Anterior patterning by synergistic activity of the early gastrula organizer and the anterior germ layer tissues of the mouse embryo.

Fragments of the germ layer tissues isolated from the early-primitive-streak (early-streak) stage mouse embryos were tested for axis induction activity by transplantation to late-gastrula (late-streak to early-bud) stage host embryos. The posterior epiblast fragment that contains the early gastrula organizer was able to recruit the host tissues to form an ectopic axis. However, the most anterior neural gene that was expressed in the ectopic axis was Krox20 that marks parts of the hindbrain, but markers of the mid- and forebrain (Otx2 and En1) were not expressed. Anterior visceral endoderm or the anterior epiblast alone did not induce any ectopic neural tissue. However, when these two anterior germ layer tissues were transplanted together, they can induce the formation of ectopic host-derived neural tissues but these tissues rarely expressed anterior neural genes and did not show any organization of an ectopic axis. Therefore, although the anterior endoderm and epiblast together may display some inductive activity, they do not act like a classical organizer. Induction of the anterior neural genes in the ectopic axis was achieved only when a combination of the posterior epiblast fragment, anterior visceral endoderm and the anterior epiblast was transplanted to the host embryo. The formation of anterior neural structures therefore requires the synergistic interaction of the early gastrula organizer and anterior germ layer tissues.     

The L5 epitope: an early marker for neural induction in the chick embryo and its involvement in inductive interactions.

The pattern of expression of the carbohydrate epitope L5 was studied during early development of the chick neuroepithelium. Immunoreactivity first appears during gastrulation, at mid-primitive streak stage, and persists until at least 3.5 days of development. The epitope is expressed on all the components of the developing nervous system, both central and peripheral. In immunoblots, the antibody recognises a major component of about Mr 500,000 and several more minor components of lower molecular mass. If a Hensen's node from a donor embryo is transplanted into the area opaca of a host embryo, L5 immunoreactivity appears in the epiblast surrounding the graft. If hybridoma cells secreting the antibody are grafted together with Hensen's node into a host chick embryo, the induction of a supernumerary nervous system is inhibited. We suggest that the L5 epitope is an early and general marker for neural induction and that it may be involved directly in inductive interactions.     

Identification of chick frizzled-10 expressed in the developing limb and the central nervous system

We have identified chick frizzled (Fz)-10, encoding a Wnt receptor, and examined the expression pattern during embryogenesis. Fz-10 is expressed in the region posterior to the Hensen's node at stage 6. Fz-10 expression is detected in the dorsal domain of the neural tube and the central nervous system of the developing embryo. In the developing limb, Fz-10 expression starts at stage 18 in the posterior-dorsal region of the distal mesenchyme, and gradually expands to the anterior-distal region. Fz-10 is also expressed in the feather bud and branchial arch. Implantation of Sonic hedgehog (Shh)-expressing cells into the anterior margin of the limb bud resulted in the induction of Fz-10 expression in anterior-dorsal mesenchyme.     

The organizer of the mouse gastrula is composed of a dynamic population of progenitor cells for the axial mesoderm

An organizer population has been identified in the anterior end of the primitive streak of the mid-streak stage embryo, by the expression of Hnf3beta, Gsc(lacZ) and Chrd, and the ability of these cells to induce a second neural axis in the host embryo. This cell population can therefore be regarded as the mid-gastrula organizer and, together with the early-gastrula organizer and the node, constitute the organizer of the mouse embryo at successive stages of development. The profile of genetic activity and the tissue contribution by cells in the organizer change during gastrulation, suggesting that the organizer may be populated by a succession of cell populations with different fates. Fine mapping of the epiblast in the posterior region of the early-streak stage embryo reveals that although the early-gastrula organizer contains cells that give rise to the axial mesoderm, the bulk of the progenitors of the head process and the notochord are localized outside the early gastrula organizer. In the mid-gastrula organizer, early gastrula organizer derived cells that are fated for the prechordal mesoderm are joined by the progenitors of the head process that are recruited from the epiblast previously anterior to the early gastrula organizer. Cells that are fated for the head process move anteriorly from the mid-gastrula organizer in a tight column along the midline of the embryo. Other mid-gastrula organizer cells join the expanding mesodermal layer and colonize the cranial and heart mesoderm. Progenitors of the trunk notochord that are localized in the anterior primitive streak of the mid-streak stage embryo are later incorporated into the node. The gastrula organizer is therefore composed of a constantly changing population of cells that are allocated to different parts of the axial mesoderm.     

Clonal and molecular analysis of the prospective anterior neural boundary in the mouse embryo

In the mouse embryo the anterior ectoderm undergoes extensive growth and morphogenesis to form the forebrain and cephalic non-neural ectoderm. We traced descendants of single ectoderm cells to study cell fate choice and cell behaviour at late gastrulation. In addition, we provide a comprehensive spatiotemporal atlas of anterior gene expression at stages crucial for anterior ectoderm regionalisation and neural plate formation. Our results show that, at late gastrulation stage, expression patterns of anterior ectoderm genes overlap significantly and correlate with areas of distinct prospective fates but do not define lineages. The fate map delineates a rostral limit to forebrain contribution. However, no early subdivision of the presumptive forebrain territory can be detected. Lineage analysis at single-cell resolution revealed that precursors of the anterior neural ridge (ANR), a signalling centre involved in forebrain development and patterning, are clonally related to neural ectoderm. The prospective ANR and the forebrain neuroectoderm arise from cells scattered within the same broad area of anterior ectoderm. This study establishes that although the segregation between non-neural and neural precursors in the anterior midline ectoderm is not complete at late gastrulation stage, this tissue already harbours elements of regionalisation that prefigure the later organisation of the head.     

Anterior neural induction by nodes from rabbits and mice

The organizer of vertebrate embryos represents the major regulatory center for the formation of the embryonic axis during gastrulation. The early blastopore lip of amphibia and Hensen's node of the chick at the full-length primitive streak stage possess both a head- and a trunk-inducing potential. In mice, a head-inducing activity was identified in the extraembryonic, anterior visceral endoderm (AVE) by tissue ablation and genetic experiments. Evidence for a similar activity in the AVE from the rabbit was obtained by transplanting below the avian epiblast. However, it was still unclear whether the AVE is the exclusive origin of anterior neural induction or if this activity is recapitulated by the node and/or its derivatives. We report here that nodes from both rabbit and mouse embryos can induce a complete neural axis including forebrain structures upon grafting to chick hosts. Thus, in rabbits and mice not only the AVE, but also the node, possesses a potential for the induction of anterior neural tissue.     

Different clonal dispersion in the rostral and caudal mouse central nervous system

We have performed a systematic clonal analysis to describe the modes of growth, dispersion and production of cells during the development of the mouse neural system. We have used mice expressing a LaacZ reporter gene under the control of the neuron specific enolase promoter to randomly generate LacZ clones in the central nervous system (CNS). We present evidence for (1) a pool of CNS founder cells that is not regionalized, i.e. give descendants dispersed along the entire A-P axis, (2) an early separation between pools of precursors for the anterior and posterior CNS and (3) distinct modes of production of progenitors in these two domains. More specifically, cell growth and dispersion of the progenitors follow a relatively coherent pattern throughout the anterior CNS, a mode that leads to a progressive regionalization of cell fates. In contrast, cell growth of progenitors of the SC appears to involve self-renewing stem cells that progress caudally during regression of the mode. Therefore, at least part of the area surrounding the node is composed of precursors with self-renewing properties and the development of the trunk is dependent on pools of stem cells regressing from A to P. Taken together with our analysis of the cell growth changes associated with neuromere formation (Mathis, L., Sieur, J., Voiculescu, O., Charnay, P. and Nicolas, J. F. (1999) Development 126, 4095-4106), our results suggest that major transitions in CNS development correspond to changes in cell behavior and may provide a link between morphogenesis and genetic patterning mechanisms (i.e. formation of the body plan).     

Mesoderm induction in the future tail region of Xenopus

Summary We have used interspecific grafts between Xenopus borealis and Xenopus laevis to study the signalling system that produces tail mesoderm. Early gastrula ectoderm grafted into the posterior neural plate region of neurulae responds to a mesodermal inducing signal in this region and forms mainly tail somites; this signal persists until at least the early tail bud stage. Ventral ectoderm grafted into the posterior neural plate loses its competence to respond to this signal after stage 10 1/2. We have established the specification of anterior and posterior neural plate ectoderm. In ectodermal sandwiches or when grafted into unusual positions, anterior regions gave rise to mainly nervous system and posterior regions to large amounts of muscle, together with some nervous system. Thus it was impossible to assess the competence of posterior neural plate ectoderm to form further mesoderm and hence to establish if mesodermal induction continues during neurulation in unmanipulated embryos.     

Fgf signalling through MAPK cascade is required for development of the subpallial telencephalon in zebrafish embryos.

The telencephalon is formed in the most anterior part of the central nervous system (CNS) and is organised into ventral subpallial and dorsal pallial domains. In mice, it has been demonstrated that Fgf signalling has an important role in induction and patterning of the telencephalon. However, the precise role of Fgf signalling is still unclear, owing to overlapping functions of Fgf family genes. To address this, we have examined, in zebrafish embryos, the activation of Ras/mitogen-activated protein kinase (MAPK), one of the major downstream targets of Fgf signalling. Immunohistochemical analysis reveals that an extracellular signal-regulated kinase (ERK), a vertebrate MAPK is activated in the anterior neural boundary (ANB) of the developing CNS at early segmentation stages. Experiments with Fgf inhibitors reveal that ERK activation at this stage is totally dependent on Fgf signalling. Interestingly, a substantial amount of ERK activation is observed in ace mutants in which fgf8 gene is mutated. We then examine the function of Fgf signalling in telencephalic development by use of several inhibitors to Fgf signalling cascade, including dominant-negative forms of Ras (Ras(N17)) and the Fgf receptor (Fgfr), and a chemical inhibitor of Fgfr, SU5402. In treated embryos, the induction of telencephalic territory normally proceeded but the development of the subpallial telencephalon was suppressed, indicating that Fgf signalling is required for the regionalisation within the telencephalon. Finally, antisense experiments with morpholino-modified oligonucleotides suggest that zebrafish fgf3, which is also expressed in the ANB, co-operates with fgf8 in subpallial development.     

Induction of anterior neural fates in the ascidian Ciona intestinalis

The sensory vesicle of ascidians is thought to be homologous to the vertebrate forebrain and midbrain (Development 125 (1998) 1113). Here we report the isolation of two sensory vesicle markers in the ascidian Ciona intestinalis, which are homologs of vertebrate otx and gsx homeobox genes. By using these markers to analyze the induction of anterior neural tissue in Ciona, we find that the restriction of anterior neural fate to the progeny of the anterior animal blastomeres is due to a combination of two factors. The vegetal blastomeres show a differential inducing activity along the anterior-posterior axis, while the competence to respond to this inducing signal is markedly higher in the anterior animal blastomeres than in the posterior animal blastomeres. This differential competence to respond is also observed in response to bFGF, a candidate neural inducer in ascidians (J. Physiol. 511.2 (1998) 347) and can be detected by the gastrula stage. Our results, however, indicate that bFGF can only induce a subset of the responses of the endogenous inducer, suggesting that additional signals in the embryo are necessary to induce a fully patterned nervous system.     

Development of neural inducing capacity in dissociated Xenopus embryos

During gastrulation in amphibians, dorsal ectoderm is converted by induction from an epidermal pathway to central nervous system (CNS). We show in this report that the ability of the embryo to manufacture this neural inducing signal is not dependent upon intercellular communication between early cleavage and early gastrula stages. This result is consistent with the interpretation that the cell lineage that induces CNS formation arises itself by a cell-autonomous mechanism, perhaps specified by materials inherited from the fertilized egg.     

Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels

Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.     

Development of embryonic cells containing serotonin, catecholamines, and FMRFamide-related peptides in Aplysia californica

This study demonstrates the presence of a relatively extensive but previously unrecognized nervous system in embryonic stages of the opisthobranch mollusc Aplysia californica. During the trochophore stage, two pairs of cells were observed to be reactive to antibodies raised against the neuropeptides FMRFamide and EFLRIamide. These cells were located in the posterior region of the embryo, and their anterior projections terminated under the apical tuft. As the embryos developed into veliger stages, serotonin-like immunoreactive (LIR) cells appeared in the apical organ and were later observed to innervate the velum. Also, aldehyde-induced fluorescence indicative of catecholamines was present in cells in the foot, oral, and possibly apical regions during late embryonic veliger stages. Just before the embryo hatches as a free-swimming veliger, additional FMRFamide-LIR and catecholamine-containing cells appeared in regions that correspond to the ganglia of what will become the adult central nervous system (CNS). Neurons and connectives that will contribute to the adult CNS appear to develop along the pathways that are pioneered by the earliest posterior FMRFamide-LIR cells. These observations are consistent with the hypothesis that, besides their presumed roles in the control of embryonic behaviors, some elements may also guide the development of the CNS. Embryonic nervous systems that develop prior to and outside of the adult CNS have also been reported in pulmonate and prosobranch species of molluscs. Therefore, the demonstration of early developing neurons and their transmitter phenotypes in A. californica presents new opportunities for a better understanding of the ontogeny and phylogeny of both behavioral and neuronal function in this important model species.     

The avian organizer

The development of avian embryos is characterized by the large amount of yolk present from the one-cell stage until late phases of organogenesis. In the chick, an axis of bilateral symmetry is established already before egg laying, when the egg rotates in the uterus. There is evidence for an active Wnt-catenin pathway in the vegetal cells in the periphery of the multi-cellular embryo. It overlaps with the posteriorly restricted expression of genes characterizing the vegetal hemisphere in amphibia. The zone of overlap bears several functional characteristics of a Nieuwkoop center, which is first apparent in the posterior marginal zone, but continues into the early primitive streak. Only the anterior part of the late streak is capable of direct neural induction, and only its tip, Hensen's node, can induce an anterior neural identity. This latter activity leaves the node together with the cells representing the anterior mesendoderm. Thus, although the constraints and dynamics of avian development make comparisons with the amphibian situation a complex undertaking, Hensen's node comes as close as possible to an organizer in Spemann and H. Mangold's definition.     

The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning

Dickkopf1 (dkk1) encodes a secreted WNT inhibitor expressed in Spemann's organizer, which has been implicated in head induction in Xenopus. Here we have analyzed the role of dkk1 in endomesoderm specification and neural patterning by gain- and loss-of-function approaches. We find that dkk1, unlike other WNT inhibitors, is able to induce functional prechordal plate, which explains its ability to induce secondary heads with bilateral eyes. This may be due to differential WNT inhibition since dkk1, unlike frzb, inhibits Wnt3a signalling. Injection of inhibitory antiDkk1 antibodies reveals that dkk1 is not only sufficient but also required for prechordal plate formation but not for notochord formation. In the neural plate dkk1 is required for anteroposterior and dorsoventral patterning between mes- and telencephalon, where dkk1 promotes anterior and ventral fates. Both the requirement of anterior explants for dkk1 function and their ability to respond to dkk1 terminate at late gastrula stage. Xenopus embryos posteriorized with bFGF, BMP4 and Smads are rescued by dkk1. dkk1 does not interfere with the ability of bFGF to induce its immediate early target gene Xbra, indicating that its effect is indirect. In contrast, there is cross-talk between BMP and WNT signalling, since induction of BMP target genes is sensitive to WNT inhibitors until the early gastrula stage. Embryos treated with retinoic acid (RA) are not rescued by dkk1 and RA affects the central nervous system (CNS) more posterior than dkk1, suggesting that WNTs and retinoids may act to pattern anterior and posterior CNS, respectively, during gastrulation.     

Neural induction in Xenopus: requirement for ectodermal and endomesodermal signals via Chordin, Noggin, beta-Catenin, and Cerberus

The origin of the signals that induce the differentiation of the central nervous system (CNS) is a long-standing question in vertebrate embryology. Here we show that Xenopus neural induction starts earlier than previously thought, at the blastula stage, and requires the combined activity of two distinct signaling centers. One is the well-known Nieuwkoop center, located in dorsal-vegetal cells, which expresses Nodal-related endomesodermal inducers. The other is a blastula Chordin- and Noggin-expressing (BCNE) center located in dorsal animal cells that contains both prospective neuroectoderm and Spemann organizer precursor cells. Both centers are downstream of the early beta-Catenin signal. Molecular analyses demonstrated that the BCNE center was distinct from the Nieuwkoop center, and that the Nieuwkoop center expressed the secreted protein Cerberus (Cer). We found that explanted blastula dorsal animal cap cells that have not yet contacted a mesodermal substratum can, when cultured in saline solution, express definitive neural markers and differentiate histologically into CNS tissue. Transplantation experiments showed that the BCNE region was required for brain formation, even though it lacked CNS-inducing activity when transplanted ventrally. Cell-lineage studies demonstrated that BCNE cells give rise to a large part of the brain and retina and, in more posterior regions of the embryo, to floor plate and notochord. Loss-of-function experiments with antisense morpholino oligos (MO) showed that the CNS that forms in mesoderm-less Xenopus embryos (generated by injection with Cerberus-Short [CerS] mRNA) required Chordin (Chd), Noggin (Nog), and their upstream regulator beta-Catenin. When mesoderm involution was prevented in dorsal marginal-zone explants, the anterior neural tissue formed in ectoderm was derived from BCNE cells and had a complete requirement for Chd. By injecting Chd morpholino oligos (Chd-MO) into prospective neuroectoderm and Cerberus morpholino oligos (Cer-MO) into prospective endomesoderm at the 8-cell stage, we showed that both layers cooperate in CNS formation. The results suggest a model for neural induction in Xenopus in which an early blastula beta-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.