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 model of primitive streak initiation in the chick embryo

Initiation of the primitive streak in avian embryos provides a well-studied example of a pattern-forming event that displays a striking capacity for regulation. The mechanisms underlying the regulative properties are, however, poorly understood and are not easily accounted for by traditional models of pattern formation, such as reaction-diffusion models. In this paper, we propose a new activator-inhibitor model for streak initiation. We show that the model is consistent with experimental observations, both in its pattern-forming properties and in its ability to form these patterns on the correct time-scales for biologically realistic parameter values. A key component of the model is a travelling wave of inhibition. We present a mathematical analysis of the speed of such waves in both diffusive and juxtacrine relay systems. We use the streak initiation model to make testable predictions. By varying parameters of the model, two very different types of patterning can be obtained, suggesting that our model may be applicable to other processes in addition to streak initiation.     

Protein synthesis in epiblast versus hypoblast during the critical stages of induction and growth of the primitive streak in the chick embryo.

In the chick the inducing power of the hypoblast for primitive streak was assumed to reach its maximum at the beginning of the primitive streak stage and to last until its completion. It was therefore of interest to trace the protein synthetic activity of the epiblast and hypoblast during five successive developmental stages and to correlate them with the known morphogenetic events.The investigation was done along two lines: 1) A quantitative survey was made of the uptake of tritiated phenylalanine into epiblasts versus hypoblasts and their incorporation into trichloroacetic acid-precipitable protein. 2) Incorporation of label into protein was followed by a comparative investigation of the electropherograms of epiblast versus hypoblast at the different stages.The quantitative survey has shown an almost uniform and rather low incorporation of label into protein in the hypoblast layer with a very short period of doubled activity between full hypoblast and initial primitive streak (p.s.). During this period the inductive capacity of the hypoblast for primitive streak was supposed to reach its maximal value.The qualitative survey indicated different patterns of incorporation in the two layers studied. Of special interest are two peaks (III and IV) which appear in the hypoblast previous to p.s. formation at the time of its augmented synthetic activity which also coincides with the onset of its inductive capacity. At later stages two similar peaks appear in the epiblast. It is suggested that a protein included in the above peaks might represent the inductor of the primitive streak.     

Scanning electron microscopy (SEM) of the chick embryo primitive streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2--5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the privitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arrangedcells with long. thin processes. Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

The chick's marginal zone and primitive streak formation. II. Quantification of the marginal zone's potencies--temporal and spatial aspects

When a posterior fragment of the chick's marginal zone (PM) was exchanged with equal sized lateral marginal zone fragment (LM), of the same blastoderm, its capacity to initiate an ectopic primitive streak (PS) was found to be both size and stage dependent. Good correlation was demonstrated between the areas of PM fragments and the number of cells they contained. In stage X blastoderms, PM fragments containing less than 1200 cells were incapable of initiating an ectopic PS. Transplanted PMs containing between 1200 and 1500 cells initiated a lateral ectopic PS in 50% of the cases, while in the other 50% a posterior PS developed from the original posterior side. PMs containing 1500 cells or more in all cases initiating an ectopic PS and inhibited the formation of a posterior PS. At stage XI, laterally transplanted PMs containing less than 1800 cells were not effective. Stage XI PMs containing 1800-2300 cells in some cases succeeded in initiating a lateral ectopic PS, in addition to the posterior one. Stage XI PMs containing 2300 cells or more invariably promoted the development of an ectopic PS, but were unable to suppress the formation of a posterior PS, so that two PSs developed in the same blastoderm, one posterior and one ectopic. When a stage XI PM fragment was laterally transplanted into a younger, stage X blastoderm, the minimal effective cell number needed to initiate an ectopic PS increased to at least 3000 cells, again without inhibiting the formation of a posterior PS. The inductive potential of a stage X PM is therefore at least twice that of a stage XI PM. The marginal zone belt of stage X blastoderms was checked for the decrease in its developmental potential from the posterior to the lateral side by evaluating its effect on the developmental expression of two competing stage X PMs, one located posteriorly and the other inserted laterally. The developmental expression of the laterally inserted PM was consistently inferior to that of the posterior PM. The developmental expression of each PM was not related to absolute size, but depended on the size ratio of lateral PM/posterior PM. When the ratio was 1.2 or less, only posterior PSs developed. When the ratio was 1.3-1.4, three different results were encountered: (1) only a posterior PS, (2) posterior plus lateral, and (3) only lateral PS. When the ratio was 1.5 or more, only a lateral PS developed, which suppressed the posterior PS.     

Mitotic cells and their microappendages in the primitive streak of the chick embryo.

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscropy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.     

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 chick's marginal zone and primitive streak formation. I. Coordinative effect of induction and inhibition

A variety of transplantation experiments of posterior and lateral marginal zone fragments at stages X, XI, and XII have been carried out in order to test their relevance to the development of a primitive streak (PS). At the stages studied the marginal zone (MZ) was shown to behave as a ring-like gradient field, the maximal value of which was at the posterior end (PM). The PM was found to be capable at the same time of promoting the development of a PS and of suppressing the inductive potential of other regions of the MZ. By systematically evaluating inductive and suppressive capacities of PMs, at different developmental stages, it was found that both features are maximal at stage X. During stages XI and XII, both properties gradually decrease in the MZ and build up in the forming hypoblast.     

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.     

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis

Summary Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[³⁵S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.     

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.     

A 'chemotactic dipole' mechanism for large-scale vortex motion during primitive streak formation in the chick embryo

Primitive streak formation in the chick embryo involves significant coordinated cell movement lateral to the streak, in addition to the posterior-anterior movement of cells in the streak proper. Cells lateral to the streak are observed to undergo 'polonaise movements', i.e. two large counter-rotating vortices, reminiscent of eddies in a fluid. In this paper, we propose a mechanism for these movement patterns which relies on chemotactic signals emitted by a dipolar configuration of cells in the posterior region of the epiblast. The 'chemotactic dipole' consists of adjacent regions of cells emitting chemo-attractants and chemo-repellents. We motivate this idea using a mathematical analogy between chemotaxis and electrostatics, and test this idea using large-scale computer simulations. We implement active cell response to both neighboring mechanical interactions and chemotactic gradients using the Subcellular Element Model. Simulations show the emergence of large-scale vortices of cell movement. The length and time scales of vortex formation are in reasonable agreement with experimental data. We also provide quantitative estimates for the robustness of the chemotaxis dipole mechanism, which indicate that the mechanism has an error tolerance of about 10% to variation in chemotactic parameters, assuming that only 1% of the cell population is involved in emitting signals. This tolerance increases for larger populations of cells emitting signals.     

Morphometry of cellular protrusions of mesodermal cells and fibrous extracellular matrix in the primitive streak stage chick embryo

Summary Chick mesodermal cells, having become invaginated and beginning to locomote prior to the formation of the mesodermal cell layer at an early primitive streak stage, extend many filopodia and flatten themselves against the basal surface of the epiblast. Morphometry on scanning electron micrographs of chick mesodermal cells revealed two statistically significant tendencies. Each cell took an extended form and protruded filopodia, preferably along its major axis, suggesting that the force extending the cell body was generated by both ends rich in filopodia. The cells also tended to protrude filopodia most frequently in a direction away from Hensen's node. The orientation of the fibrous extracellular matrix (fECM), running on the basal surface of the epiblast, was assessed quantitatively, and it was proved statistically that the orientation of the fECM was radial around the primitive streak: With an immunogold staining technique, fECM, to which the filopodia of the mesodermal cells attached frequently and closely, was confirmed to be rich in fibronectin (FN). These results lead us to conclude that the mesodermal cells in chick gastrula were guided to locomote towards the periphery of the area pellucida by FN-rich fECM laid on the basal surface of the epiblast, and that this movement was due to an in vivo locomotive mechanism using filopodia.Offprint requests to: R. Toyoizumi     

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.