Interactions between Wnt and Vg1 signalling pathways initiate primitive streak formation in the chick embryo

The posterior marginal zone (PMZ) of the chick embryo has Nieuwkoop centre-like properties: when transplanted to another part of the marginal zone, it induces a complete embryonic axis, without making a cellular contribution to the induced structures. However, when the PMZ is removed, the embryo can initiate axis formation from another part of the remaining marginal zone. Chick Vg1 can mimic the axis-inducing ability of the PMZ, but only when misexpressed somewhere within the marginal zone. We have investigated the properties that define the marginal zone as a distinct region. We show that the competence of the marginal zone to initiate ectopic primitive streak formation in response to cVg1 is dependent on Wnt activity. First, within the Wnt family, only Wnt8C is expressed in the marginal zone, in a gradient decreasing from posterior to anterior. Second, misexpression of Wnt1 in the area pellucida enables this region to form a primitive streak in response to cVg1. Third, the Wnt antagonists Crescent and Dkk-1 block the primitive streak-inducing ability of cVg1 in the marginal zone. These findings suggest that Wnt activity defines the marginal zone and allows cVg1 to induce an axis. We also present data suggesting some additional complexity: first, the Vg1 and Wnt pathways appear to regulate the expression of downstream components of each other's pathway; and second, misexpression of different Wnt antagonists suggests that different classes of Wnts may cooperate with each other to regulate axis formation in the normal embryo.     

The hypoblast of the chick embryo positions the primitive streak by antagonizing nodal signaling

The hypoblast (equivalent to the mouse anterior visceral endoderm) of the chick embryo plays a role in regulating embryonic polarity. Surprisingly, hypoblast removal causes multiple embryonic axes to form, suggesting that it emits an inhibitor of axis formation. We show that Cerberus (a multifunctional antagonist of Nodal, Wnt, and BMP signaling) is produced by the hypoblast and inhibits primitive streak formation. This activity is mimicked by Cerberus-Short (CerS), which only inhibits Nodal. Nodal misexpression can initiate an ectopic primitive streak, but only when the hypoblast is removed. We propose that, during normal development, the primitive streak forms only when the hypoblast is displaced away from the posterior margin by the endoblast, which lacks Cerberus.     

A hierarchy of gene expression accompanying induction of the primitive streak by Vg1 in the chick embryo

In the chick embryo, two secreted factors have recently be shown to cooperate in inducing the first axial structure, the primitive streak: cWnt8C (normally expressed around the circumference of the embryo, in the marginal zone) and the TGF beta superfamily member cVg1 (expressed in the posterior part of the marginal zone) (Development 128 (2001) 2915). Misexpression of Vg1 in the anterior marginal zone induces an ectopic primitive streak and recapitulates the morphological changes associated with normal primitive streak formation. Here, we analyse the time-course of appearance and disappearance of expression of 12 genes (cVg1, Lef1, Nodal, FGF8, cWnt8C, cBra, cNot1, goosecoid, HNF3 beta, Chordin, Otx2 and Sox3, whose normal expression is also polarized at early stages of development) in response to cVg1 misexpression in the anterior marginal zone. We show that a hierarchy of gene expression accompanies induction of the ectopic axis, reminiscent of the order in which the same genes begin to be expressed in the normal embryo.     

Correct patterning of the primitive streak requires the anterior visceral endoderm

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.     

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     

Cell movement during chick primitive streak formation

Gastrulation in amniotes begins with extensive re-arrangements of cells in the epiblast resulting in the formation of the primitive streak. We have developed a transfection method that enables us to transfect randomly distributed epiblast cells in the Stage XI-XIII chick blastoderms with GFP fusion proteins. This allows us to use time-lapse microscopy for detailed analysis of the movements and proliferation of epiblast cells during streak formation. Cells in the posterior two thirds of the embryo move in two striking counter-rotating flows that meet at the site of streak formation at the posterior end of the embryo. Cells divide during this rotational movement with a cell cycle time of 6-7 h. Daughter cells remain together, forming small clusters and as result of the flow patterns line up in the streak. Expression of the cyclin-dependent kinase inhibitor, P21/Waf inhibits cell division and severely limits embryo growth, but does not inhibit streak formation or associated flows. To investigate the role off cell-cell intercalation in streak formation we have inhibited the Wnt planar-polarity signalling pathway by expression of a dominant negative Wnt11 and a Dishevelled mutant Xdd1. Both treatments do not result in an inhibition of streak formation, but both severely affect extension of the embryo in later development. Likewise inhibition of myosin II which as been shown to drive cell-cell intercalation during Drosophila germ band extension, has no effect on streak formation, but also effectively blocks elongation after regression has started. These experiments make it unlikely that streak formation involves known cell-cell intercalation mechanisms. Expression of a dominant negative FGFR1c receptor construct as well as the soluble extracellular domain of the FGFR1c receptor both effectively block the cell movements associated with streak formation and mesoderm differentiation, showing the importance of FGF signalling in these processes.     

Effects of truncated activin and FGF receptors and of follistatin on the inducing activities of BVg1 and activin: does activin play a role in mesoderm induction?

Activin and Vg1, two members of the TGF-beta family, are believed to play roles in mesoderm induction and axis formation in the amphibian embryo. Both molecules are provided maternally, either as protein (activin) or as RNA and protein (Vg1), and experiments with a truncated form of a type IIB activin receptor have led to the conclusion that activin is required for induction of mesoderm in vivo. In this paper we first show that truncated versions of two different Xenopus activin receptors also have severe effects on the activity of the mature region of Vg1, suggesting that such receptors may block the function of several members of the TGF-beta family. We go on to demonstrate that follistatin, a secreted protein which binds activin and blocks its activity, does not interfere with Vg1 signalling. Furthermore, overexpression of follistatin mRNA in Xenopus embryos does not perturb mesoderm formation. Taken together, our data show that the effects of truncated activin receptors on Xenopus development can be explained by the inhibition of Vg1 activity, while the lack of effect of follistatin argues against a function for activin in mesoderm induction.     

Tsukushi cooperates with VG1 to induce primitive streak and Hensen's node formation in the chick embryo

Three classes of signaling molecule, VG1, WNT and BMP, play crucial roles in axis formation in the chick embryo. Although VG1 and WNT signals have a pivotal function in inducing the primitive streak and Hensen's node in the embryo midline, their action is complemented by that of BMP antagonists that protect the prospective axial tissue from the inhibitory influence of BMPs secreted from the periphery. We have previously reported that a secreted factor, chick Tsukushi (TSK), is expressed in the primitive streak and Hensen's node, where it works as a BMP antagonist. Here, we describe a new crucial function for TSK in promoting formation of the primitive streak and Hensen's node by positively regulating VG1 activity. We provide evidence that TSK directly binds VG1 in vitro, and that TSK and VG1 functionally interact in axis formation, as shown by biological assays performed in chick and Xenopus embryos. Furthermore, we show that alternative splicing of TSK RNA leads to the formation of two isoforms (TSKA, originally designated as TSK, and TSKB) that differ in their C-terminal region. Biochemical and biological assays indicate that TSKB is a much weaker BMP antagonist than TSKA, although both isoforms efficiently interact with VG1. Remarkably, although both TSKA and TSKB are expressed throughout the early extending primitive streak, their expression patterns diverge during gastrulation. TSKA expression concentrates in Hensen's node, a well-known source of anti-BMP signals, whereas TSKB accumulates in the middle primitive streak (MPS), a region known to work as a node-inducing center where VG1 expression is also specifically localized. Loss-of-function experiments demonstrate that TSKB, but not TSKA, function is required in the MPS for induction of Hensen's node. Taken together, these results indicate that TSK isoforms play a crucial role in chick axis formation by locally modulating VG1 and BMP activities during gastrulation.     

Activation of epiblast gene expression by the hypoblast layer in the prestreak chick embryo

Axis formation is a highly regulated process in vertebrate embryos. In mammals, inductive interactions between an extra-embryonic layer, the visceral endoderm, and the embryonic layer before gastrulation are critical both for anterior neural patterning and normal primitive streak formation. The role(s) of the equivalent extra-embryonic endodermal layer in the chick, the hypoblast, is still less clear, and dramatic effects of hypoblast on embryonic gene expression have yet to be demonstrated. We present evidence that two genes later associated with the gastrula organizer (Gnot-1 and Gnot-2) are induced by hypoblast signals in prestreak embryos. The significance of this induction by hypoblast is discussed in terms of possible hypoblast functions and the regulation of axis formation in the early embryo. Several factors known to be expressed in hypoblast, and retinoic acid, synergistically induce Gnot-1 and Gnot-2 expression in blastoderm cell culture. The presence of retinoic acid in prestreak embryos has not yet been directly demonstrated, but exogenous retinoic acid appears to mimic the effects of hypoblast rotation on primitive streak extension, raising the possibility that retinoid signaling plays some role in the pregastrula embryo.     

Molecular interactions continuously define the organizer during the cell movements of gastrulation.

The organizer is a unique region in the gastrulating embryo that induces and patterns the body axis. It arises before gastrulation under the influence of the Nieuwkoop center. We show that during gastrulation, cell movements bring cells into and out of the chick organizer, Hensen's node. During these movements, cells acquire and lose organizer properties according to their position. A "node inducing center," which emits Vg1 and Wnt8C, is located in the middle of the primitive streak. Its activity is inhibited by ADMP produced by the node and by BMPs at the periphery. These interactions define the organizer as a position in the embryo, whose cellular makeup is constantly changing, and explain the phenomenon of organizer regeneration.     

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.     

FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity.

We have investigated the cell interactions and signalling molecules involved in setting up and maintaining the border between the neural plate and the adjacent non-neural ectoderm in the chick embryo at primitive streak stages. msx-1, a target of BMP signalling, is expressed in this border at a very early stage. It is induced by FGF and by signals from the organizer, Hensen's node. The node also induces a ring of BMP-4, some distance away. By the early neurula stage, the edge of the neural plate is the only major site of BMP-4 and msx-1 expression, and is also the only site that responds to BMP inhibition or overexpression. At this time, the neural plate appears to have a low level of BMP antagonist activity. Using in vivo grafts and in vitro assays, we show that the position of the border is further maintained by interactions between non-neural and neural ectoderm. We conclude that the border develops by integration of signals from the organizer, the developing neural plate, the paraxial mesoderm and the non-neural epiblast, involving FGFs, BMPs and their inhibitors. We suggest that BMPs act in an autocrine way to maintain the border state.     

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.     

HNF3beta and Lim1 interact in the visceral endoderm to regulate primitive streak formation and anterior-posterior polarity in the mouse embryo.

Recent embryological and genetic experiments have suggested that the anterior visceral endoderm and the anterior primitive streak of the early mouse gastrula function as head- and trunk-organising centers, respectively. Here, we report that HNF3beta and Lim1 are coexpressed in both organising centers suggesting synergistic roles of these genes in regulating organiser functions and hence axis development in the mouse embryo. To investigate this possibility, we generated compound HNF3beta and Lim1 mutant embryos. An enlarged primitive streak and a lack of axis formation were observed in HNF3beta (-)(/)(-);Lim1(-)(/)(-), but not in single homozygous mutant embryos. Chimera experiments indicate that the primary defect in these double homozygous mutants is due to loss of activity of HNF3beta and Lim1 in the visceral endoderm. Altogether, these data provide evidence that these genes function synergistically to regulate organiser activity of the anterior visceral endoderm. Moreover, HNF3beta (-)(/)(-);Lim1(-)(/)(-) mutant embryos also exhibit defects in mesoderm patterning that are likely due to lack of specification of anterior primitive streak cells.     

Formation of the avian primitive streak from spatially restricted blastoderm: evidence for polarized cell division in the elongating streak

Gastrulation in the amniote begins with the formation of a primitive streak through which precursors of definitive mesoderm and endoderm ingress and migrate to their embryonic destinations. This organizing center for amniote gastrulation is induced by signal(s) from the posterior margin of the blastodisc. The mode of action of these inductive signal(s) remains unresolved, since various origins and developmental pathways of the primitive streak have been proposed. In the present study, the fate of chicken blastodermal cells was traced for the first time in ovo from prestreak stages XI-XII through HH stage 3, when the primitive streak is initially established and prior to the migration of mesoderm. Using replication-defective retrovirus-mediated gene transfer and vital dye labeling, precursor cells of the stage 3 primitive streak were mapped predominantly to a specific region where the embryonic midline crosses the posterior margin of the epiblast. No significant contribution to the early primitive streak was seen from the anterolateral epiblast. Instead, the precursor cells generated daughter cells that underwent a polarized cell division oriented perpendicular to the anteroposterior embryonic axis. The resulting daughter cell population was arranged in a longitudinal array extending the complete length of the primitive streak. Furthermore, expression of cVg1, a posterior margin-derived signal, at the anterior marginal zone induced adjacent epiblast cells, but not those lateral to or distant from the signal, to form an ectopic primitive streak. The cVg1-induced epiblast cells also exhibited polarized cell divisions during ectopic primitive streak formation. These results suggest that blastoderm cells located immediately anterior to the posterior marginal zone, which secretes an inductive signal, undergo spatially directed cytokineses during early primitive streak formation.     

Establishment of anterior-posterior polarity in avian embryos.

In pre-streak chick embryos, the extraembryonic posterior marginal zone is able to induce an embryonic axis at an ectopic site without contributing cells to the induced primitive streak. This region expresses mesoderm-inducing factors that are capable of inducing an ectopic streak. Downstream of these events, chordin and bone morphogenetic protein acting within the central disc may play mutually opposing roles influencing streak formation. Although extraembryonic regions are important in establishing the embryonic axis, there does not appear to be an anterior region with head-inducing activity similar to that of the anterior visceral endoderm of the mammalian embryo.     

Acquisition of Hox codes during gastrulation and axial elongation in the mouse embryo

Early sequential expression of mouse Hox genes is essential for their later function. Analysis of the relationship between early Hox gene expression and the laying down of anterior to posterior structures during and after gastrulation is therefore crucial for understanding the ontogenesis of Hox-mediated axial patterning. Using explants from gastrulation stage embryos, we show that the ability to express 3' and 5' Hox genes develops sequentially in the primitive streak region, from posterior to anterior as the streak extends, about 12 hours earlier than overt Hox expression. The ability to express autonomously the earliest Hox gene, Hoxb1, is present in the posterior streak region at the onset of gastrulation, but not in the anterior region at this stage. However, the posterior region can induce Hoxb1 expression in these anterior region cells. We conclude that tissues are primed to express Hox genes early in gastrulation, concomitant with primitive streak formation and extension, and that Hox gene inducibility is transferred by cell to cell signalling. Axial structures that will later express Hox genes are generated in the node region in the period that Hox expression domains arrive there and continue to spread rostrally. However, lineage analysis showed that definitive Hox codes are not fixed at the node, but must be acquired later and anterior to the node in the neurectoderm, and independently in the mesoderm. We conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node.     

Nodal specifies embryonic visceral endoderm and sustains pluripotent cells in the epiblast before overt axial patterning

Anteroposterior (AP) polarity in the mammalian embryo is specified during gastrulation when naive progenitor cells in the primitive ectoderm are recruited into the primitive streak to form mesoderm and endoderm. At the opposite pole, this process is inhibited by signals previously induced in distal visceral endoderm (DVE). Both DVE and primitive streak formation, and hence positioning of the AP axis, rely on the TGFbeta family member Nodal and its proprotein convertases Furin and Pace4. Here, we show that Nodal and Furin are initially co-expressed in the primitive endoderm together with a subset of DVE markers such as Lefty1 and Hex. However, with the appearance of extra-embryonic ectoderm (ExE), DVE formation is transiently inhibited. During this stage, Nodal activity is essential to specify embryonic VE and restrict the expression of Furin to the extra-embryonic region. Activation of Nodal is also necessary to maintain determinants of pluripotency such as Oct4, Nanog and Foxd3 during implantation, and to stimulate elongation of the egg cylinder, before inducing DVE and germ layer formation. We conclude that Nodal is already activated in primitive endoderm, but induces a functional DVE only after promoting the expansion of embryonic VE and pluripotent progenitor cells in the epiblast.