Date Published: May 11, 2004
Publisher: Public Library of Science
Author(s): Hiroki Kuroda, Oliver Wessely, E. M. De Robertis
Abstract: 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.
Partial Text: Vertebrate development results from a series of cell–cell interactions in which groups of cells induce their neighbors to acquire new cell differentiation fates. This process, known as embryonic induction, was first reported for the induction of the lens in surface ectoderm by the optic vesicles originating from the brain (Spemann 1901; Lewis 1904). Subsequent work showed that the surface ectoderm itself also plays an important role (reviewed by Grainger 1992). From the analysis of lens induction, Spemann (1938) proposed that a double assurance mechanism (doppelte Sicherung) could provide a way of explaining the robustness of vertebrate development via reciprocal interactions between two layers of cells. Lens induction is an example of a secondary embryonic induction. Most experimental embryologists concentrated their research on the induction of the neural plate, which is considered the primary embryonic induction (Spemann 1938; Saxén and Toivonen 1962; Harland 2000; Gilbert 2001; Stern 2002). In the classical organizer transplantation experiment, Spemann and Mangold (1924) demonstrated that dorsal lip mesoderm is sufficient to induce the differentiation of a complete central nervous system (CNS) in responding ectoderm. Spemann devoted an entire chapter of his book to the discussion of whether a double assurance mechanism existed in the case of neural plate induction (Chapter 8 in Spemann 1938) and concluded that the evidence supported a role for the underlying mesoderm, but not for the prospective neuroectoderm.
The results presented here are consistent with the following sequence of events during CNS development in Xenopus. A dorsal β-Catenin signal triggered by the early cortical rotation of the egg (Gerhart et al. 1991; De Robertis et al. 2000) induces the expression of anti-BMP molecules such as Chd and Nog in a group of cells located in the dorsal animal region at the blastula stage (see Figure 8). The dorsal prospective neuroectoderm is already specified to form CNS at blastula (see Figure 3). Remarkably, transplantation studies showed that this BCNE center was required for brain formation in vivo (see Figure 4). The normal fate of these dorsal animal cap cells during development is to give rise to anterior CNS, floor plate, and notochord (see Figure 2). We note that our previous term “preorganizer” (Wessely et al. 2001) was somewhat inadequate. Since BCNE grafts are unable to induce CNS in neighboring cells after transplantation, they lack organizer activity.