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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.

There has long been controversy concerning the role of the axial mesoderm in the induction and rostrocaudal patterning of the vertebrate nervous system. Here we investigate the neural inducing and regionalising properties of defined rostrocaudal regions of head process/prospective notochord in the chick embryo by juxtaposing these tissues with extraembryonic epiblast or neural plate explants. We localise neural inducing signals to the emerging head process and using a large panel of region-specific neural markers, show that different rostrocaudal levels of the head process derived from headfold stage embryos can induce discrete regions of the central nervous system. However, we also find that rostral and caudal head process do not induce expression of any of these molecular markers in explants of the neural plate. During normal development the head process emerges beneath previously induced neural plate, which we show has already acquired some rostrocaudal character. Our findings therefore indicate that discrete regions of axial mesendoderm at headfold stages are not normally responsible for the establishment of rostrocaudal pattern in the neural plate. Strikingly however, we do find that caudal head process inhibits expression of rostral genes in neural plate explants. These findings indicate that despite the ability to induce specific rostrocaudal regions of the CNS de novo, signals provided by the discrete regions of axial mesendoderm do not appear to establish regional differences, but rather refine the rostrocaudal character of overlying neuroepithelium.

Transformation of an epithelial sheet into a migrating mesenchymal cell population implies the destruction of the basal lamina underlying the epithelium, and the subsequent localized digestion of the extracellular matrix by the migrating cells. Proteases are involved in these processes. Among them, molecules containing both a serine protease domain and at least one kringle domain have been identified as possible important effectors. Interestingly, related proteins containing an inactive serine protease domain also seem to play a role, suggesting that the function of these molecules in epithelial-mesenchymal transformation is not confined to proteolytic digestion of cell attachments. Instead, these molecules act through specific tyrosine kinase receptors in the membrane of the responding cells. In this review, we summarize data implicating this family of molecules in various epithelial-mesenchymal transitions during embryonic development. Our major focus of attention are: hepatocyte growth factor/scatter factor (HGF/SF), its tyrosine kinase receptor proto-oncogene c-met, and the related peptide factor HGF-like/macrophage-stimulating protein (HGF1/MSP), whose receptor is the Ron tyrosine kinase. c-met and Ron also have another close homolog in the chick, called Sea, whose ligand remains unknown. Interestingly, HGF/SF is activated by other plasminogen-related molecules which, apart from a specific activator, include the protease urokinase. HGF/SF, c-met and HGF/MSP are expressed in dynamic ways during early embryonic development, correlating with regions undergoing epithelial/mesenchymal transformations. Moreover, several assays are now starting to reveal great pleiotropism of function during development, including both the loss and the acquisition of epithelial morphology according to the cell type and assay used, as well as angiogenesis, kidney tubule morphogenesis, cell motility, the maintenance of competence for neural induction and some aspects of the later development of the musculoskeletal and nervous systems.

We have cloned chick homologues of the type-II activin receptor, which we have designated cActR-IIA and -IIB. Binding assays show that the two receptors are indistinguishable in their ability to bind activin-A, with comparable kds. Injection of mRNAs encoding these receptors into Xenopus embryos causes axial duplications. Expression of both receptors can first be detected in the primitive streak by in situ hybridization. This suggests that these genes may be activated in response to mesoderm induction. In agreement with this, we find that treatment of preprimitive streak chick embryos with activin-A leads to rapid induction of the expression of cActR-IIB. At later stages, cActR-IIA transcripts become localized mainly in the notochord and myotome and cActR-IIB in the dorsal neural tube, proximal-anterior part of the limb bud, sensory placodes, and specific regions of the fore- and midbrain. To test the response of early chick embryonic tissues to activin, we designed a new in vitro assay for differentiation. We find that explants of area opaca epiblast or posterior primitive streak from various stages can respond to activin treatment by differentiating into a variety of mesodermal cell types in a dose-dependent manner. These results suggest that the importance of activin-related signaling pathways is not confined to pregastrulation stages and that these receptors may be involved in mediating the effects of inducing signals during later stages of development of the mesoderm, limbs, and nervous system.

Cell lineage analysis has revealed that the amniote organizer, Hensen's node, is subdivided into distinct regions, each containing a characteristic subpopulation of cells with defined fates. Here, we address the question of whether the inducing and regionalising ability of Hensen's node is associated with a specific subpopulation. Quail explants from Hensen's node are grafted into an extraembryonic site in a host chick embryo allowing host- and donor-derived cells to be distinguished. Cell-type- and region-specific markers are used to assess the fates of the mesodermal and neural cells that develop. We find that neural inducing ability is localised in the epiblast layer and the mesendoderm (deep portion) of the medial sector of the node. The deep portion of the posterolateral part of the node does not have neural inducing ability. Neural induction also correlates with the presence of particular prospective cell types in our grafts: chordamesoderm (notochord/head process), definitive (gut) endoderm or neural tissue. However, only grafts that include the epiblast layer of the node induce neural tissue expressing a complete range of anteroposterior characteristics, although prospective prechordal plate cells may also play a role in specification of the forebrain.

The mechanisms controlling the formation of the embryonic axis, and specifically those that give rise to the mesoderm, have received renewed attention recently. In the frog, some of these mechanisms have begun to be elucidated, and several factors have been found to cause uncommitted ectoderm cells to differentiate into mesoderm. All of the factors identified to date are related either to fibroblast growth factor (FGF) or to transforming growth factor beta (TGF-beta). Do the mechanisms that generate the embryonic axis of amphibians also operate in chick and mouse embryos? Here I address how amphibian and amniote embryos might provide complementary pieces of a puzzle.

The hypoblast is generally thought to be responsible for inducing the mesoderm in the chick embryo because the primitive streak, and subsequently the embryonic axis, form according to the orientation of the hypoblast. However, some cells become specified as embryonic mesoderm very late in development, towards the end of the gastrulation period and long after the hypoblast has left the embryonic region. We argue that induction of embryonic mesoderm and of the embryonic axis are different and separable events, both in amniotes and in amphibians. We also consider the relationships between the dorsoventral and anteroposterior axes in both groups of vertebrates.

We have investigated whether the developing spinal cord is intrinsically segmented in its rostrocaudal (anteroposterior) axis by mapping the spread of clones derived from single labelled cells within the neural tube of the chick embryo. A single cell in the ventrolateral neural tube of the trunk was marked in situ with the fluorescent tracer lysinated rhodamine dextran (LRD) and its descendants located after two days of further incubation. We find that clones derived from cells labelled before overt segmentation of the adjacent mesoderm do not respect any boundaries within the neural tube. Those derived from cells marked after mesodermal segmentation, however, never cross an invisible boundary aligned with the middle of each somite, and tend to be elongated along the mediolateral axis of the neural tube. When the somite pattern is surgically disturbed, neighbouring clones derived from neuroectodermal cells labelled after somite formation behave like clones derived from younger cells: they no longer respect any boundaries, and are not elongated mediolaterally. These results indicate that periodic lineage restrictions do exist in the developing spinal cord of the chick embryo, but their maintenance requires the presence of the adjacent somite mesoderm.

We have used microsurgical techniques to investigate the effects of embryonic mesodermal tissues on the pattern of chick neural crest cell migration in the trunk. Segmental plate or lateral plate mesenchyme was transplanted into regions encountered by neural crest cells. We found that neural crest cells are able to migrate through lateral plate mesenchyme but not through segmental plate tissue until this tissue differentiates into a sclerotome. After this stage, segmental migration is controlled by the subdivision of the sclerotome into a rostral and a caudal half; when the rostrocaudal orientation of the sclerotomes is reversed by rotating the segmental plate 180 degrees about its rostrocaudal axis, neural crest cells migrate through the portion of the sclerotome that was originally rostral.

In amniotes, all of the tissues of the adult arise from the epiblast, one of the two layers of cells present in the early embryo; the mesoderm and gut endoderm arise from an epiblast-derived structure known as the primitive streak. The monoclonal antibody HNK-1 recognizes the cells of the primitive streak in the chick embryo. Before streak formation, HNK-1 identifies cells that are randomly distributed within the epiblast. We have now used two novel ways to study cell lineage and commitment to show that the epiblast of the early chick embryo contains two distinct populations of cells with different developmental fates at a stage during which 'mesodermal induction' is believed to occur. One cell population, recognized by monoclonal antibody HNK-1, is destined to form mesoderm and endoderm; the rest of the epiblast is unable to give rise to mesoderm if this population of cells is removed.

We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral half-sclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite. It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed. Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.

Experiments have been carried out on explanted chick embryos to test certain widely accepted concepts about the role of Hensen's node in somite formation. The relationship between elongation of the area pellucida and regression of Hensen's node has also been investigated. We conclude from these experiments that: (a) The timing of somite formation is not controlled by the regression of Hensen's node, nor by the shearing of the mesoderm into right and left halves. (b) Somite size and shape are probably controlled by local conditions in the chick embryo. (c) Elongation and regression are two different events. (d) The position of the probably depends on mechanical tensions in the area pellucida. (e) The notochord is not required for the stability of somites in vivo.

After neural processes emerge from the neural tube in the chick embryo, their growth is restricted to the cranial halves of the neighbouring somites. In this study we have developed an in vitro system to model the interactions between these tissue types. Pioneer neurites display a hierarchy of preferences in terms of the substrates they can grow on. As expected, tissue culture plastic does not support neural outgrowth, but this can be overcome by coating the plastic substrate with either collagen or poly-L-lysine. Neural crest, cranial half somite, and a number of other tissues support growth well, while caudal half somite and tail bud mesenchyme do so to a much smaller extent. The binding pattern of a variety of lectins was assessed in cryostat sections of embryos and in cultured cells of the above tissues. It was found that peanut agglutinin can discriminate between cranial and caudal sclerotome both in vitro and in the embryo, since it binds preferentially to caudal sclerotome in both cases. This difference is expressed as soon as the sclerotome forms. The significance of these findings is twofold: first, they show that the interactions that take place during peripheral neural segmentation can be modelled in vitro; second, they represent the first instance of a molecular difference between the cranial and caudal halves of the sclerotome, detectable both in culture and in the 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.

This study provides evidence that cells destined to segment together into somites have a degree of cell division synchrony. We have measured the duration of the cell division cycle in somite and segmental plate cells of the chick embryo as 9.5 h using [3H]thymidine pulse- and-chase. Treatment of embryos with any of a variety of inhibitors known to affect the cell division cycle causes discrete periodic segmental anomalies: these anomalies appear about 6-7 somites after treatment and, in some cases, a second anomaly is observed 6 to 7 somites after the first. Since somites take 1.5 h to form, the 6- to 7- somite interval corresponds to about 9-10 h, which is the duration of the cell cycle as determined in these experiments. The anomalies are similar to those seen after heat shock of 2-day chick embryos. Heat shock and some of the other treatments induce the expression of heat-shock proteins (hsp); however, since neither the expression nor the distribution of these proteins relate to the presence or distribution of anomalies seen, we conclude that hsps are not responsible for the pattern of segmental anomalies observed. The production of periodic segmental anomalies appears to be linked to the cell cycle. A simple model is proposed, in which we suggest that the cell division cycle is involved directly in gating cells that will segment together.

In all higher vertebrate embryos the sensory ganglia of the trunk develop adjacent to the neural tube, in the cranial halves of the somite-derived sclerotomes. It has been known for many years that ganglia do not develop in the most cranial (occipital) sclerotomes, caudal to the first somite. Here we have investigated whether this is due to craniocaudal variation in the neural tube or crest, or to an unusual property of the sclerotomes at occipital levels. Using the monoclonal antibody HNK-1 as a marker for neural crest cells in the chick embryo, we find that the crest does enter the cranial halves of the occipital sclerotomes. Furthermore, staining with zinc iodide/osmium tetroxide shows that some of these crest-derived cells sprout axons within these sclerotomes. By stage 23, however, no dorsal root ganglia are present within the five occipital sclerotomes, as assessed both by haematoxylin/eosin and zinc iodide/osmium tetroxide staining. Moreover, despite this loss of sensory cells, motor axons grow out in these segments, many of them later fasciculating to form the hypoglossal nerve. The sclerotomes remain visible until stages 27/28, when they dissociate to form the base of the skull and the atlas and axis vertebrae. After grafting occipital neural tube from quail donor embryos in place of trunk neural tube in host chick embryos, quail-derived ganglia do develop in the trunk sclerotomes. This shows that the failure of occipital ganglion development is not the result of some fixed local property of the neural crest or neural tube at occipital levels. We therefore suggest that in the chick embryo the cranial halves of the five occipital sclerotomes lack factors essential for normal sensory ganglion development, and that these factors are correspondingly present in all the more caudal sclerotomes.

It is known that both neural crest cell migration and motor axon outgrowth in most vertebrate embryos are segmented because of restrictions imposed upon their distribution by the neighbouring sclerotomes, each of which is divided into a rostral and a caudal half. The caudal half does not allow crest migration or axon outgrowth, while the rostral half does. In this paper, we investigate the expression of proteins and glycoproteins in the two halves of the sclerotome of the chick embryo at stages between 20 and 32 pairs of somites by two-dimensional SDS-polyacrylamide gel electrophoresis. We find that the patterns of expression are complex, and that polypeptides and glycoproteins vary both spatially and temporally: of those that are expressed differentially by the sclerotome, some differ quantitatively and others qualitatively. Some macromolecules change their spatial distribution with developmental age, and some appear or disappear as the embryos become older.

We have studied the lineage history of the progenitors of the somite mesoderm and of the neural tube in the chick embryo by injecting single cells with the fluorescent tracer, rhodamine-lysine-dextran. We find that, although single cells within the segmental plate give rise to discrete clones in the somites to which they contribute, neither the somites nor their component parts (sclerotome, dermatome, myotome or their rostral and caudal halves) are 'compartments' in the sense defined in insects. Cells in the rostral two thirds or so of the segmental plate contribute only to somite tissue and divide about every 10 h, while those in the caudal portions of this structure contribute both to the somites and to intermediate and lateral plate mesoderm derivatives. In the neural tube, the descendants of individual prospective ventral horn cells remain together within the horn, with a cycle time of 10 h. We have also investigated the role of the cell division cycle in the formation and subsequent development of somites. A single treatment of 2-day chick embryos with heat shock or a variety of drugs that affect the cell cycle all produce repeated anomalies in the pattern of somites and vertebrae that develop subsequent to the treatment. The interval between anomalies is 6-7 somites (or a multiple of this distance), which corresponds to 10 h. This interval is identical to that measured for the cell division cycle. Given that cell division synchrony is seen in the presomitic mesoderm, we suggest that the cell division cycle plays a role in somite formation. Finally, we consider the mechanisms responsible for regionalization of derivatives of the somite, and conclude that it is likely that both cell interactions and cell lineage history are important in the determination of cell fates.

A single heat shock, given to 2-day-old chick embryos, can generate multiple but discrete somite and skeletal anomalies. Each of these anomalies is restricted to one, or at the most two, consecutive segments. The anomalies are separated from each other by a distance of 6-7 somites or vertebrae, or a multiple of this distance. These results argue against the 'clock and wavefront' model; while they support the idea of a cellular clock, they are not consistent with a single propagating wave gating cells destined to form each segment. Heat shock also alters the size and number of segments, as well as the rostrocaudal proportions of the sclerotome. The results are consistent with the rostrocaudal fate of sclerotome cells being determined during segmentation. From our observations, we speculate on the implications for regionalization of the vertebral column.