Tracing the lineage of tracing cell lineages.

The study of cell lineages has been, and remains, of crucial importance in developmental biology. It requires the identification of a cell or group of cells and of all of their descendants during embryonic development. Here, we provide a brief survey of how different techniques for achieving this have evolved over the last 100 years.

Strong electrical currents leave the primitive streak of chick embryos.

The electrical fields above chick embryos were explored with a vibrating probe. These fields indicate that steady currents with exit densities of the order of 100 microamperes per square centimeter leave the whole streak and return elsewhere through the epiblast. The epicenter of these strong exit currents lies near Hensen's node. They are probably pumped into the intraembryonic space by the epiblast and then leak out of the streak because it is a zone of junctional disruption.

Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones.

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.

Vertebrate gastrulation.

Our understanding of the mechanisms that control gastrulation is still in its infancy. One problem is that gastrulation is a complex set of coordinated behaviours involving directional cell movements, several types of cell interactions, changes in cell fate and gene expression. Therefore, the successful analysis of its control mechanisms requires simultaneous analysis of more than one of these, or at least some way of separating them. Although progress has been slow, some recent studies have made significant advances in the field and we can probably look forward to some major breakthroughs in the near future.

Cerberus regulates left-right asymmetry of the embryonic head and heart.

BACKGROUND: Most of the molecules known to regulate left-right asymmetry in vertebrate embryos are expressed on the left side of the future trunk region of the embryo. Members of the protein family comprising Cerberus and the putative tumour suppressor Dan have not before been implicated in left-right asymmetry. In Xenopus, these proteins have been shown to antagonise members of the transforming growth factor beta (TGF-beta) and Wnt families of signalling proteins. RESULTS: Chick Cerberus (cCer) was found to be expressed in the left head mesenchyme and in the left flank of the embryo. Expression on the left side of the head was controlled by Sonic hedgehog (Shh) acting through the TGF-beta family member Nodal; in the flank, cCer was also regulated by Shh, but independently of Nodal. Surprisingly, although no known targets of Cerberus are expressed asymmetrically on the right side of the embryo at these stages, misexpression of cCer on this side of the embryo led to upregulation of the transcription factor Pitx2 and reversal of the direction of heart and head turning, apparently as independent events. Consistent with the possibility that cCer may be acting on bilaterally expressed TGF-beta family members such as the bone morphogenetic proteins (BMPs), this result was mimicked by right-sided misexpression of the BMP antagonist, Noggin. CONCLUSIONS: Our findings suggest that cCer maintains a delicate balance of different TGF-beta family members involved in laterality decisions, and reveal the existence of partially overlapping molecular pathways regulating left-right asymmetry in the head and trunk of the embryo.

Mesoderm induction and development of the embryonic axis in amniotes.

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.

Neural induction. A bird's eye view.

Since the discovery of the phenomenon of neural induction by Spemann and Mangold in 1924, considerable effort has been invested in identifying the signals produced by the organizer that are responsible for diverting the fate of cells from epidermal to neural. Substantial progress has been made only recently by the finding in amphibians that BMP4 is a neural inhibitor and epidermal inducer, and that endogenous antagonists of BMPs are secreted by the organizer. However, recent results in the chick point to the existence of other, upstream events required before BMP inhibition stabilizes neural fates. Here we take a critical view of the evidence for and against the view that BMP inhibition is a sufficient trigger for neural induction in different vertebrates.

Expression of mouse HES-6, a new member of the Hairy/Enhancer of split family of bHLH transcription factors.

We studied the expression of mouse HES-6, a new member of the Hairy/Enhancer of split family of basic helix-loop-helix transcription factors. HES-6 is expressed in all neurogenic placodes and their derivatives and in the brain, where it is patterned along both the anteroposterior and dorsoventral axes. HES-6 is also expressed in the trunk, in the dorsal root ganglia and in the myotomes. In the limb buds HES-6 is expressed in skeletal muscle and presumptive tendons.

Mesoderm patterning and somite formation during node regression: differential effects of chordin and noggin.

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.

Gata2 and Gata3: novel markers for early embryonic polarity and for non-neural ectoderm in the chick embryo.

We have investigated in detail the expression patterns of two Gata genes, cGata2 and cGata3, during early chick development. In addition to confirming previously described expression of these two genes in developing brain, kidney and blood islands, this study reveals several important novel expression domains during very early stages of development. cGata2 is expressed in the area opaca in pre-primitive streak stages, forming a gradient along the A-P axis (strongest anteriorly). Both genes are expressed strongly in the entire non-neural ectoderm from stage 4+, and neither is expressed in prospective neural plate at any stage. Unlike other previously described non-neural markers, neither gene is expressed in the dorsal neural tube. We also describe dynamic expression of cGata2 and cGata3 during eye, ear and gut development.

Establishment and maintenance of the border of the neural plate in the chick: involvement of FGF and BMP activity.

We have investigated the cell interactions and signalling molecules involved in setting up and maintaining the border between the neural plate and the adjacent non-neural ectoderm in the chick embryo at primitive streak stages. msx-1, a target of BMP signalling, is expressed in this border at a very early stage. It is induced by FGF and by signals from the organizer, Hensen's node. The node also induces a ring of BMP-4, some distance away. By the early neurula stage, the edge of the neural plate is the only major site of BMP-4 and msx-1 expression, and is also the only site that responds to BMP inhibition or overexpression. At this time, the neural plate appears to have a low level of BMP antagonist activity. Using in vivo grafts and in vitro assays, we show that the position of the border is further maintained by interactions between non-neural and neural ectoderm. We conclude that the border develops by integration of signals from the organizer, the developing neural plate, the paraxial mesoderm and the non-neural epiblast, involving FGFs, BMPs and their inhibitors. We suggest that BMPs act in an autocrine way to maintain the border state.

SWiP-1: novel SOCS box containing WD-protein regulated by signalling centres and by Shh during development.

We describe a novel chick WD-protein, cSWiP-1, expressed in somitic mesoderm and developing limb buds as well as in other embryonic structures where Hedgehog signalling has been shown to play a role. Using embryonic manipulations we show that in somites cSWiP-1 expression integrates two signals originating from structures adjacent to the segmental mesoderm: a positive signal from the notochord and a negative signal from intermediate and/or lateral mesoderm. In explant cultures of somitic mesoderm, Shh protein induces cSWiP-1, while a blocking antibody to Shh inhibits the induction of cSWiP-1 by the notochord. These results show that the positive signal from the notochord is mediated by Shh. We also show that in limb buds cSWiP-1 is upregulated by ectopic Shh. This occurs in about the same time period as upregulation of BMP2, placing cSWiP-1 among the earliest markers for the change of limb pattern caused by ectopic Shh. We also describe a human homologue of cSWiP-1 and a mouse gene, mSWiP-2, that is more distantly related to SWiP-1, suggesting that SWiP-1 belongs to a novel subfamily of WD-proteins.

Goosecoid misexpression alters the morphology and Hox gene expression of the developing chick limb bud.

The homeobox-containing gene goosecoid (gsc) has been implicated in a variety of embryonic processes from gastrulation to rib patterning. We have analyzed the role it plays during chick limb development. Expression is initially observed at stage 20 in a proximal-anterior-ventral domain of the early limb bud which expands during subsequent stages. Later in limb development a second domain of expression appears distally which resolves to regions which surround the condensing cartilage. In order to understand the function of gsc in limb development, we have examined the effect of misexpressing gsc throughout the limb. Two striking phenotypes are observed. The first, evident at stage 24, is an alteration in the angle of femur outgrowth from the main body axis. The second, which can be detected at day 10 of development, is an overall decrease in the size of the limb with bones that are small, misshapen and bent. These phenotypes correlate with a decrease in levels of Hox gene expression in gsc-infected limb buds. From these results we suggest that gsc may normally function to regulate growth and patterning of the limb, perhaps through regulation of Hox gene expression.

Early chick embryos in vitro.

In 1955, Denis New described a technique for the in vitro culture of early avian embryos that has formed the basis for nearly all of the experimental embryological studies performed on these species since that day. Many modifications to this technique have also been described in these four decades for specific experimental purposes. Here, we review the effects of some parameters that appear to be important for different aspects of the growth of embryos in this type of culture, and conduct a small experimental comparison between different modifications of the technique as described by various authors. We conclude that the original technique still compares favorably with its alternatives.

Formation and maintenance of the organizer among the vertebrates.

The organizer is established at the blastula stage of development, under the influence of a special region of cells known as the Nieuwkoop center in amphibians, where Vg1/activin-like signals overlap with activity of the Wnt-pathway. Despite differences in their mode of early development, a similar region can be identified in other vertebrates. It has widely been assumed that once the organizer property is assigned to cells at this early stage, it is fixed so that by the gastrula stage, no new cells acquire organizer properties. However, when the organizer is ablated, it can regenerate for a limited period during gastrulation, a process regulated by both positive and negative signals emanating from various domains in the embryo. Here we compare the mechanisms that initially establish the organiser in the blastula with those that maintain it during gastrulation in different vertebrate classes, and argue that similar molecular mechanisms may be involved in the two processes. We also suggest that these mechanisms are required to ensure the appropriate location of the organizer property in the gastrula, where cells are continuously moving.

HGF-SF: a neural inducing molecule in vertebrate embryos?

Neural induction is the process, during early embryonic development, by which cells of the mesoderm cause the overlying ectoderm cells to differentiate into neural structures, rather than epidermis. The phenomenon was discovered over 80 years ago in Hans Spemann's laboratory, and has since attracted much interest. However, we are still ignorant about the signals that elicit such a change in the direction of ectodermal differentiation, and about the mechanisms involved in the response of the ectoderm. Here, we report that HGF-SF can cause cultured chick ectodermal cells to become neural. We also discuss preliminary evidence suggesting that a homolog of this factor is expressed in Hensen's node, the inducing tissue, at about the stage at which neural induction occurs. We speculate that HGF-SF, or a related factor, could be a neural inducing signal during the early development of vertebrate embryos.

A re-examination of mitotic activity in the early chick embryo.

As a result of extensive mitotic index analysis in colchicine-arrested chick embryos during gastrulation, it was ascertained that the primitive streak is a region of elevated mitotic index as compared to the surrounding tissue. Along the cephalo-caudal axis, the embryo displays two large peaks of mitotic index, one at the posterior end of the primitive streak and the other just anterior to Hensen's node. The length of the various phases of the mitotic period was determined in vitro by time-lapse filming, and the colchicine-arrested mitotic indices in vivo and in vitro were determined and compared for various regions. Some observations regarding the orientation of mitotic spindles and abnormal mitosis in vitro are also included, and the relevance of the above observations to early embryonic development is discussed.

Mitotic activity during somite segmentation in the early chick embryo.

The mitotic activity of the somites, segmental plate and posterior mesoderm were investigated in colchicine-treated and untreated chick embryos at st. 7-14. The mitotic figures in the somites are restricted to the proximity of the lumen and have their spindles orientated predominantly tangentially to the cavity. In the segmental plate there is no pattern in terms of the position or orientation of the mitotic spindles, but there is a single region, often found close to the cranial end of the segmental plate, with an elevated mitotic index. This may indicate a certain degree of synchrony among groups of segmental plate cells. These results are discussed in relation to the process of somite segmentation.