Primitive streak-forming cells of the chick invaginate through a basement membrane

Summary Recently fibronectin was shown to appear in the development of the chick for the first time as a thin band on the epiblastic side facing the hypoblast just prior to primitive streak formation. It was thus suggested that fibronectin might be instrumental in the migration of cells that lead to axis formation during primitive streak formation. In the present work we have examined simultaneously for the presence of fibronectin and the specific basement membrane glycoprotein laminin during primitive streak formation using immunofluorescence methods. Laminin was found to be expressed between the epiblast and the hypoblast of stage XIII¹ chick blastoderms. During the immediately following process of streak formation the laminin was found to be continuously detectable throughout the area covered by the hypoblast, but disrupted on the streak area. Fibronectin was found to co-distribute with laminin in stage XIII and in the early primitive streak chick blastoderms. It is concluded that at stage XIII laminin and fibronectin form part of a basement membrane that is partially disrupted during the immediately following process of primitive streak formation in order to allow the migration of the streak-forming epiblastic cells during this morphogenetic process.     

Mesoblastic Cells in the Early Phase of Primitive Streak Formation in Chick Embryos. Observations by Scanning Electron Microscopy in ovo and time-Lapse Cinematography in vitro.

During primitive streak formation in the chick embryo, mesoblastic cells were observed by SEM after removal of the hypoblast layer. Before the primitive streak began to develop, numbers of bleb cells and bleb-like protrusions were seen on the ventral surface of the epiblast. From optical observation on the process of change of epiblastic cells into bleb cells in vitro , it was concluded that cells that had elongated became bleb cells when they emerged from the epiblast. Cell behavior during primitive streak formation is discussed on the basis of these findings.     

Distribution of acid phosphatase in the chicken blastoderm]

The distribution of acid phosphatase in the chick blastoderm (stages 2--4 by HH) has been studied using cytochemistry. A marked increase of enzymatic activity all over the blastoderm was shown to coincide with the beginning of primitive streak formation. A part of the cells after their immigration are characterized by the fall of acid phosphatase activity. The percentage of such cells increases in the cranio-caudal direction of the definitive primitive streak. The patterns of yolk utilization upon the separation of individual embryonic rudiments are discussed.     

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.     

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.     

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.     

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。     

Fate maps of the primitive streak in chick and quail embryo: ingression timing of progenitor cells of each rostro-caudal axial level of somites

Developmental fates of cells emigrating from the primitive streak were traced by a fluorescent dye Dil both in chick and in quail embryos from the fully grown streak stage to 12-somite stage, focusing on the development of mesoderm and especially on the timing of ingression of each level of somitic mesoderm. The fate maps of the chick and quail streak were alike, although the chick streak was longer at all stages examined. The anterior part of the primitive streak predominantly produced somites. The thoracic and the lumbar somites were shown to begin to ingress at the 5 somite-stage and 10 somite-stage in a chick embryo, and 6 somite-stage and 9 somite-stage in a quail embryo, respectively. The posterior part of the streak served mainly as the origin of more lateral or extra embryonic mesoderm. As development proceeded, the fate of the posterior part of the streak changed from the lateral plate mesoderm to the tail bud mesoderm and then to extra embryonic, allantois mesoderm. The fate map of the primitive streak in chick and quail embryo presented here will serve as basic data for studies on mesoderm development with embryo manipulation, especially for transplantation experiments between chick and quail embryos.     

Epithelial scatter factor and development of the chick embryonic axis

Scatter factor, a recently characterised protein secreted by certain embryonic fibroblasts, affects cultured epithelial by increasing cell motility, the breakdown of cell junctions and cell scattering. The process of gastrulation in higher vertebrate embryos, during which the primitive streak forms, involves an epithelial-to-mesenchymal transformation resembling the effects of the factor on cultured cells. The factor was applied locally to chick embryos, using both scatter-factor-secreting cell lines and inert carriers. We found that scatter factor can generate local supernumerary axial structures resembling primitive streak and/or neural plate and conclude that it may have primitive-streak and/or neural-inducing activity in chick embryos.     

Restriction of cloning potential in agarose of early chick embryonic cells as development progresses

Early chick embryonic cells, prior to the formation of the primitive streak, form colonies when cultured in soft agarose [Mitrani, E.: Exp. Cell Res. 152, 148-153 (1984)]. The present work is an attempt to determine at which stages of development this ability is expressed and which areas of the chick embryo harbour the colony-forming cells. We found that the capacity to form colonies decreases as development progresses and cells enter alternative differentiation pathways. At pre-primitive streak stages, the capacity is concentrated to the peripheral areas of the embryo and decreases towards the centre. With the onset of hypoblast formation only cells from Area Opaca and, to a lesser degree, the Marginal Zone, can form colonies in agarose. At post-primitive streak stages only extra-embryonic cells can form colonies in agarose. By 48 h of incubation all cells of the chick blastoderm seem to have lost the capacity to form colonies in agarose.     

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.     

The dynamics of antibody-induced redistribution of viral envelope antigens in the plasma membranes of avian tumour virus-infected chick embryo fibroblasts.

Avian tumour virus-infected chick embryo fibroblasts express new antigens, identical with the viral envelope antigens, in their plasma membranes. Electron-microscopic examination of carbon-platinum replicas of cells labelled with haemocyanin-marked antibody has shown the distribution of these antigens to be diffuse over the cell surface with an increased concentration on peripheral cell processes. However, antigen-antibody complexes (AAC), resulting from reaction with specific antibody, may be redistributed into a variety of patterns. Observation of the time course of antibody-induced antigen mobility revealed a rapid and a delayed phase of redistribution. During the rapid phase (10 min or less) some of the antigen-bearing cells reorganized AAC into patches, while the remainder maintained a diffuse distribution. A fraction of the cells with either diffuse or patchy distribution also redistributed AAC into a pattern of 'marginal redistribution (MR)', consisting of linear aggreagation of AAC, at the cell edge. During the 'late' phase of redistribution (after about 20 min), AAC began to condense into one or more foci of coalescence (FC) on each cell. As the number of cells with FC increased with time, the fraction of cells which were labelled decreased. Electron-microscopic observation of thin sections of ferritin-labelled specimens indicated that AAC were lost by endocytosis and that this process was probably related to FC formation. Inhibitors of oxidative phosphorylation, protein synthesis, RNA synthesis, or microtubule assembly had no significant effect on the patterns or the course of redistribution. Iodoacetic acid (IAA), which depletes cellular ATP, and cytochalasin B (CB), which is believed to depolymerize microfilaments, partially inhibited MR and completely prevented FC formation and endocytosis. Paradoxically, IAA or CB-treated cells lost AAC very rapidly by some alternate mechanism not involving FC formation but which may entail a centrifugal migration of complexes to the cell extremities during the process of AAC disposal.     

PTEN抑制胚胎原肠胚形成期EMT的过程

PTEN(Phosphatase and tensin homolog)是一种重要的抑癌基因,具有非常广泛的生物学活性,例如在细胞的生长发育、迁移、凋亡和信号传导等均发挥重要作用。PTEN基因表达始于在胚胎早期的上胚层,而后主要出现在神经外胚层和胚胎中胚层结构,表明PTEN可能参与胚胎早期发育过程的细胞迁移、增殖和分化。文章主要应用在体改变早期胚胎PTEN的表达水平来观察其对上胚层至中胚层细胞转换—EMT(Epithe-lial-mesenchymal transition)的作用。首先,原位杂交结果提示,内源性PTEN表达在原条以及之后的中胚层细胞结构如体节等。在体PTEN转染实验,体外培养至HH3期的鸡胚胎,转染Wt PTEN-GFP或移植Wt PTEN-GFP原条组织至未转染的同时期的宿主胚胎相同部位后,观察到PTEN转染细胞大都由上胚层迁移至原条并滞留于原条,不再参与中胚层细胞形成。移植实验也得到相似结果,发现在Wt PTEN-GFP阳性原条组织移植后很少细胞迁移出原条。另外在原肠胚期PTEN siRNA降调胚胎一侧PTEN基因后,降调侧中胚层细胞数明显少于正常侧。上述研究结果均提示PTEN基因在胚胎原肠胚期三胚层形成过程中可能具有抑制EMT的作用。     

Onset of the segmentation clock in the chick embryo: evidence for oscillations in the somite precursors in the primitive streak

Vertebrate somitogenesis is associated with a molecular oscillator, the segmentation clock, which is defined by the periodic expression of genes related to the Notch pathway such as hairy1 and hairy2 or lunatic fringe (referred to as the cyclic genes) in the presomitic mesoderm (PSM). Whereas earlier studies describing the periodic expression of these genes have essentially focussed on later stages of somitogenesis, we have analysed the onset of the dynamic expression of these genes during chick gastrulation until formation of the first somite. We observed that the onset of the dynamic expression of the cyclic genes in chick correlated with ingression of the paraxial mesoderm territory from the epiblast into the primitive streak. Production of the paraxial mesoderm from the primitive streak is a continuous process starting with head mesoderm formation, while the streak is still extending rostrally, followed by somitic mesoderm production when the streak begins its regression. We show that head mesoderm formation is associated with only two pulses of cyclic gene expression. Because such pulses are associated with segment production at the body level, it suggests the existence of, at most, two segments in the head mesoderm. This is in marked contrast to classical models of head segmentation that propose the existence of more than five segments. Furthermore, oscillations of the cyclic genes are seen in the rostral primitive streak, which contains stem cells from which the entire paraxial mesoderm originates. This indicates that the number of oscillations experienced by somitic cells is correlated with their position along the AP axis.     

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation

The expression pattern of the receptor tyrosine kinase gene EphB3 was examined during the early stages of chick embryogenesis, and is described in this report. In the gastrula, EphB3 is expressed in epiblast cells adjacent to and entering the anterior portion of the primitive streak; expression is extinguished once cells have ingressed. At headfold stages, EphB3 is strongly transcribed in the floor of the foregut and in anterior lateral endoderm, and is expressed in the subjacent cardiogenic mesoderm. EphB3 is transiently expressed in the lateral ectoderm, neural tube, and neural crest during these stages. Later neural expression is localized to the mesencephalon. In the somitic mesoderm, EphB3 is initially expressed in the sclerotome, but later is expressed predominantly in the dermatome. Prominent expression is also detected in the developing heart, liver, posterior ventral limb bud mesenchyme, pharyngeal arches, and head mesenchyme.     

Intermediate filament protein expression and mesoderm formation in the rabbit embryo

Summary This study aims to describe the regulation of vimentin and cytokeratin expression during differentiation of primary mesenchymal cells in the 7 day old rabbit embryo; unusual intermediate filament protein expression patterns have already been found in this species at later embryonic stages. Double-labelling indirect immunofluorescence assays with a panel of monoclonal intermediate filament antibodies are performed on frozen sections and compared with aldehyde-fixed plastic-embedded tissues. The histological part of the study, serving as a basis for the topographical orientation in the immunostained frozen sections, emphasises many similarities between the primitive streak embryos of the rabbit and the chick. The immunohistochemical analysis reveals cytokeratin expression to varying degrees in all germ layers. Vimentin expression, always in combination with cytokeratin expression, is found in a few cells of the ectoderm, endoderm and lateral mesoderm, but not in the primary mesenchymal cells of either the primitive node or the primitive streak. The results are discussed in relation to recent experimental findings on differentiation and morphogenetic processes in the primitive streak embryo. While these complex expression patterns make it seem unlikely that intermediate filament protein subtypes are expressed independently of cellular function during development, no indication can be found for a relation between vimentin expression and the morphogenetic changes thought to be important during mesoderm formation.Supported by the Deutsche Forschungsgemeinschaft (Wa 359-9) and by the Netherlands Cancer Foundation Offprint requests to: C. Viebahn     

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.     

Cardiac progenitor migration and specification

The heart is the first organ to function during vertebrate development and cardiac progenitors, are among the first cell lineages to be established from mesoderm cells emerging from the primitive streak during gastrulation. Cardiac progenitors have been mapped in the epiblast of pre-streak embryos. In the early chick gastrula they are located in the mid-primitive streak, from which they enter the mesoderm bilaterally. However, migration routes of cardiac progenitors have never been directly observed within the embryo and the factor(s) controlling their movement are not known. Furthermore, it is not understood how signals controlling cell movement are integrated with those that determine cell fate. Long-term video microscopy combined with GFP labelling and image processing enabled us to observe the movement patterns of prospective cardiac cells in whole embryos in real time. Embryo manipulations and the analysis of explants suggest that Wnt3a plays a crucial role in guiding these cells through a RhoA dependent mechanism involving negative chemotaxis. Wnt3a is expressed at high levels in the amniote primitive streak and ectopic signalling activity caused wider movement trajectories resulting in cardia bifida, which was rescued by dominant-negative Wnt3a. Our studies revealed Wnt3a-RhoA mediated chemo-repulsion as a novel mechanism guiding cardiac progenitors. This activity can act at long-range and does not interfere with cardiac cell fate specification.     

Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with chordin

During chick gastrulation, inhibition of BMP signaling is required for primitive streak formation and induction of Hensen's node. We have identified a unique secreted protein, Tsukushi (TSK), which belongs to the Small Leucine-Rich Proteoglycan (SLRP) family and is expressed in the primitive streak and Hensen's node. Grafts of cells expressing TSK in combination with the middle primitive streak induce an ectopic Hensen's node, while electroporation of TSK siRNA inhibits induction of the node. In Xenopus embryos, TSK can block BMP function and induce a secondary dorsal axis, while it can dorsalize ventral mesoderm and induce neural tissue in embryonic explants. Biochemical analysis shows that TSK binds directly to both BMP and chordin and forms a ternary complex with them. These observations indicate that TSK is an essential dorsalizing factor involved in the induction of Hensen's node.