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.     

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.     

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.     

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.     

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     

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.     

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.     

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.     

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.     

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 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.     

Nuclear beta-catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos.

Studies in Xenopus laevis and zebrafish suggest a key role for beta-catenin in the specification of the axis of bilateral symmetry. In these organisms, nuclear beta-catenin demarcates the dorsalizing centers. We have asked whether beta-catenin plays a comparable role in the chick embryo and how it is adapted to the particular developmental constraints of chick development. The first nuclear localization of beta-catenin is observed in late intrauterine stages of development in the periphery of the blastoderm, the developing area opaca and marginal zone. Obviously, this early, radially symmetric domain does not predict the future organizing center of the embryo. During further development, cells containing nuclear beta-catenin spread under the epiblast and form the secondary hypoblast. The onset of hypoblast formation thus demarcates the first bilateral symmetry in nuclear beta-catenin distribution. Lithium chloride exposure also causes ectopic nuclear localization of beta-catenin in cells of the epiblast in the area pellucida. Embryos treated before primitive streak formation become completely radialized, as shown by the expression of molecular markers, CMIX and GSC. Lithium treatments performed during early or medium streak stages cause excessive development of the anterior primitive streak, node and notochord, and lead to a degeneration of prospective ventral and posterior structures, as shown by the expression of the molecular markers GSC, CNOT1, BMP2 and Ch-Tbx6L. In summary, we found that in spite of remarkable spatiotemporal differences, beta-catenin acts in the chick in a manner similar to that in fish and amphibia.     

O-fucosylation of thrombospondin type 1 repeats restricts epithelial to mesenchymal transition (EMT) and maintains epiblast pluripotency during mouse gastrulation

Thrombospondin type 1 repeat (TSR) superfamily members regulate diverse biological activities ranging from cell motility to inhibition of angiogenesis. In this study, we verified that mouse protein O-fucosyltransferase-2 (POFUT2) specifically adds O-fucose to TSRs. Using two Pofut2 gene-trap lines, we demonstrated that O-fucosylation of TSRs was essential for restricting epithelial to mesenchymal transition in the primitive streak, correct patterning of mesoderm, and localization of the definitive endoderm. Although Pofut2 mutant embryos established anterior/posterior polarity, they underwent extensive mesoderm differentiation at the expense of maintaining epiblast pluripotency. Moreover, mesoderm differentiation was biased towards the vascular endothelial cell lineage. Localization of Foxa2 and Cer1 expressing cells within the interior of Pofut2 mutant embryos suggested that POFUT2 activity was also required for the displacement of the primitive endoderm by definitive endoderm. Notably, Nodal, BMP4, Fgf8, and Wnt3 expression were markedly elevated and expanded in Pofut2 mutants, providing evidence that O-fucose modification of TSRs was essential for modulation of growth factor signaling during gastrulation. The ability of Pofut2 mutant embryos to form teratomas comprised of tissues from all three germ layer origins suggested that defects in Pofut2 mutant embryos resulted from abnormalities in the extracellular environment. This prediction is consistent with the observation that POFUT2 targets are constitutive components of the extracellular matrix (ECM) or associate with the ECM. For this reason, the Pofut2 mutants represent a valuable tool for studying the role of O-fucosylation in ECM synthesis and remodeling, and will be a valuable model to study how post-translational modification of ECM components regulates the formation of tissue boundaries, cell movements, and signaling.     

Signaling by FGF4 and FGF8 is required for axial elongation of the mouse embryo

Fibroblast growth factor (FGF) signaling has been shown to play critical roles in vertebrate segmentation and elongation of the embryonic axis. Neither the exact roles of FGF signaling, nor the identity of the FGF ligands involved in these processes, has been conclusively determined. Fgf8 is required for cell migration away from the primitive streak when gastrulation initiates, but previous studies have shown that drastically reducing the level of FGF8 later in gastrulation has no apparent effect on somitogenesis or elongation of the embryo. In this study, we demonstrate that loss of both Fgf8 and Fgf4 expression during late gastrulation resulted in a dramatic skeletal phenotype. Thoracic vertebrae and ribs had abnormal morphology, lumbar and sacral vertebrae were malformed or completely absent, and no tail vertebrae were present. The expression of Wnt3a in the tail and the amount of nascent mesoderm expressing Brachyury were both severely reduced. Expression of genes in the NOTCH signaling pathway involved in segmentation was significantly affected, and somite formation ceased after the production of about 15-20 somites. Defects seen in the mutants appear to result from a failure to produce sufficient paraxial mesoderm, rather than a failure of mesoderm precursors to migrate away from the primitive streak. Although the epiblast prematurely decreases in size, we did not detect evidence of a change in the proliferation rate of cells in the tail region or excessive apoptosis of epiblast or mesoderm cells. We propose that FGF4 and FGF8 are required to maintain a population of progenitor cells in the epiblast that generates mesoderm and contributes to the stem cell population that is incorporated in the tailbud and required for axial elongation of the mouse embryo after gastrulation.     

The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

The PAM-1 aminopeptidase regulates centrosome positioning to ensure anterior-posterior axis specification in one-cell C. elegans embryos

In the one-cell Caenorhabditis elegans embryo, the anterior-posterior (A-P) axis is established when the sperm donated centrosome contacts the posterior cortex. While this contact appears to be essential for axis polarization, little is known about the mechanisms governing centrosome positioning during this process. pam-1 encodes a puromycin sensitive aminopeptidase that regulates centrosome positioning in the early embryo. Previously we showed that pam-1 mutants fail to polarize the A-P axis. Here we show that PAM-1 can be found in mature sperm and in cytoplasm throughout early embryogenesis where it concentrates around mitotic centrosomes and chromosomes. We provide further evidence that PAM-1 acts early in the polarization process by showing that PAR-1 and PAR-6 do not localize appropriately in pam-1 mutants. Additionally, we tested the hypothesis that PAM-1's role in polarity establishment is to ensure centrosome contact with the posterior cortex. We inactivated the microtubule motor dynein, DHC-1, in pam-1 mutants, in an attempt to prevent centrosome movement from the cortex and restore anterior-posterior polarity. When this was done, the aberrant centrosome movements of pam-1 mutants were not observed and anterior-posterior polarity was properly established, with proper localization of cortical and cytoplasmic determinants. We conclude that PAM-1's role in axis polarization is to prevent premature movement of the centrosome from the posterior cortex, ensuring proper axis establishment in the embryo.     

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.     

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.