Neural Induction in Xenopus: Requirement for Ectodermal and Endomesodermal Signals via Chordin, Noggin, β-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 β-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 β-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 β-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

Neural Induction in Xenopus: Requirement for Ectodermal and Endomesodermal Signals via Chordin, Noggin, β-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 β-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 β-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 β-Catenin signal predisposes the prospective neuroectoderm to neural induction by endomesodermal signals emanating from Spemann's organizer.     

Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals

At the border of the neural plate, the induction of the neural crest can be achieved by interactions with the epidermis, or with the underlying mesoderm. Wnt signals are required for the inducing activity of the epidermis in chick and amphibian embryos. Here, we analyze the molecular mechanisms of neural crest induction by the mesoderm in Xenopus embryos. Using a recombination assay, we show that prospective paraxial mesoderm induces a panel of neural crest markers (Slug, FoxD3, Zic5 and Sox9), whereas the future axial mesoderm only induces a subset of these genes. This induction is blocked by a dominant negative (dn) form of FGFR1. However, neither dnFGFR4a nor inhibition of Wnt signaling prevents neural crest induction in this system. Among the FGFs, FGF8 is strongly expressed by the paraxial mesoderm. FGF8 is sufficient to induce the neural crest markers FoxD3, Sox9 and Zic5 transiently in the animal cap assay. In vivo, FGF8 injections also expand the Slug expression domain. This suggests that FGF8 can initiate neural crest formation and cooperates with other DLMZ-derived factors to maintain and complete neural crest induction. In contrast to Wnts, eFGF or bFGF, FGF8 elicits neural crest induction in the absence of mesoderm induction and without a requirement for BMP antagonists. In vivo, it is difficult to dissociate the roles of FGF and WNT factors in mesoderm induction and neural patterning. We show that, in most cases, effects on neural crest formation were parallel to altered mesoderm or neural development. However, neural and neural crest patterning can be dissociated experimentally using different dominant-negative manipulations: while Nfz8 blocks both posterior neural plate formation and neural crest formation, dnFGFR4a blocks neural patterning without blocking neural crest formation. These results suggest that different signal transduction mechanisms may be used in neural crest induction, and anteroposterior neural patterning.     

Endodermal Nodal-related signals and mesoderm induction in Xenopus

In Xenopus, mesoderm induction by endoderm at the blastula stage is well documented, but the molecular nature of the endogenous inductive signals remains unknown. The carboxy-terminal fragment of Cerberus, designated Cer-S, provides a specific secreted antagonist of mesoderm-inducing Xenopus Nodal-Related (Xnr) factors. Cer-S does not inhibit signalling by other mesoderm inducers such as Activin, Derrière, Vg1 and BMP4, nor by the neural inducer Xnr3. In the present study we show that Cer-S blocks the induction of both dorsal and ventral mesoderm in animal-vegetal Nieuwkoop-type recombinants. During blastula stages Xnr1, Xnr2 and Xnr4 are expressed in a dorsal to ventral gradient in endodermal cells. Dose-response experiments using cer-S mRNA injections support the existence of an endogenous activity gradient of Xnrs. Xnr expression at blastula can be activated by the vegetal determinants VegT and Vg1 acting in synergy with dorsal (beta)-catenin. The data support a modified model for mesoderm induction in Xenopus, in which mesoderm induction is mediated by a gradient of multiple Nodal-related signals released by endoderm at the blastula stage.     

Endogenous Cerberus activity is required for anterior head specification in Xenopus

We analyzed the endogenous requirement for Cerberus in Xenopus head development. 'Knockdown' of Cerberus function by antisense morpholino oligonucleotides did not impair head formation in the embryo. In contrast, targeted increase of BMP, Nodal and Wnt signaling in the anterior dorsal-endoderm (ADE) resulted in synergistic loss of anterior head structures, without affecting more posterior axial ones. Remarkably, those head phenotypes were aggravated by simultaneous depletion of Cerberus. These experiments demonstrated for the first time that endogenous Cerberus protein can inhibit BMP, Nodal and Wnt factors in vivo. Conjugates of dorsal ectoderm (DE) and ADE explants in which Cerberus function was 'knocked down' revealed the requirement of Cerberus in the ADE for the proper induction of anterior neural markers and repression of more posterior ones. This data supports the view that Cerberus function is required in the leading edge of the ADE for correct induction and patterning of the neuroectoderm.     

Borrowing thy neighbour's genetics: Neural induction and a Brachyury mutant in Xenopus

A recent article by Rao⁽¹⁾ exemplifies a number of new trends in developmental biology, both of technical strategy and approach to the problem of neural induction. Rao introduced into frog embryos a mutant form of a mesodermal gene, Brachyury, and caused ectopic neural differentiation. This essay traces the route from the original Brachyury mutation in mouse to the most likely conclusion of Rao's experiments — suggested previously⁽²⁾ — that neural fate is a default pathway.     

Neural and head induction by insulin-like growth factor signals.

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.     

Tissues and signals involved in the induction of placodal Six1 expression in Xenopus laevis

Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.     

Planar and vertical signals in the induction and patterning of the Xenopus nervous system.

The cellular mechanisms responsible for the formation of the Xenopus nervous system have been examined in total exogastrula embryos in which the axial mesoderm appears to remain segregated from prospective neural ectoderm and in recombinates of ectoderm and mesoderm. Posterior neural tissue displaying anteroposterior pattern develops in exogastrula ectoderm. This effect may be mediated by planar signals that occur in the absence of underlying mesoderm. The formation of a posterior neural tube may depend on the notoplate, a midline ectodermal cell group which extends along the anteroposterior axis. The induction of neural structures characteristic of the forebrain and of cell types normally found in the ventral region of the posterior neural tube requires additional vertical signals from underlying axial mesoderm. Thus, the formation of the embryonic Xenopus nervous system appears to involve the cooperation of distinct planar and vertical signals derived from midline cell groups.     

Two-step induction of primitive erythrocytes in Xenopus laevis embryos: signals from the vegetal endoderm and the overlying ectoderm

Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.     

Distinct roles for Distal-less genes Dlx3 and Dlx5 in regulating ectodermal development in Xenopus

In vertebrates, there are six or more copies of genes related to the Drosophila pattern formation homeodomain gene Distal-less. Among this family, Dlx3 and Dlx5 share extensive sequence homology and have similar, but distinctive, expression patterns, suggesting that these two factors may have substantially redundant developmental functions. Here we show that at the earliest phases of embryogenesis in Xenopus, there are significant differences between Dlx3 and Dlx5 expression and that this correlates with different functions in the restriction of neural crest and neural plate boundaries, respectively.     

Xoom is required for epibolic movement of animal ectodermal cells in Xenopus laevis gastrulation

Gastrulation is the most dynamic cell movement and initiates the body plan in amphibian development. In contrast to numerous molecular studies on mesodermal induction, the driving force of gastrulation is as yet poorly understood. A novel transmembrane protein, Xoom, was previously reported, which is required for Xenopus gastrulation. In the present study, the role of Xoom during Xenopus gastrulation was further examined in detail. Overexpression and misexpression of Xoom induced overproduction of Xoom protein, but not a changed phenotype. However, Xoom antisense ribonucleic acid (RNA) injection reduced the Xoom protein and caused gastrulation defects without any influence on the involution and translation levels of mesodermal marker genes. Normal migrating activity of dorsal mesodermal cells was recognized in the antisense RNA-injected explant. Morphological examination using artificial exogastrulation showed that convergent extension of mesodermal cells occurred normally, but the ectodermal cell layer significantly shrank in the antisense RNA-injected embryo. Comparison of cell shape among various experimental conditions showed that inhibition of cell spreading occurs specifically in the outer ectodermal layer of the antisense RNA-injected embryo. Cytochemical examination indicated disorganization of F-actin in the ectodermal cells of the antisense RNA-injected embryo. These results suggest that Xoom plays an important role in the epibolic movement of ectodermal cells through some regulation of actin filament organization.     

The apparent requirement of two hormones, alpha- and beta-ecdysone, for molting induction in insects

Puff formations at loci I-18-C and IV-2-B of the salivary gland chromosomes are early indications of a beginning molting process in Chironomus tentans larvae. The effectiveness of the two ecdysone analogs, α- and β-ecdysone, in inducing these puffs was compared. Incubation of salivary glands in vitro with β-ecdysone causes only puff IV-2-B to appear; incubation with α-ecdysone stimulates initially puffing at only I-18-C. After an injection of α-ecdysone, puffing at I-18-C begins within less than 15 min, whereas puffing at IV-2-B is delayed for more than 30 min. Following an injection of β-ecdysone, puffing at IV-2-B begins within less than 15 min, whereas puffing at I-18-C is delayed. Injected ³H-α-ecdysone is converted to β-ecdysone and a polar compound. Injected ³H-β-ecdysone is converted to a compound less polar than α-ecdysone and a polar metabolite which stimulates puffing at I-18-C, like α-ecdysone. It is suggested that the two ecdysones have different targets in the cell, that they can be rapidly converted to compounds with the activity of the other analog, and that the induction of a complete molt requires the action of both hormones.