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.     

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.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.     

Primitive streak formation in mice is preceded by localized activation of Brachyury and Wnt3

The prevalent model for the generation of axial polarity in mouse embryos proposes that a radial to a linear transition in the expression of primitive streak markers precedes the formation of the primitive streak on one side of the epiblast. This model contrasts with the models of mesoderm formation in other vertebrates as it suggests that the primitive streak is initially established in a radial pattern rather than a localized region of the epiblast. Here, we examine the proposed correlation between the expression of Brachyury and Wnt3, two genes reported as expressed radially in the proximal epiblast, with the movements of proximal anterior epiblast cells at stages leading to the formation of the primitive streak. Our results reveal that neither Brachyury nor Wnt3 forms a ring of expression in the proximal epiblast as previously thought. In embryos dissected between 5.5 and 6.5 dpc, Brachyury is first expressed in the distal extra-embryonic ectoderm and subsequently on one side of the epiblast. Wnt3 expression is evident first in the posterior visceral endoderm of 5.5 dpc embryos and later in the posterior epiblast. Lineage analysis shows that the movements of the proximal epiblast do not restrict Brachyury expression to the posterior epiblast. Our data suggest a model whereby the localized expression of these genes in the posterior epiblast, and hence the formation of the primitive streak, is the result of local cell-cell interactions in the future posterior portion of the egg cylinder rather than regionalization of a radial pattern of expression in proximal epiblast cells.     

The marginal zone and its contribution to the hypoblast and primitive streak of the chick embryo

The marginal zone of the chick embryo has been shown to play an important role in the formation of the hypoblast and of the primitive streak. In this study, time-lapse filming, fate mapping, ablation and transplantation experiments were combined to study its contribution to these structures. It was found that the deep (endodermal) portion of the posterior marginal zone contributes to the hypoblast and to the junctional endoblast, while the epiblast portion of the same region contributes to the epiblast of the primitive streak and to the definitive (gut) endoderm derived from it. Within the deep part of the posterior marginal zone, a subpopulation of HNK-1-positive cells contributes to the hypoblast. Removal of the deep part of the marginal zone prevents regeneration of the hypoblast but not the formation of a primitive streak. Removal of both layers of the marginal zone leads to a primitive streak of abnormal morphology but mesendodermal cells nevertheless differentiate. These results show that the two main properties of the posterior marginal zone (contributing to the hypoblast and controlling the site of primitive streak formation) are separable, and reside in different germ layers. This conclusion does not support the idea that the influence of the posterior marginal zone on the development of axial structures is due to it being the source of secondary hypoblast cells.     

5-Bromodeoxyuridine inhibition of the epiblast competence for primitive streak formation in the young chick blastoderm

The competence of stage XIII chick epiblast which under the influence of an inductive hypoblast is directed to form a normal primitive streak, is affected by 5-bromodeoxyuridine (BUdR). The BUdR-treated epiblast forms an atypical primitive streak and no axial mesoderm. However, a nonorganized mesenchymal layer is formed between the epiblast and the hypoblast, and atypical neural tissue in the epiblast. BUdR interferes neither with hypoblast formation nor with its inductivity even when blastoderms are treated with BUdR as early as uterine stage VIII and later.     

Analysis of tissue flow patterns during primitive streak formation in the chick embryo

We have investigated the patterns of tissue flow underlying the formation of the primitive streak in the chick embryo. Analysis of time-lapse sequences of brightfield images to extract the tissue velocity field and of fluorescence images of small groups of DiI-labelled cells have shown that epiblast cells move in two large-scale counter-rotating streams, which merge at the site of streak formation. Despite the large-scale tissue flows, individual cells appear to move little relative to their neighbours. As the streak forms, it elongates in both the anterior and posterior directions. Inhibition of actin polymerisation via local application of the inhibitor latrunculin A immediately terminates anterior extension of the streak tip, but does not prevent posterior elongation. Inhibition of actin polymerisation at the base of the streak completely inhibits streak formation, implying that continuous movement of cells into the base of the forming streak is crucial for extension. Analysis of cycling cells in the early embryo shows that cell-cycle progression in the epiblast is quite uniform before the primitive streak forms then decreases in the central epiblast and incipient streak and increases at the boundary between the area pellucida and area opaca during elongation. The cell-cycle inhibitor aphidicolin, at concentrations that completely block cell-cycle progression, permits initial streak formation but arrests development during extension. Our analysis suggests that cell division maintains the cell-flow pattern that supplies the streak with cells from the lateral epiblast, which is critical for epiblast expansion in peripheral areas, but that division does not drive streak formation or the observed tissue flow.     

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.     

Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo

We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.     

Epithelial and basement membrane responses to chick embryo primitive streak grafts

Chick embryo primitive streak grafts, placed beneath the epiblast of host embryos, tend to result in the formation of either a neural plate in response to anterior streak grafts, or in de-epithelialization in response to posterior grafts. Ultrastructural and immunocytochemical examination shows that both reactions are preceded by basement membrane disruption and early removal of fibronectin therefrom. This disruption does not occur in response to non-streak grafts. It is suggested that the disruption, evoked by primitive streak cells, is a prerequisite first step, allowing direct graft-epiblast cell contact. This contact elicits a specific cytoskeletal reaction determining the epiblast response.     

Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation

The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal-distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. © 1995 Wiley-Liss, Inc.     

鸡胚原肠胚期原条细胞命运决定于其所处的局部微环境

在果蝇、斑马鱼、鸡等三胚层动物胚胎早期发育的原肠胚期,原条两侧的上胚层细胞进入原条经历上皮-间充质转化(EMT),迁移进入囊胚腔,形成松散的中胚层细胞,位于原条不同部位的细胞其迁移路线和分化命运不同,如前部原条细胞贡献于体节和心脏等,而后部原条细胞则迁移至胚外形成血岛。为了研究细胞的迁移途径及分化命运是否会随着细胞所处不同部位微环境的改变而改变,利用传统的移植技术,将宿主鸡胚原条前部的一部分细胞用GFP阳性的相同时期鸡胚原条组织替换,培养一段时间后,用荧光体视显微镜追踪GFP阳性细胞的迁移途径。结果发现,从供体原条后部移植到宿主原条前部的细胞遵循原条前部细胞迁移的路线,反之亦然;原位杂交结果显示移植后的GFP阳性细胞分化为所处部位的细胞类型。上述结果表明:鸡胚原肠胚期原条细胞迁移和分化的命运决定于细胞所处的微环境或者说局部基因表达的时空性。     

The enigmatic primitive streak: prevailing notions and challenges concerning the body axis of mammals

The primitive streak establishes the antero‐posterior body axis in all amniote species. It is thought to be the conduit through which mesoderm and endoderm progenitors ingress and migrate to their ultimate destinations. Despite its importance, the streak remains poorly defined and one of the most enigmatic structures of the animal kingdom. In particular, the posterior end of the primitive streak has not been satisfactorily identified in any species. Unexpectedly, and contrary to prevailing notions, recent evidence suggests that the murine posterior primitive streak extends beyond the embryo proper. In its extraembryonic site, the streak creates a node‐like cell reservoir from which the allantois, a universal caudal appendage of all amniotes and the future umbilical cord of placental mammals, emerges. This new insight into the fetal/umbilical relationship may explain the etiology of a large number of umbilical‐associated birth defects, many of which are correlated with abnormalities of the embryonic midline.     

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.     

Mesoderm patterning and somite formation during node regression: differential effects of chordin and noggin.

In Xenopus, one of the properties defining Spemann's organizer is its ability to dorsalise the mesoderm. When placed ajacent to prospective lateral/ventral mesoderm (blood, mesenchyme), the organizer causes these cells to adopt a more axial/dorsal fate (muscle). It seems likely that a similar property patterns the primitive streak of higher vertebrate embryos, but this has not yet been demonstrated clearly. Using quail/chick chimaeras and a panel of molecular markers, we show that Hensen's node (the amniote organizer) can induce posterior primitive streak (prospective lateral plate) to form somites (but not notochord) at the early neurula stage. We tested two BMP antagonists, noggin and chordin (both of which are expressed in the organizer), for their ability to generate somites and intermediate mesoderm from posterior streak, and find that noggin, but not chordin, can do this. Conversely, earlier in development, chordin can induce an ectopic primitive streak much more effectively than noggin, while neither BMP antagonist can induce neural tissue from extraembryonic epiblast. Neurulation is accompanied by regression of the node, which brings the prospective somite territory into a region expressing BMP-2, -4 and -7. One function of noggin at this stage may be to protect the prospective somite cells from the inhibitory action of BMPs. Our results suggest that the two BMP antagonists, noggin and chordin, may serve different functions during early stages of amniote development.     

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.     

Low proliferative and high migratory activity in the area of Brachyury expressing mesoderm progenitor cells in the gastrulating rabbit embryo

General mechanisms initiating the gastrulation process in early animal development are still elusive, not least because embryonic morphology differs widely among species. The rabbit embryo is revived here as a model to study vertebrate gastrulation, because its relatively simple morphology at the appropriate stages makes interspecific differences and similarities particularly obvious between mammals and birds. Three approaches that centre on mesoderm specification as a key event at the start of gastrulation were chosen. (1) A cDNA fragment encoding 212 amino acids of the rabbit Brachyury gene was cloned by RT-PCR and used as a molecular marker for mesoderm progenitors. Whole-mount in situ hybridisation revealed single Brachyury-expressing cells in the epiblast at 6.2 days post conception, i.e. several hours before the first ingressing mesoderm cells can be detected histologically. With the anterior marginal crescent as a landmark, these mesoderm progenitors are shown to lie in a posterior quadrant of the embryonic disc, which we call the posterior gastrula extension (PGE), for reasons established during the following functional analysis. (2) Vital dye (DiI) labelling in vitro suggests that epiblast cells arrive in the PGE from anterior parts of the embryonic disc and then move within this area in a complex pattern of posterior, centripetal and anterior directions to form the primitive streak. (3) BrdU labelling shows that proliferation is reduced in the PGE, while the remaining anterior part of the embryonic disc contains several areas of increased proliferation. These results reveal similarities with the chick with respect to Brachyury expression and cellular migration. They differ, however, in that local differences in proliferation are not seen in the pre-streak avian embryo. Rather, rabbit epiblast cells start mesoderm differentiation in a way similar to Drosophila, where a transient downregulation of proliferation initiates mesoderm differentiation and, hence, gastrulation.     

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     

Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.