Initiation of gastrulation in the mouse embryo is preceded by an apparent shift in the orientation of the anterior-posterior axis

BACKGROUND: It is generally assumed that the migration of anterior visceral endoderm (AVE) cells from a distal to a proximal position at embryonic day (E)5.5 breaks the radial symmetry of the mouse embryo, marks anterior, and conditions the formation of the primitive streak on the opposite side at E6.5. Transverse sections of a gastrulating mouse embryo fit within the outline of an ellipse, with the primitive streak positioned at one end of its long axis. How the establishment of anterior-posterior (AP) polarity relates to the morphology of the postimplantation embryo is, however, unclear. RESULTS: Transverse sections of prestreak E6.0 embryos also reveal an elliptical outline, but the AP axis, defined by molecular markers, tends to be perpendicular to the long axis of the ellipse. Subsequently, the relative orientations of the AP axis and of the long axis change so that when gastrulation begins, they are closer to being parallel, albeit not exactly aligned. As a result, most embryos briefly lose their bilateral symmetry when the primitive streak starts forming in the epiblast. CONCLUSIONS: The change in the orientation of the AP axis is only apparent and results from a dramatic remodeling of the whole epiblast, in which cell migrations take no part. These results reveal a level of regulation and plasticity so far unsuspected in the mouse gastrula.     

Distinct functions of the major Fgf8 spliceform, Fgf8b, before and during mouse gastrulation

The vertebrate Fgf8 gene produces multiple protein isoforms by alternative splicing. Two evolutionarily conserved spliceforms, Fgf8a and Fgf8b, exhibit distinct bioactivities, with Fgf8b having a more potent inductive activity due to higher affinity for Fgf receptors. To investigate the in vivo requirement for Fgf8b, we created a splice-site mutation abolishing Fgf8b expression in mice. Analysis of this mutant has uncovered a novel function of Fgf8 signaling before the onset of gastrulation. We show that the loss of Fgf8b disrupts the induction of the brachyury gene in the pregastrular embryo and, in addition, disrupts the proper alignment of the anteroposterior axis with the shape of the embryo and the uterine axes at embryonic day (E) 6.5. Importantly, Fgf8-null embryos display the same phenotype as Fgf8b-deficient embryos at E6.5, demonstrating that signaling by Fgf8b is specifically required for development of the pregastrular embryo. By contrast, during gastrulation, Fgf8a can partially compensate for the loss of Fgf8b in mesoderm specification. We show that an increased level of Fgf8a expression, which leads to Fgf4 expression in the primitive streak, can also promote mesoderm migration in the absence of Fgf8b. Therefore, different Fgf signals may have distinct requirements for the morphogenesis and gene regulation before and during gastrulation. Importantly, our findings implicate Fgf8 in the morphogenetic process that establishes the defined relationship between the axes of the embryo and the uterus at the beginning of gastrulation, a perplexing phenomenon discovered two decades ago.     

FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo

At the end of gastrulation in avians and mammals, the endoderm germ layer is an undetermined sheet of cells. Over the next 24-48 h, endoderm forms a primitive tube and becomes regionally specified along the anterior-posterior axis. Fgf4 is expressed in gastrulation and somite stage embryos in the vicinity of posterior endoderm that gives rise to the posterior gut. Moreover, the posterior endoderm adjacent to Fgf4-expressing mesoderm expresses the FGF-target genes Sprouty1 and 2 suggesting that endoderm respond to an FGF signal in vivo. Here, we report the first evidence suggesting that FGF4-mediated signaling is required for establishing gut tube domains along the A-P axis in vivo. At the gastrula stage, exposing endoderm to recombinant FGF4 protein results in an anterior shift in the Pdx1 and CdxB expression domains. These expression domains remain sensitive to FGF4 levels throughout early somite stages. Additionally, FGF4 represses the anterior endoderm markers Hex1 and Nkx2.1 and disrupts foregut morphogenesis. FGF signaling directly patterns endoderm and not via a secondary induction from another germ layer, as shown by expression of dominant-active FGFR1 specifically in endoderm, which results in ectopic anterior expression of Pdx1. Loss-of-function studies using the FGF receptor antagonist SU5402 demonstrate that FGF signaling is necessary for establishing midgut gene expression and for maintaining gene expression boundaries between the midgut and hindgut from gastrulation through somitogenesis. Moreover, FGF signaling in the primitive streak is necessary to restrict Hex1 expression to anterior endoderm. These data show that FGF signaling is critical for patterning the gut tube by promoting posterior and inhibiting anterior endoderm cell fate.     

Is the anterior-posterior axis of the fetus specified before implantation in the mouse?

The mouse conceptus is generally held to be radially symmetrical about its embryonic-abembryonic axis from the blastocyst stage until the primitive streak appears at the beginning of gastrulation. However, this notion has been challenged by recent observations on conceptuses sectioned in utero which suggest that the blastocyst is already bilaterally symmetrical when it begins to implant. Accordingly, the blastocyst has been assigned an anterior-posterior axis which appears to persist through gastrulation and is claimed to coincide with the anterior-posterior axis of the future fetus in both orientation and polarity. In the present investigation the relationship between these two axes was examined in conceptuses dissected from the uterus early in gastrulation so that it could be determined more accurately than is possible in situ. The anterior-posterior axis of the conceptus and nascent fetus were found to be either parallel or antiparallel to each other, suggesting that while the orientation of the fetal axis may be specified at the blastocyst stage its polarity is not.     

Control of early anterior-posterior patterning in the mouse embryo by TGF-beta signalling

Prior to gastrulation the mouse embryo exists as a symmetrical cylinder consisting of three tissue layers. Positioning of the future anterior-posterior axis of the embryo occurs through coordinated cell movements that rotate a pre-existing proximal-distal (P-D) axis. Overt axis formation becomes evident when a discrete population of proximal epiblast cells become induced to form mesoderm, initiating primitive streak formation and marking the posterior side of the embryo. Over the next 12-24 h the primitive streak gradually elongates along the posterior side of the epiblast to reach the distal tip. The most anterior streak cells comprise the 'organizer' region and include the precursors of the so-called 'axial mesendoderm', namely the anterior definitive endoderm and prechordal plate mesoderm, as well as those cells that give rise to the morphologically patent node. Signalling pathways controlled by the transforming growth factor-beta ligand nodal are involved in orchestrating the process of axis formation. Embryos lacking nodal activity arrest development before gastrulation, reflecting an essential role for nodal in establishing P-D polarity by generating and maintaining the molecular pattern within the epiblast, extraembryonic ectoderm and the visceral endoderm. Using a genetic strategy to manipulate temporal and spatial domains of nodal expression reveals that the nodal pathway is also instrumental in controlling both the morphogenetic movements required for orientation of the final axis and for specification of the axial mesendoderm progenitors.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Origin of body axes in the mouse embryo

How and at what stage of development are the axes of the body determined? The left-right axis of the mouse embryo is generated de novo at embryonic day (E) 8.0 in a manner dependent on pre-existing positional cues. The anterior-posterior (A-P) axis becomes apparent earlier when distally located visceral endoderm migrates toward the future anterior side at E5.5. The direction of this migration is predetermined by asymmetric expression of Lefty1 and Cerl1(Cerberus-like 1). Asymmetric expression of Lefty1 takes place even earlier, in the primitive endoderm of the implanting blastocyst, pushing back the origin of the A-P axis to the peri-implantation stage. Although its functional significance remains to be seen, studies on how this molecular asymmetry emerges may provide insight into the origin of A-P polarity. The first cell fate decision occurs by the morula stage. Although blastomeres at the two-cell or four-cell stage may have biased fates, it is currently unknown whether this bias has any causal relation to later fate.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Impact of node ablation on the morphogenesis of the body axis and the lateral asymmetry of the mouse embryo during early organogenesis.

The node of the mouse gastrula is the major source of the progenitor cells of the notochord, the floor plate, and the gut endoderm. The node may also play a morphogenetic role since it can induce a partial body axis following heterotopic transplantation. The impact of losing these progenitor cells and the morphogenetic activity on the development of the body axes was studied by the ablation of the node at late gastrulation. In the ablated embryo, an apparently intact anterior-posterior body axis with morphologically normal head folds, neural tube, and primitive streak developed during early organogenesis. Cell fate analysis revealed that the loss of the node elicits de novo recruitment of neural ectoderm and somitic mesoderm from the surrounding germ-layer tissues. This leads to the restoration of the neural tube and the paraxial mesoderm. However, the body axis of the embryo was foreshortened and somite formation was retarded. Histological and gene expression studies reveal that in most of the node-ablated embryos, the notochord in the trunk was either absent or interrupted, and the floor plate was absent in the ventral region of the reconstituted neural tube. The loss of the node did not affect the differentiation of the gut endoderm or the formation of the mid- and hindgut. In the node-ablated embryo, expression of the Pitx2 gene in the lateral plate mesoderm was no longer restricted to the left side but was found on both sides of the body or was completely absent from the lateral plate mesoderm. Therefore, the loss of the node results in the failure to delineate the laterality of the body axis. The node and its derivatives therefore play a critical role in the patterning of the ventral neural tube and lateral body axis but not of the anterior-posterior axis during early organogenesis.     

Inhibition of heart formation by lithium is an indirect result of the disruption of tissue organization within the embryo

Lithium is a commonly used drug for the treatment of bipolar disorder. At high doses, lithium becomes teratogenic, which is a property that has allowed this agent to serve as a useful tool for dissecting molecular pathways that regulate embryogenesis. This study was designed to examine the impact of lithium on heart formation in the developing frog for insights into the molecular regulation of cardiac specification. Embryos were exposed to lithium at the beginning of gastrulation, which produced severe malformations of the anterior end of the embryo. Although previous reports characterized this deformity as a posteriorized phenotype, histological analysis revealed that the defects were more comprehensive, with disfigurement and disorganization of all interior tissues along the anterior-posterior axis. Emerging tissues were poorly segregated and cavity formation was decreased within the embryo. Lithium exposure also completely ablated formation of the heart and prevented myocardial cell differentiation. Despite the complete absence of cardiac tissue in lithium treated embryos, exposure to lithium did not prevent myocardial differentiation of precardiac dorsal marginal zone explants. Moreover, precardiac tissue freed from the embryo subsequent to lithium treatment at gastrulation gave rise to cardiac tissue, as demonstrated by upregulation of cardiac gene expression, display of sarcomeric proteins, and formation of a contractile phenotype. Together these data indicate that lithium's effect on the developing heart was not due to direct regulation of cardiac differentiation, but an indirect consequence of disrupted tissue organization within the embryo.     

Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3

The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFβ target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.     

Retinoic acid can mimic endogenous signals involved in transformation of the Xenopus nervous system.

In the frog Xenopus laevis, signals from the mesoderm divert part of the ectoderm from an epidermal to a neural fate. In the course of neural induction, the neurectoderm also acquires anterior-posterior polarity. In this report, the early expression of two genes, XlHbox6 and the neurofilament gene XIF6, is examined. The pattern of expression of the two genes seen in the tailbud embryo develops progressively over a 4 hr period following gastrulation. Physiological concentrations of retinoic acid can mimic this effect in isolated embryonic explants, consistent with the involvement of retinoic acid, or a closely related molecule, in localizing gene expression along the anterior-posterior axis of the neural tube.     

Building the mouse gastrula: signals, asymmetry and lineages

The mouse embryo is built by assembling the progenitors of various tissue types into a body plan. Early postimplantation development involves the establishment of anatomical asymmetries and regionalized gene expression in the conceptus, the specification of tissue lineages, and the coordination of cell movement for correct positioning of the lineage progenitors before and at gastrulation. Recent findings reveal that Wnt and Tgfbeta signalling function is instrumental in delineating the anterior-posterior embryonic axis by defining the site of primitive streak formation and by directing the movement of the visceral endoderm. These signalling activities are also required for the specification of anterior and posterior fates of the epiblast cells and for the induction and navigation of the primordial germ cells.     

Predictability of dorso-ventral asymmetry in the cleavage stage zebrafish embryo: an analysis using lithium sensitivity as a dorso-ventral marker.

The dorso-ventral axis in zebrafish first becomes apparent at gastrulation, when the future ventral side appears thinner than the dorsal side. The exact time of establishment of the dorso-ventral axis is not known. We show here that the dorso-ventral axis is specified as early as the 32 cell stage. Using lithium as a marker for dorso-ventral asymmetry, we show that lithium-sensitivity is a characteristic of future ventral cell, but not future dorsal cells, and that there is an asymmetric lithium-sensitivity along the long axis of the 32 cell stage embryo. Consequently, the dorso-ventral axis corresponds to the long axis of the embryo. Because the effect of lithium treatment is short-lived, the dorso-ventral axis must be specified in zebrafish already at the 32 cell stage.     

Asymmetric expression of the BMP antagonists chordin and gremlin in the sea anemone Nematostella vectensis: implications for the evolution of axial patterning

The evolutionary origin of the anterior-posterior and the dorsoventral body axes of Bilateria is a long-standing question. It is unclear how the main body axis of Cnidaria, the sister group to the Bilateria, is related to the two body axes of Bilateria. The conserved antagonism between two secreted factors, BMP2/4 (Dpp in Drosophila) and its antagonist Chordin (Short gastrulation in Drosophila) is a crucial component in the establishment of the dorsoventral body axis of Bilateria and could therefore provide important insight into the evolutionary origin of bilaterian axes. Here, we cloned and characterized two BMP ligands, dpp and GDF5-like as well as two secreted antagonists, chordin and gremlin, from the basal cnidarian Nematostella vectensis. Injection experiments in zebrafish show that the ventralizing activity of NvDpp mRNA is counteracted by NvGremlin and NvChordin, suggesting that Gremlin and Chordin proteins can function as endogenous antagonists of NvDpp. Expression analysis during embryonic and larval development of Nematostella reveals asymmetric expression of all four genes along both the oral-aboral body axis and along an axis perpendicular to this one, the directive axis. Unexpectedly, NvDpp and NvChordin show complex and overlapping expression on the same side of the embryo, whereas NvGDF5-like and NvGremlin are both expressed on the opposite side. Yet, the two pairs of ligands and antagonists only partially overlap, suggesting complex gradients of BMP activity along the directive axis but also along the oral-aboral axis. We conclude that a molecular interaction between BMP-like molecules and their secreted antagonists was already employed in the common ancestor of Cnidaria and Bilateria to create axial asymmetries, but that there is no simple relationship between the oral-aboral body axis of Nematostella and one particular body axis of Bilateria.     

Building divergent body plans with similar genetic pathways

Deuterostome animals exhibit widely divergent body plans. Echinoderms have either radial or bilateral symmetry, hemichordates include bilateral enteropneust worms and colonial pterobranchs, and chordates possess a defined dorsal-ventral axis imposed on their anterior-posterior axis. Tunicates are chordates only as larvae, following metamorphosis the adults acquire a body plan unique for the deuterostomes. This paper examines larval and adult body plans in the deuterostomes and discusses two distinct ways of evolving divergent body plans. First, echinoderms and hemichordates have similar feeding larvae, but build a new adult body within or around their larvae. In hemichordates and many direct-developing echinoderms, the adult is built onto the larva, with the larval axes becoming the adult axes and the larval mouth becoming the adult mouth. In contrast, indirect-developing echinoderms undergo radical metamorphosis where adult axes are not the same as larval axes. A second way of evolving a divergent body plan is to become colonial, as seen in hemichordates and tunicates. Early embryonic development and gastrulation are similar in all deuterostomes, but, in chordates, the anterior-posterior axis is established at right angles to the animal-vegetal axis, in contrast to hemichordates and indirect-developing echinoderms. Hox gene sequences and anterior-posterior expression patterns illuminate deuterostome phylogenetic relationships and the evolution of unique adult body plans within monophyletic groups. Many genes that are considered vertebrate 'mesodermal' genes, such as nodal and brachyury T, are likely to ancestrally have been involved in the formation of the mouth and anus, and later were evolutionarily co-opted into mesoderm during vertebrate development.     

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.     

Axial patterning interactions in the sea urchin embryo: suppression of nodal by Wnt1 signaling

Wnt and Nodal signaling pathways are required for initial patterning of cell fates along anterior-posterior (AP) and dorsal-ventral (DV) axes, respectively, of sea urchin embryos during cleavage and early blastula stages. These mechanisms are connected because expression of nodal depends on early Wnt/β-catenin signaling. Here, we show that an important subsequent function of Wnt signaling is to control the shape of the nodal expression domain and maintain correct specification of different cell types along the axes of the embryo. In the absence of Wnt1, the posterior-ventral region of the embryo is severely altered during early gastrulation. Strikingly, at this time, nodal and its downstream target genes gsc and bra are expressed ectopically, extending posteriorly to the blastopore. They override the initial specification of posterior-ventral ectoderm and endoderm fates, eliminating the ventral contribution to the gut and displacing the ciliary band dorsally towards, and occasionally beyond, the blastopore. Consequently, in Wnt1 morphants, the blastopore is located at the border of the re-specified posterior-ventral oral ectoderm and by larval stages it is in the same plane near the stomodeum on the ventral side. In normal embryos, a Nodal-dependent process downregulates wnt1 expression in dorsal posterior cells during early gastrulation, focusing Wnt1 signaling to the posterior-ventral region where it suppresses nodal expression. These subsequent interactions between Wnt and Nodal signaling are thus mutually antagonistic, each limiting the range of the other's activity, in order to maintain and stabilize the body plan initially established by those same signaling pathways in the early embryo.     

Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation

Background  

To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region.