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.     

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.     

Urodeles remove mesoderm from the superficial layer by subduction through a bilateral primitive streak

Urodeles begin gastrulation with much of their presumptive mesoderm in the superficial cell layer, all of which must move into the deep layers during development. We studied the morphogenesis of superficial mesoderm in the urodeles Ambystoma maculatum, Ambystoma mexicanum, and Taricha granulosa. In all three species, somitic, lateral, and ventral mesoderm move into the deep layer during gastrulation, ingressing through a "bilateral primitive streak" just inside the blastopore. The mesodermal epithelium appears to slide under the endodermal epithelium by a mechanism we term "subduction." Subduction removes the large expanse of superficial presumptive somitic and lateral-ventral mesoderm that initially separates the sub-blastoporal endoderm from the notochord, leaving the endoderm bounding the still epithelial notochord along the gastrocoel roof. Subduction may be a common feature of urodele gastrulation, differing in this regard from anurans. Subducting cells constrict their apices and become bottle-shaped as they approach the junction of the mesodermal and endodermal epithelia. Subducting bottle cells endocytose apical membrane and withdraw the tight junctional component cingulin from the contracting circumferential tight junctions. Either in conjunction with or immediately after subducting, the mesodermal cells undergo an epithelial-to-mesenchymal transition. The mechanism by which epithelial cells release their apical junctions to become mesenchymal, without disrupting the integrity of the epithelium, remains mysterious, but this system should prove useful in understanding this process in a developmental context.     

STELLA-positive subregions of the primitive streak contribute to posterior tissues of the mouse gastrula

The developmental relationship between the posterior embryonic and extraembryonic regions of the mammalian gastrula is poorly understood. Although many different cell types are deployed within this region, only the primordial germ cells (PGCs) have been closely studied. Recent evidence has suggested that the allantois, within which the PGCs temporarily take up residence, contains a pool of cells, called the Allantoic Core Domain (ACD), critical for allantoic elongation to the chorion. Here, we have asked whether the STELLA-positive cells found within this region, thought to be specified PGCs, are actually part of the ACD and to what extent they, and other ACD cells, contribute to the allantois and fetal tissues. To address these hypotheses, STELLA was immunolocalized to the mouse gastrula between Early Streak (ES) and 12-somite pair (-s) stages (~6.75-9.0 days post coitum, dpc) in histological sections. STELLA was found in both the nucleus and cytoplasm in a variety of cell types, both within and outside of the putative PGC trajectory. Fate-mapping the headfold-stage (~7.75-8.0 dpc) posterior region, by which time PGCs are thought to be segregated into a distinct lineage, revealed that the STELLA-positive proximal ACD and intraembryonic posterior primitive streak (IPS) contributed to a wide range of somatic tissues that encompassed derivatives of the three primary germ layers. This contribution included STELLA-positive cells localizing to tissues both within and outside of the putative PGC trajectory. Thus, while STELLA may identify a subpopulation of cells destined for the PGC lineage, our findings reveal that it may be part of a broader niche that encompasses the ACD and through which the STELLA population may contribute cells to a wide variety of posterior tissues of the mouse gastrula.     

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.     

Effects of ethanol on the primitive streak stage mouse embryo

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.     

A role for the hypoblast (AVE) in the initiation of neural induction, independent of its ability to position the primitive streak

The mouse anterior visceral endoderm (AVE) has been implicated in embryonic polarity: it helps to position the primitive streak and some have suggested that it might act as a "head organizer", inducing forebrain directly. Here we explore the role of the hypoblast (the chick equivalent of the AVE) in the early steps of neural induction and patterning. We report that the hypoblast can induce a set of very early markers that are later expressed in the nervous system and in the forebrain, but only transiently. Different combinations of signals are responsible for different aspects of this early transient induction: FGF initiates expression of Sox3 and ERNI, retinoic acid can induce Cyp26A1 and only a combination of low levels of FGF8 together with Wnt- and BMP-antagonists can induce Otx2. BMP- and Wnt-antagonists and retinoic acid, in different combinations, can maintain the otherwise transient induction of these markers. However, neither the hypoblast nor any of these factors or combinations thereof can induce the definitive neural marker Sox2 or the formation of a mature neural plate or a forebrain, suggesting that the hypoblast is not a head organizer and that other signals remain to be identified. Interestingly, FGF and retinoids, generally considered as caudalizing factors, are shown here to play a role in the induction of a transient "pre-neural/pre-forebrain" state.     

Formation of the chick primitive streak as studied in computer simulations

We have used a computer simulation system to examine formation of the chick primitive streak and to test the proposal (Wei and Mikawa Development 127 (2000) 87) that oriented cell division could account for primitive streak elongation. We find that this proposal is inadequate to explain elongation of the streak. In contrast, a correctly patterned model streak can be generated if two putative mechanisms are operative. First, a subpopulation of precursor cells that is known to contribute to the streak is assigned a specific, but simple, movement pattern. Second, additional cells within the epiblast are allowed to incorporate into the streak based on near-neighbor relations. In this model, the streak is cast as a steady-state system with continuous recruitment of neighboring epiblast cells, egress of cells into deeper layers and an internal pattern of cell movement. The model accurately portrays elongation and maintenance of a robust streak, changes in the composition of the streak and defects in the streak after experimental manipulation.     

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.