two pathways 221 (Fig. 3B) it became clear that, from the ventral to the dorsal 222 margin, there is a continuous variation in the ratio of BMP to 223 Nodal activity: high at the ventral side, low dorso-laterally and 224 null in the dorsal most domain where only Nodal is active. 225 Therefore, each domain of the margin from the ventral to the 226 dorsal side is characterized by a specific BMP/Nodal ratio of 227 activity. 228 The role of the combined activity of these two signaling path229 ways has been probed by stimulating the animal pole territory by 230 injection into a single blastomere at the 128-cell stage of various 231 ratios of BMP and Nodal mRNAs (Fig. 3A). In these experiments, it 232 is assumed that, within the range of mRNAs injected (3–250 pg), 233 the amount of ligands (BMP or Nodal) translated and secreted is 234 proportional to the amount of mRNA injected. 235 Injection of a mixture of mRNAs with a high BMP/Nodal ratio 236 (20–25× more BMP4 than Nodal related 2 – Ndr2 – mRNA for 237 example) induces formation of an ectopic tail growing at the ani238 mal pole from animal pole cells. It contains cells derived from 239 the injected blastomere (labeled in green) as well as animal pole 240 cells recruited in response to the stimulation by these two mor241 phogens ([5,6]; Fig. 3E). This ectopic tail is similar to the tail that 242 is induced by grafting a piece of ventral margin at the animal pole 243 of a host blastula. It comprises epidermis, mesoderm and neural 244 tube but lacks the axial tissues (notochord, floor plate and the ven245 tral spinal cord). Therefore, stimulation of animal pole cells with 246 this ratio of BMP/Nodal activity fully reproduces the organizing 247 activity of ventral marginal cells leading to organization of the 248 tail. 249 Injection of BMP and Nodal mRNAs with a decreasing BMP to 250 Nodal ratio reproduces conditions of signaling present at the lat251 eral and dorso-lateral margin and induces formation of a secondary 252 trunk (for 5× more BMP than Nodal mRNAs – Fig. 3F) or of a sec253 ondary posterior head (when BMP and Nodal mRNAs are injected 254 in the same quantity – Fig. 3G). 255 Altogether, the organizing properties of the different por256 tions of the margin identified in grafting experiments can be 257 fully mimicked by stimulating animal pole cells with different 258 ratios of BMP and Nodal activity that reproduce, in this presump259 tive cephalic region, the conditions of relative stimulation for 260 these two signaling pathways at the margin of the blastula/early 261 gastrula. 262 Most importantly, injection of serial dilutions (up to 16×) of 263 BMP/Nodal RNA mixes with, for example, 25 times more BMP4 264 mRNA that Ndr2 mRNA (a ratio that gives rise to ectopic tail, Fig. 3E) 265 always gives rise to formation of a tail. This demonstrates that only 266 the ratio between BMP and Nodal activity matters and not their 267 absolute levels of activity [6]. 202 203

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4. Organization of a complete embryonic axis with opposing gradients of BMP and Nodal Analyzes described above clearly show that the organization of the embryonic axis results from intrinsic properties distributed along the blastula/gastrula margin and depends on the activity of two gradients of TGF␤ superfamily members, BMP and Nodal. A given ratio of BMP/Nodal activity can reproduce at the animal pole the conditions that exist at a particular position along the dorsoventral axis of the endogenous blastula/gastrula margin. However, the organization of the embryo as a whole requires the complete set of BMP/Nodal activities with the BMP/Nodal ratio progressively decreasing from ventral to dorsal (Fig. 3B). Therefore, every condition reproducing a continuous variation of the BMP/Nodal ratio of activity within a field of naive pluripotent cells able to respond to these signals results in their organization into a complete embryonic axis. The Spemann–Mangold grafting experiment in which the dorsal blastopore lip of a donor embryo is transplanted into the ventral position of a host gastrula is the first experimental condition that meets this requirement. As illustrated in Fig. 4A, grafting the embryonic shield (the zebrafish Spemann organizer) of one embryo into the ventral marginal territory of a host gastrula stage embryo corresponds to moving cells secreting BMP and Wnt antagonists (such as Chordin or Noggin1 for BMP and Frzb [13,24] for Wnt) into a territory of high BMP and Wnt activity. This results in a local inhibition of the canonical Wnt/␤-catenin signaling and in the formation of a BMP gradient. This generates ventrally a mirror image duplication of the Wnt and BMP gradients of activity present on the dorsal half of a wild-type gastrula. In the grafted embryos, the lateral marginal territory is the place where the BMP/Nodal ratio of activity is the highest and this ratio decreases progressively and continuously both toward the dorsal and the ventral margin. Consequently, a complete secondary embryonic axis is organized on the ventral side of the gastrula. It contains head and trunk territories as well as axial mesoderm but is fused with the primary axis at its posterior end. A continuous variation of the BMP/Nodal ratio of activity can also be obtained by injecting BMP and Nodal mRNAs into two different animal pole blastomeres at the 128-cell stage (Fig. 4B). This results in the formation of two gradients of these morphogens, centered on the clones secreting each factor. In the domain where these two gradients overlap, the BMP/Nodal ratio of activity varies continuously, decreasing from the BMP secreting center toward the Nodal-expressing clone. This recapitulates, at the animal pole, conditions observed at the blastula/gastrula margin and results in forming a complete secondary embryonic axis organized at the animal pole from animal pole cells ([15]; Fig. 4B). Because the BMP signaling pathway is active at the animal pole, a clone of cells secreting Nodal in that territory generates a gradient of activity of this factor that should overlap with the endogenous BMP activity. Nodal signaling, however, strongly induces transcription of BMP antagonists such as Chordin. Chordin diffuses away from the Nodal clone and antagonizes the endogenous activity of BMP at long distance preventing interaction between the Nodal secreting center and the endogenous BMP activity (Fig. 4C). This is why a strong overexpression of BMP close to the Nodal-secreting clone is needed to allow interaction between both signaling pathways (Fig. 4B). In this case, the excess of BMP ligand is able to overcome the antagonistic activity of Chordin and allows for an overlap between the activity gradients of the two signaling pathways. However, in a homozygous chordin mutant embryo, expression of Nodal at the animal pole is sufficient to induce formation of an ectopic embryonic axis ([6]; Fig. 4D). In this mutant, in the absence of Chordin activity, the other BMP antagonists induced by Nodal (Noggin1 and Follistatin1b) inhibit BMP activity at a short


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Fig. 4. Organization of embryonic axes from interaction between two opposing gradients of BMP and Nodal. (A) BMP and Nodal activities in a Spemann–Mangold grafting experiment. Drawing on the left depicts the graft of dorsal margin of a donor embryo (expressing the GFP) to the ventral margin of a host gastrula. Curves on the left represent the activity of BMP (green) and of Nodal (red) at the margin in a wild-type embryo from ventral (V) to dorsal (D). The BMP/Nodal ratio of activity varies continuously, decreasing from ventral to dorsal (med.: medium). Grafting dorsal marginal tissue in ventral marginal territory (blue arrow) moves cells secreting BMP antagonists such as Chordin (Chd) and Wnt antagonists such as Frzb into a territory of high BMP and Wnt activities. This results in a local inhibition of Wnt and in the formation of a gradient of BMP (curves on the right). Activity of BMP and Nodal (curve on the right) appear as a mirror image duplication (open arrows) of the curves of BMP and Nodal activity in the dorsal half of a wild-type embryo. Because both halves of the embryo display a continuous variation of the Nodal to BMP ratio of activity, this results in formation of two identical embryonic axes (far right image) fused in their posterior end. The secondary axis contains cells derived from the graft that express the GFP as well as unlabeled cells recruited from the ventral domain of the host. (B) Experimental engineering of opposing gradients of BMP and Nodal by injection of two separate animal pole blastomeres at the 128-cell stage (left). At late blastula, clones of cells secreting these factors generate two gradients of these morphogens that recapitulate, in the domain where they overlap, the continuous variation of the Nodal to BMP ratio of activity existing at the endogenous embryonic margin. This leads to the formation of a complete secondary embryonic axis, organized at the animal pole from animal pole cells, that contains all organs and tissues present in the primary embryonic axis, in particular the axial mesendoderm: notochord (not) and floor plate (fp) visualized (far right image) for an in situ hybridization using sonic hedgehog a (shha) as a probe. (C) Injection of a single blastomere of the animal pole of a wild-type (WT) embryo at the 128-cell stage with Nodal mRNA gives rise to a clone of cells secreting Nodal at the early gastrula stage (drawing on the left in animal pole view). The Nodal clone (red) induces expression and secretion of Chordin, which antagonizes at a long distance BMP ligands present at the animal pole (endogenous – endog. – BMP). This prevents interaction between Nodal and BMP signaling pathways as illustrated by the curve in the center for the activity of BMP and Nodal between the ventral side (V) and the animal pole (AP). In this situation, Nodal signaling acting alone induces only formation of an ectopic notochord (not; green, picture on the right). (D) The same experiment shown in Panel C but performed using a chordin homozygous mutant embryo (chd−/−). In the absence of Chordin, BMP activity present at the animal pole is antagonized at a short distance from the center of the Nodal clone by Noggin 1 (Nog1) and Follistatin like 1b (Fstl1). They generate a gradient of BMP that overlaps the ectopic gradient of Nodal activity. This recapitulates the continuous variation of BMP/Nodal ratio of activity (curves on the center) observed for the endogenous margin and results in the formation of a complete secondary embryonic axis organized at the animal pole, from the animal pole cells, that includes axial mesodermal tissue. (i) primary axis, (ii) secondary axis.

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distance from the center of the Nodal-secreting clone. In this condition, the gradient of Nodal activity formed at the animal pole overlaps with the endogenous BMP activity and a continuous variation of the BMP/Nodal ratio of activity can be observed between the Nodal secreting clone and the ventral part of the animal pole where BMP signaling is active. The respective contribution of each signaling pathway to the formation of the ectopic secondary axis has been analyzed in details [15]. As mentioned before, addition of BMP activity at the animal pole has no effect on embryo morphology (Fig. 3C) while expression of Nodal in that territory induces formation of axial mesendoderm (Fig. 3D). At early developmental stages, stimulation of animal pole cells by Nodal results in the activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway that provides guidance cues for convergence movements in the zebrafish gastrula [25,26]. This results in formation of a protrusion (Fig. 5A, left), which is equivalent to the thickening of the dorsal margin that forms at the late blastula stage and gives rise to the embryonic shield. At the gastrula stage the central part of the protrusion, where Nodal signaling is highest, internalizes and forms a blastopore with a radially symmetrical blastopore lip where

the mesoderm involutes (Fig. 5A, right). At late blastula and early gastrula stages, Nodal induces expression of genes from the dorsal and dorso-lateral margin (Fig. 5B). Because Nodal is a morphogen, its target genes are expressed in concentric circles with the genes induced for the highest level of activity in the center and genes whose induction requires less stimulation induced in circles progressively more distant from the center (Fig. 5B). Notably, amongst the genes induced by Nodal is the posteriorizing factor Wnt8a. Expression of this gene within the blastopore lip induces formation of a Wnt8a gradient that establishes the antero-posterior polarity of the structure induced by Nodal (Fig. 5C). Altogether, Nodal induces radially symmetrical structures of dorsal and dorso-lateral identities, which are patterned along an antero-posterior axis. A radial symmetry is also observed for cell movements (Fig. 5D). Activation of the JAK/STAT3 pathway in the center of the Nodal-secreting clone creates an attraction center toward which surrounding cells converge and radial convergent extension movements elongate the notochord straight out of the animal pole along the vegetal-animal axis of the embryo. Addition of a clone secreting BMP next to the Nodal-expressing domain, breaks the radial symmetry leading to a bilateral


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Fig. 5. Contribution of Nodal and BMP to the organization of the embryonic axis. (A–D) Injection of Nodal mRNA into an animal pole blastomere at the 128-cell stage results (A) in formation of a protrusion (pr) at late blastula stage. At early gastrula, the central part of this protrusion internalizes and generates a blastopore (bp) surrounded by a radially symmetrical blastopore lip. (B) Double color in situ hybridization for genes induced by Nodal at the onset of gastrulation in the blastopore lip (bpl). notail (ntl, red) labels the whole blp. The prechordal plate (pp) marker frzb and the endoderm (endo) marker sox32 are expressed in the first cells that internalize and which are located in the center of the notail-expressing ring. The notochord (not) marker, sonic hedgehog a (shha), is expressed in the inner part of the bpl while expression of the posteriorization factor wnt8a is observed in the outer part of the bpl that corresponds to the dorso-lateral margin (dlm). (C) Wnt8a defines the antero (a) – posterior (p) polarity of the primary embryonic axis (green) and of the bpl structure induced by Nodal (red). The posterior territory is where Wnt8a activity is maximal. (D) Because cell movements are radially symmetric, at the late gastrula stage, the ectopic notochord induced by Nodal at the animal pole elongates straight out of the animal pole along the vegetal-animal axis (arrow). (E–G) Presence of a clone secreting BMP (brown) close to the blastopore lip induced by Nodal patterns this structure, restricting (E) expression of dorsal markers, frzb, chordin (chd) and shha to the portion of the blastopore lip opposite to the BMP secreting clone and inducing expression of eve1, a marker for the ventral and lateral margin (vlm) in the portion the blp close to the BMP secreting cells. (F) Blastopore lip seen in lateral view with position of the BMP (green) and Nodal (red) clones. BMP signaling polarizes cell movements (green open arrow, picture on the right) and cells converge toward the side of the bpl opposite to the BMP center. As a result the bpl becomes asymmetrical (double arrow) with the dorsal bpl (away from BMP) becoming thicker and the ventral bpl (close to the BMP center) becoming thinner. (G) Secondary embryonic axis growing at the animal pole seen in lateral view. Due to polarization of cell movements by BMP, the prechordal plate (pp) and the notochord (not) migrate anteriorly (a) away from the BMP secreting center. Consequently, when the axis elongates posteriorly (p) at the end of gastrulation the BMP expressing clone will be in posterior ventral position. (H) The animal pole region of an embryo, which had been instructed with two clones of BMP and Nodal, is explanted at the 512-cell stage and cultured in vitro, where it becomes spherical after 1 h in culture medium. Nodal induces gastrulation of the explant while BMP patterns and polarizes the gastrulating explant, which is very similar to an amphibian gastrula and displays clear antero-posterior (a–p) and dorso-ventral (d–v) axes as revealed in an in situ hybridization for the epidermal marker foxi1. When allowed to develop for a day in culture medium, the gastrulating explants form embryoids (far right image shows one such embryoid in lateral view, anterior to the left, dorsal to the top), which are very similar to wild-type embryos and in which organs are easily recognizable. bc: blastocoel; dbp: dorsal blastopore lip; ep: epidermis; hb: hindbrain; sc: spinal cord; fb: forebrain; np: neural plate; m: mesoderm. (I) Dorsal view of the head of an embryoid resulting from a fusion of an animal pole explant instructed with Nodal and BMP with a non instructed explant (that increases the number of cells that can be instructed by the morphogen gradients) showing a clear bilateral symmetry with well differentiated eyes. ret: retina; le: lens.

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symmetry. First, BMP signaling directly induces expression of the transcriptional repressors vent and vox (our unpublished observation). This limits the expression territory of dorsal specific genes such as chordin, frzb or sonic hedgehog (Fig. 5E) to the side of the blastopore lip opposite to the BMP activity center. Therefore cells close to the BMP secreting clone acquire ventral and lateral identity (Fig. 5E). Finally, by negatively regulating Ca(2+)/Cadherindependent cell–cell adhesiveness, the BMP gradient directs the migration of lateral mesodermal cells toward the dorsal part of the blastopore lip as it does in a normal gastrula stage embryo [27]. As a result (Fig. 5F), the blastopore lip becomes thicker on its dorsal side (away from the BMP signaling center) and thinner in proximity to the BMP secreting cells (the ventral side). These polarized cell movements affect the orientation of the growing embryonic axis. The prechordal plate, followed by the notochord, extends anteriorly away from the BMP signaling center and, when the embryonic axis elongates posteriorly after the end of gastrulation, this places the BMP secreting cells in a ventral posterior position (Fig. 5G). In consequence, the secondary embryonic axis organized by the two opposing gradients of Nodal and BMP always elongates posteriorly in the direction of the BMP signaling center.

The role of BMP and Nodal in organizing early embryonic cells has been further established in experiments that have been performed with animal pole explants cultured in vitro in order to rule out any contribution to the organizing activity from signals that may come from the yolk, the yolk syncytial layer or the endogenous blastula margin (Fig. 5H and I). Animal pole cells of zebrafish early blastula embryos do not contain any localized instructing signals at or before the midblastula transition (512-cell stage). In particular, nuclei of animal pole blastomeres are completely devoid of ␤-catenin [28] and only a few cells from the dorsal yolk syncytial layer and the dorsal margin display ␤-catenin in their nucleus at these early stages [29,30]. This is different from what is observed in amphibian embryos that display a preexistent pattern at the animal pole of the blastula [31] that depends on the endogenous dorso-ventral gradient of ␤-catenin [30]. Therefore, animal pole explants of a zebrafish early blastula are not equivalent to Xenopus animal caps and the animal pole blastomeres of zebrafish early blastulae are truly naive, pluripotent cells. Instruction of animal pole explants by injection of BMP and Nodal mRNAs into two different blastomeres (Fig. 5H) induces patterning and morphogenetic movements. After 1 h in culture,


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explants are spherical and differentiate an enveloping layer at their periphery. Three hours after explantation, surprisingly, they form a blastocoel, which is a fluid-filled central cavity within the blastula, observed in most vertebrate species but absent in zebrafish [32]. Two hours later, the initially spherical explants form a protrusion then internalization of the mesendoderm induced by Nodal occurs. Surprisingly, the gastrulating explants appear strikingly similar to the gastrula of amphibian embryos, with a blastocoel, a blastopore with an asymmetrical blastopore lip and the formation of the three germ layers suggesting that the zebrafish animal pole explant follows the prototypical gastrulation of the vertebrates that the presence of a huge unsegmented yolk makes difficult to recognize in a gastrulating zebrafish embryo. Analyzes by in situ hybridization with tissue specific molecular markers reveal the presence of clear antero-posterior and dorso-ventral axes. Interestingly, in these gastrulating embryoids the blastopore is in a dorsal position. This has long been known to be a characteristic of vertebrate gastrulae [33], the blastopore of other deuterostomes being in a vegetal/posterior position. If allowed to differentiate in culture these gastrulating explants give rise to embryoids that display tissues and organs very similar to those of a normal embryo (Fig. 5I) but with only 1/3 of the wildtype size. By fusing two explants, one which has not been instructed and the other that has been instructed with two clones secreting BMP and Nodal, embryoids of bigger size can be formed (about 2/3 of the size of a normal embryo) that frequently display clear bilateral symmetry (Fig. 5I). In optimal conditions well-differentiated organs such as eyes, otic vesicles, a notochord, and somites can be observed. Some embryoids contain a beating heart and show spontaneous muscle contractions indicative of a functional nervous system. Altogether, this in vitro approach confirms that the BMP and Nodal gradients are sufficient to instruct a group of naive, pluripotent embryonic cells to organize a complete embryonic axis without any contribution of spatially localized maternal determinants and independent from any influence of the endogenous signals from the blastula/gastrula margin, the yolk or the yolk syncytial layer.

5. Conclusion The studies performed during the past 25 years aiming at elucidating the origin of the organizing activity in vertebrate embryos have largely disregarded the role of Nodal. Nevertheless, this signaling is the primary zygotic signal responsive for the organization of the embryonic axis. Nodal can fully mimic the activity of the Spemann organizer [6,20]; however, its activity extends far beyond this. Nodal expression is induced at the dorsal margin as the result of the activity of the maternal ␤-catenin signaling pathway, which is activated by the dorsal determinant (Wnt8a in zebrafish [28]). In this dorsal territory, Nodal controls the initiation of dorsal convergence movements through the activation of the STAT3 signaling pathway. However, at the blastula stage, the initial dorso-marginal expression of Nodal rapidly spreads all around the margin where Nodal induces and patterns the mesendoderm and promotes internalization of endoderm and mesoderm germ layers at gastrulation. In addition, Nodal controls the expression of posteriorization factors. Therefore it acts as the upstream signal that defines the initial vegetal-animal polarity of the embryo, which is progressively converted into a definitive posterior to anterior axis. Finally, through induction of the expression of BMP antagonists, of FGF ligands and of transcriptional repressors at the dorsal margin, Nodal prevents the transcription of ventral morphogens in dorsal territories and controls the establishment of the BMP activity gradient.

BMP is the secondary signal. It patterns the embryo from ventral to dorsal providing positional information to ventral and lateral territories. It promotes dorsal convergence of lateral cells during gastrulation and defines the direction toward which the posterior part of the embryonic axis elongates after gastrulation. Combinations of these two signals are sufficient to pattern the embryo, instructing cells about their identities along both dorsoventral and antero-posterior axes and regulating their adhesivity and migration behavior. Definition of these different properties by Nodal and BMP activity gradients is the true origin of the organizing activities present along the blastula/gastrula margin. Therefore, the function of the Spemann organizer corresponds to a subset of the activities controlled by Nodal, that is those that prevent expression of ventral morphogens on the dorsal side of the embryo and that shape the gradient of activity of the BMP signaling pathway. In consequence, the Spemann organizer can be more accurately described as the dorsal patterning center of the embryo. Finally, the recent studies performed using the zebrafish model provide amazing and exciting results. Despite the high number of players shown to be necessary for early embryonic development and to be involved in the various mechanisms regulating patterning and morphogenesis (see Fig. 1, for example), only two initial signals, BMP and Nodal, acting at the top of a cascade of regulatory and inductive signals, are sufficient to trigger all necessary downstream developmental programs leading to the formation of a fully differentiated embryo. All vertebrates are derived from a common ancestor, which appeared about 540 million years ago in the early Cambrian era. Remarkably, genes responsible for the control of early embryonic development have been conserved among all vertebrate species and the expression pattern of the early embryonic genes is strikingly similar across the entire vertebrate subphylum. We anticipate that the observations made using the zebrafish embryo will be extended to all vertebrates’ species. Therefore, we predict that any field of naive, pluripotent cells able to respond to both BMP and Nodal signals, such as embryonic stem cells, can be organized by opposing two gradients of these morphogens, resulting in the formation of an embryonic axis. If this prediction is true, it should be possible to induce morphogenesis in vitro and therefore to take control of these pluripotent cells, organizing them into fully functional embryonic structures. This holds great implications for the future of regenerative medicine.

Acknowledgements We thank A. Agathon, J.-D. Fauny, K. Ferri-Lagneau, P.-F. Xu and N. Houssin for their participation to the different steps of the characterization of the organizing activities of the fish embryo. We also thank R.A. Bloodgood for his careful reading of the manuscript. This work was supported by funds from the University of Virginia. Q4

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Estrogen formation in the early rabbit embryo.

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Effective LARC Providers: Moving Beyond Training.

Pattern formation in the flowering plant embryo.