[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Development 09/2001; 128(15):2915-27. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The whole-mount in situ hybridization process has revolutionized the study of gene expression in the embryo. This procedure allows extremely sensitive detection of RNA transcripts and excellent spatial resolution. Numerous experiments benefit from the detection of more than one marker molecule in the same experimental embryo. While antisense RNA probes are extremely useful and methods for two-color in situ hybridization are available, antibodies recognizing specific protein species can help to expand the range of markers detected. Here we present a protocol that permits the simultaneous localization of RNA transcripts and immunocytochemical localization of proteins in the chick embryo.
[show abstract][hide abstract] ABSTRACT: For three-quarters of a century, developmental biologists have been asking how the nervous system is specified as distinct from the rest of the ectoderm during early development, and how it becomes subdivided initially into distinct regions such as forebrain, midbrain, hindbrain and spinal cord. The two events of 'neural induction' and 'early neural patterning' seem to be intertwined, and many models have been put forward to explain how these processes work at a molecular level. Here I consider early neural patterning and discuss the evidence for and against the two most popular models proposed for its explanation: the idea that multiple signalling centres (organizers) are responsible for inducing different regions of the nervous system, and a model first articulated by Nieuwkoop that invokes two steps (activation/transformation) necessary for neural patterning. As recent evidence from several systems challenges both models, I propose a modification of Nieuwkoop's model that most easily accommodates both classical and more recent data, and end by outlining some possible directions for future research.
[show abstract][hide abstract] ABSTRACT: 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.
The International Journal of Developmental Biology 02/2001; 45(1):165-75. · 2.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Over the past 50 years and more, many models have been proposed to explain how the nervous system is initially induced and how it becomes subdivided into gross regions such as forebrain, midbrain, hindbrain and spinal cord. Among these models is the 2-signal model of Nieuwkoop & Nigtevecht (1954), who suggested that an initial signal ('activation') from the organiser both neuralises and specifies the forebrain, while later signals ('transformation') from the same region progressively caudalise portions of this initial territory. An opposing idea emerged from the work of Otto Mangold (1933) and other members of the Spemann laboratory: 2 or more distinct organisers, emitting different signals, were proposed to be responsible for inducing the head, trunk and tail regions. Since then, evidence has accumulated that supports one or the other model, but it has been very difficult to distinguish between them. Recently, a considerable body of work from mouse embryos has been interpreted as favouring the latter model, and as suggesting that a 'head organiser', required for the induction of the forebrain, is spatially separate from the classic organiser (Hensen's node). An extraembryonic tissue, the 'anterior visceral endoderm' (AVE), was proposed to be the source of forebrain-inducing signals. It is difficult to find tissues that are directly equivalent embryologically or functionally to the AVE in other vertebrates, which led some (e.g. Kessel, 1998) to propose that mammals have evolved a new way of patterning the head. We will present evidence from the chick embryo showing that the hypoblast is embryologically and functionally equivalent to the mouse AVE. Like the latter, the hypoblast also plays a role in head development. However, it does not act like a true organiser. It induces pre-neural and pre-forebrain markers, but only transiently. Further development of neural and forebrain phenotypes requires additional signals not provided by the hypoblast. In addition, the hypoblast plays a role in directing cell movements in the adjacent epiblast. These movements distance the future forebrain territory from the developing organiser (Hensen's node), and we suggest that this is a mechanism to protect the forebrain from caudalising signals from the node. These mechanisms are consistent with all the findings obtained from the mouse to date. We conclude that the mechanisms responsible for setting up the forebrain and more caudal regions of the nervous system are probably similar among different classes of higher vertebrates. Moreover, while reconciling the two main models, our findings provide stronger support for Nieuwkoop's ideas than for the concept of multiple organisers, each inducing a distinct region of the CNS.
Journal of Anatomy 01/2001; 199(Pt 1-2):35-52. · 2.36 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mechanisms of Development 12/2000; 98(1-2):133-7. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Several models have been proposed for the generation of the rostral nervous system. Among them, Nieuwkoop's activation/transformation hypothesis and Spemann's idea of separate head and trunk/tail organizers have been particularly favoured recently. In the mouse, the finding that the visceral endoderm (VE) is required for forebrain development has been interpreted as support for the latter model. Here we argue that the chick hypoblast is equivalent to the mouse VE, based on fate, expression of molecular markers and characteristic anterior movements around the time of gastrulation. We show that the hypoblast does not fit the criteria for a head organizer because it does not induce neural tissue from naïve epiblast, nor can it change the regional identity of neural tissue. However, the hypoblast does induce transient expression of the early markers Sox3 and Otx2. The spreading of the hypoblast also directs cell movements in the adjacent epiblast, such that the prospective forebrain is kept at a distance from the organizer at the tip of the primitive streak. We propose that this movement is important to protect the forebrain from the caudalizing influence of the organizer. This dual role of the hypoblast is more consistent with the Nieuwkoop model than with the notion of separate organizers, and accommodates the available data from mouse and other vertebrates.
Development 10/2000; 127(17):3839-54. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: During neural induction, the 'organizer' of the vertebrate embryo instructs neighbouring ectodermal cells to become nervous system rather than epidermis. This process is generally thought to occur around the mid-gastrula stage of embryogenesis. Here we report the isolation of ERNI, an early response gene to signals from the organizer (Hensen's node). Using ERNI as a marker, we present evidence that neural induction begins before gastrulation--much earlier in development than previously thought. We show that the organizer and some of its precursor cells produce a fibroblast growth factor signal, which can initiate, and is required for, neural induction.
[show abstract][hide abstract] ABSTRACT: Segmentation, or metamerism, consists of the subdivision of the body into discrete units that subsequently acquire regional specializations. In vertebrates, the most obvious manifestation of this phenomenon is seen during the formation of the mesodermal somites and their derivatives. This review surveys three different models for how somites form, and how they relate to recent molecular data suggesting the involvement of transcription factors and cell surface molecules. A new model (the "Morse code" model) is proposed to convey positional information to somitogenic cells. Finally, the molecular events of boundary formation (during the initial epithelialization of somites) and boundary maintenance (between adjacent somite halves as well as in resegmentation) are discussed.
Current Topics in Developmental Biology 02/2000; 47:107-29. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: The homeobox gene goosecoid was the first specific genetic marker of Spemann's organizer in vertebrate embryos to be discovered. In the frog, misexpression of this gene by RNA injection produces duplication of the posterior axis. For these reasons, the recent finding that mice lacking goosecoid function have no early axial defects was rather surprising. Here we assay the neural inducing strength of wild-type and goosecoid-mutant mouse nodes by transplantation into primitive streak stage chick embryos. Wild-type mouse nodes strongly induce the neural-specific transcription factors Sox2 and Sox3 in the chick host. Homozygous goosecoid(-/- )nodes are severely impaired in their ability to induce both genes. Heterozygous goosecoid(+/-) nodes induce Sox3 as well as wild-type nodes, but resemble -/- nodes in their limited ability to induce Sox2. We propose that goosecoid does play a role in regulating the neural inducing strength of the node and that regulative mechanisms exist which mask the early phenotypic consequences of goosecoid mutations in the intact mouse embryo.
[show abstract][hide abstract] ABSTRACT: 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.
Mechanisms of Development 10/1999; 87(1-2):213-6. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
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.
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.
Current Biology 10/1999; 9(17):931-8. · 9.49 Impact Factor
[show abstract][hide abstract] ABSTRACT: The organizer is a unique region in the gastrulating embryo that induces and patterns the body axis. It arises before gastrulation under the influence of the Nieuwkoop center. We show that during gastrulation, cell movements bring cells into and out of the chick organizer, Hensen's node. During these movements, cells acquire and lose organizer properties according to their position. A "node inducing center," which emits Vg1 and Wnt8C, is located in the middle of the primitive streak. Its activity is inhibited by ADMP produced by the node and by BMPs at the periphery. These interactions define the organizer as a position in the embryo, whose cellular makeup is constantly changing, and explain the phenomenon of organizer regeneration.
[show abstract][hide abstract] ABSTRACT: 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.
Development 08/1999; 126(13):2921-34. · 6.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mechanisms of Development 08/1999; 85(1-2):85-96. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mechanisms of Development 05/1999; 82(1-2):51-66. · 2.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Mechanisms of Development 05/1999; 82(1-2):79-94. · 2.38 Impact Factor