Xenopus Embryo: Neural Induction

Abstract

In vertebrate embryos, the future nervous system forms during early development by a series of molecular events named neural induction. This includes the early action of mesoderm‐derived fibroblast growth factors (FGFs), followed by the blockade of bone morphogenetic protein (BMP) signalling by the dorsal mesoderm during gastrulation, and finally comprises the regionalisation of the neural plate along the anterior‐posterior and medial‐lateral axes, under the complex and combined actions of FGFs, retinoids and Wnts. Founding experiments using frog embryos have initiated intensive research on neural induction 90 years ago. Molecular approaches combined with classical experimental embryology strategies provide a powerful way of deciphering those intricate regulations in frogs and more recently in other vertebrates. The current model of neural induction in the frog model system Xenopus, is largely conserved between aquatic species and amniotes. This model was recently updated to describe neural induction in the posterior spinal cord.

Key Concepts

  • The formation of neural precursors from the embryonic ectoderm is one of the earliest embryonic inductions.
  • Neural induction results in the formation of a neural plate regionalised along its anterior‐posterior and medial‐lateral axis.
  • Neural induction is subdivided into at least three steps: acquisition of neural competence, induction of anterior neural tissue and posteriorisation of the neural tissue.
  • Almost a century after the discovery of the Spemann–Mangold organiser in frogs, the exploration of the molecular mechanisms driving neural induction remains a very active field of research.
  • The models and molecular mechanisms discovered in frogs are largely conserved in other vertebrates including amniotes.

Keywords: nervous system; neural tube; organiser; BMP; Noggin; Chordin; Cerberus; fibroblast growth factors; Wnt; retinoic acid; brain; spinal cord

Figure 1. Neural induction is initiated during gastrulation. Schematics depict dorsal views of gastrula and neurula stage Xenopus laevis embryos, with the blastopore (B), the neural plate (NP) and the neural folds (NF). Matching stages are shown after whole mount in situ hybridization detecting the messenger RNAs of the pan‐neural gene sox2 (black staining seen on the dorsal views of white embryos). Sox2 is detected in the future neural ectoderm as early at initiation of gastrulation, in a broad domain, which is progressively refined into neural plate and neural tube during gastrulation and neurulation.
Figure 2. The Spemann–Mangold experiment (1924). (a) The dorsal lip of the blastopore, together with the tissue located immediately dorsal to it, was taken from a lightly pigmented donor embryo and (b) grafted on the ventral side of a darkly pigmented recipient embryo (the original experiment was performed using two species of the newt Triturus). (c) Alternatively, the grafted fragment was inserted into the blastocoel cavity on the ventral side of the recipient embryo, in contact with the ventral ectoderm (variation called the Einsteck technique). In (a), (b) and (c), the asterisks indicates the dorsal side of the embryo. (d–e) These experiments resulted into the development of twinned embryos, a second twin developing from the ventral side of the recipient embryo. (f) The difference in cell pigmentation allowed tracking the progeny of the donor tissue (in white) in the resulting conjoined twin larva. Donor cells formed a minor part of the secondary axis, which demonstrates the induction of the neural tube and part of the dorsal mesoderm (excepting the notochord) formed by recipient tissues, under the influence of the dorsal lip of the blastopore (also named later on the Spemann's organiser). Moreover, the induced secondary neural tube was patterned along the anterior‐posterior axis and formed brain and spinal cord structures.
Figure 3. Common experimental strategies to study neural induction in frog embryos. Our understanding of neural induction in frogs relies on experimental strategies using late blastula and early gastrula stages. Although these early developmental stages are delicate to manipulate, such experiments were successfully developed using frog embryos, while they remain extremely challenging in other animal models. (a) Keller explants. The prospective neural plate and its adjacent dorsal mesoderm, including the dorsal blastopore lip, are peeled off at early gastrula stage and flattened under a coverslip. These explants allow testing the interactions between mesoderm and ectoderm in the plane of one tissue layer (planar induction) and allow studying the cell dynamics of convergence and extension. (b) Animal cap. At the end of cleavage, the blastocoel roof cells have not yet received mesoderm or neural inducing signals. They are used to test various inducing activities experimentally. When explanted and grown in vitro (1), the blastocoel roof ectoderm heals into a ball of cells, the animal cap (2), and differentiates into ciliated epidermis. Upon short cell dissociation (3), the blastocoel roof cells form neural tissue, suggesting that dilution of a negative signal by dissociation allows neural induction. This is the basis of the ‘default model’ for neural induction. Animal cap being equivalent to pluripotent stem cells, inducing properties of various molecular activities can also be tested. To do so, cDNA or mRNA encoding various proteins or molecular variants are injected into whole embryos at the one‐ or two‐cell stage and animal caps are dissected and grown in vitro. Their differentiation is analysed, for example, anterior neural differentiation and posterior neural differentiation. dl, dorsal lip of the blastopore; D, Dorsal side.
Figure 4. Multiple signalling pathways interact during neural induction. BMPs are secreted molecules of the transforming growth factor β (TGFβ) superfamily, acting via Smad molecules in target cells. BMP signalling antagonises neural induction using Smad1‐5‐8/Smad4 transcriptional activity. BMP signalling can be inhibited by BMP interacting molecules acting outside the cell (e.g. Noggin, Chordin and Follistatin), or by the intracellular modulation of Smad1‐5‐8 phosphorylation. For instance, Smad1 phosphorylation by MAPK primes for Smad1 phosphorylation by GSK‐3. This results in blocking Smad1/Smad4 association and nuclear translocation of the complex. Active Wnt signalling inhibits GSK‐3 and promotes β‐catenin nuclear translocation. Consequently, Wnt signals antagonise neural induction. These signalling activities are highly stage‐dependent and context‐dependent. At later stages of neural induction, FGF and Wnt signalling contribute to neural posteriorisation.
Figure 5. Long‐range BMP antagonism allows neural induction dorsally. (a) Schematics of a gastrula‐stage frog embryo, seen in sagittal section. Chordin (Chd), Noggin, Follistatin and Xnr3 are secreted by Spemann's organiser cells. They antagonise BMP4, produced ventrally. The ventral side secretes Xenopus Tolloid‐related protein (Xlr), which prevents Chd from acting ventrally. ar, archenteron; BL, blastocoel; B, bottle cells. Scheme based on Plouhinec JL, Zakin L, Moriyama Y and De Robertis EM Chordin forms a self‐organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo. Proceedings of the National Academy of Sciences of the United States of America 110: 20372–9. (b) Immunohistochemistry detecting endogenous Chd protein (in white) in a late gastrula‐stage Xenopus laevis embryo, seen in cross section. Chd is produced by Spemann's organiser cells (So) and diffuses ventrally through Brachet's cleft (Bc), the thin space filled with extracellular matrix that separates ectoderm from anterior endoderm and mesoderm. Source: Adapted from Plouhinec JL , Zakin L , Moriyama Y and De Robertis EM Chordin forms a self‐organizing morphogen gradient in the extracellular space between ectoderm and mesoderm in the Xenopus embryo. Proceedings of the National Academy of Sciences of the United States of America 110: 20372–9. (c) J.L. Plouhinec.
Figure 6. Neural regionalisation shortly follows neural induction. As anterior endoderm and axial mesoderm migrate along the blastocoel roof, they induce anterior‐type neural tissue. More posterior mesoderm induces neural tissue with a more posterior fate. (a,b) At the end of gastrulation, the neural plate is subdivided into anterior and posterior neural ectoderm. It is also subdivided along the medial‐lateral axis. (c,d) As neurulation proceeds, the neural plate elongates and rolls up into brain and spinal cord with complex anterior‐posterior and dorsal‐ventral subdivisions. ar, archenteron; yp, yolk plug. Colour code for the different tissues is indicated. Schematics are drawn after side views or cross‐sections of gastrula and neurula‐stage frog embryos.
Figure 7. (a) Neural induction follows the Nieuwkoop ‘Activation‐Transformation’ model (1952). The neural tissue induced by young dorsal blastopore lip is fated to anterior character, while older dorsal lips induce posterior neural tissue. To test directly the hypothesis of an initial anterior neural induction (the ‘activation’) followed by posterior ‘transformation’ of the anterior tissue by posterior inducers, P. Nieuwkoop inserted several pieces of competent young blastula ectoderm at the midline of a young neurula recipient. Neural tissue was formed in the part of the grafts located close to the host neural plate. Moreover, the graft located anteriorly adopted a forebrain fate, while the grafts located at hindbrain and spinal cord levels formed more posterior neural tissues close to the host neural plate, and more anterior neural structures farther from the host (see text for details). This demonstrated the existence of a posteriorising activity, acting in a graded manner, diffusing from the posterior end of the host embryo. Note that in the original report, the third fragment, implanted in the posterior spinal cord area, did not form anterior neural tissue. ar, archenteron; blp, blastopore. Colour code for the different tissues is indicated. Dotted red indicates prechordal mesoderm. (b) Recently, the model has been amended to include recent results in frog, fish and mouse embryos. In this updated model, the ‘activation‐transformation’ applies to the patterning of brain and anterior spinal cord, while the posterior spinal cord (purple) responds to different signal combinations.
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Further Reading

Demagny H, Araki T and De Robertis EM (2014) The tumor suppressor Smad4/DPC4 is regulated by phosphorylations that integrate FGF, Wnt and TGFβ signalling. Cell Reports 9: 688–700.

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Min TH, Kriebel M, Hou S and Pera EM (2011) The dual regulator Sufu integrates Hedgehog and Wnt signals in the early Xenopus embryo. Developmental Biology 358: 262–276.

Pera EM, Acosta H, Gouignard N, Climent M and Arregi I (2014) Active signals, gradient formation and regional specificity in neural induction. Experimental Cell Research 321: 25–31.

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Monsoro‐Burq, Anne‐Hélène(Jul 2020) Xenopus Embryo: Neural Induction. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000731.pub3]