Xenopus Embryo: Neural Induction

Abstract

In embryos, the prospective 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 to classical experimental embryology strategies provide a powerful way of deciphering those complex regulations in frogs and more recently in other vertebrates. This article describes the current model of neural induction in the frog model system Xenopus, a model largely conserved between aquatic species and amniotes.

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

Figure 1. The Spemann–Mangold experiment (1924). The dorsal lip of the blastopore was taken from a darkly pigmented donor embryo and grafted on the ventral side of a lightly pigmented recipient embryo (the original experiment was performed using two species of the newt Triturus). The difference in cell pigmentation allowed tracking the progeny of the donor tissue 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 dorsal mesoderm (excepting the notochord) by the dorsal lip of the blastopore (also named 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 2. 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 remain delicate to manipulate, such experiments were successfully developed using frog embryos, while they are extremely challenging in other animal models. (a) As a variant to the Spemann–Mangold graft, the donor tissue is inserted into the blastocoel of the recipient embryo, towards the ventral side (Einsteck technique). This was used to evaluate the neural inductive power of various tissues, such as dorsal blastopore lips taken at older gastrula stages. This also allows testing if inductive signals travel across embryonic tissue layers (vertical induction). (b) 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 (Keller explant). This tests the interactions between mesoderm and ectoderm in the plane of one tissue layer (planar induction) and allows studying the cell dynamics of convergence and extension. (c) At the end of cleavage, the blastocoel roof cells have not 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. blc = blastocoel; dl = dorsal lip of the blastopore.
Figure 3. 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 4. 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 orthologue (Xlr), which prevents Chd from acting ventrally. ar = archenteron; blc = blastocoel. Modified from eLS article Cleavage and Gastrulation in Xenopus laevis Embryos, with permission. (b) Immunohistochemistry detecting endogenous Chd protein 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. Reproduced with permission from Plouhinec et al., 2013. (c) J.L. Plouhinec.
Figure 5. 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.
Figure 6. Neural induction follows the Nieuwkoop ‘Activation‐Transformation’ model (). 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 (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. ar = archenteron; blp = blastopore. Colour code for the different tissues is indicated. Dotted red indicates prechordal mesoderm.
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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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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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Pla, Patrick, and Monsoro‐Burq, Anne‐Hélène(Jun 2015) Xenopus Embryo: Neural Induction. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000731.pub2]