Eddy M. De Robertis

Dorsoventral Patterning in Xenopus: Inhibition of Ventral Signals by Direct Binding of Chordin to BMP-4

Chordin (Chd) is an abundant protein secreted by Spemann organizer tissue during gastrulation. Chd antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Recombinant Xenopus Chd binds to BMP-4 with high affinity (KD, 3 x 10(-10) M), binding specifically to BMPs but not to activin or TGF-beta1. Chd protein is able to dorsalize mesoderm and to neuralize ectoderm in Xenopus gastrula explants at 1 nM. We propose that the noncell-autonomous effects of Spemanns organizer on dorsoventral patterning are executed in part by diffusible signals that directly bind to and neutralize ventral BMPs during gastrulation.

Xenopus chordin: A Novel Dorsalizing Factor Activated by Organizer-Specific Homeobox Genes

A Xenopus gene whose expression can be activated by the organizer-specific homeobox genes goosecoid and Xnot2 was isolated by differential screening. The chordin gene encodes a novel protein of 941 amino acids that has a signal sequence and four Cys-rich domains. The expression of chordin starts in Spemanns organizer subsequent to that of goosecoid, and its induction by activin requires de novo protein synthesis. Microinjection of chordin mRNA induces twinned axes and can completely rescue axial development in ventralized embryos. This molecule is a potent dorsalizing factor that is expressed at the right time and in the right place to regulate cell-cell interactions in the organizing centers of head, trunk, and tail development.

Molecular nature of Spemann's organizer: the role of the Xenopus homeobox gene goosecoid

This study analyzes the function of the homeobox gene goosecoid in Xenopus development. First, we find that goosecoid mRNA distribution closely mimics the expected localization of organizer tissue in normal embryos as well as in those treated with LiCl and UV light. Second, goosecoid mRNA accumulation is induced by activin, even in the absence of protein synthesis. It is not affected by bFGF and is repressed by retinoic acid. Lastly, microinjection of goosecoid mRNA into the ventral side of Xenopus embryos, where goosecoid is normally absent, leads to the formation of an additional complete body axis, including head structures and abundant notochordal tissue. The results suggest that the goosecoid homeodomain protein plays a central role in executing Spemanns organizer phenomenon.

Frzb-1 Is a Secreted Antagonist of Wnt Signaling Expressed in the Spemann Organizer

Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

Cerberus-like is a secreted factor with neuralizing activity expressed in the anterior primitive endoderm of the mouse gastrula

We report the isolation of mouse cerberus-like (cer-l), a gene encoding a novel secreted protein that is specifically expressed in the anterior visceral endoderm during early gastrulation. Expression in the primitive endoderm starts before the appearance of the primitive streak and lasts until the head-fold stage. In later stages, a second region of expression is found in newly formed somites. Mouse cer-l shares some sequence similarity with Xenopus cerberus (Xcer). In Xenopus assays cer-l, like Xcer, mRNA acts as a potent neuralizing factor that induces forebrain markers and endoderm, but is unable to induce ectopic head-like structures as Xcer does. In addition to cer-l, anterior visceral endoderm was found to express the transcription factors Lim1, goosecoid and HNF-3beta that are also present in trunk organizer cells. A model of how head and trunk development might be regulated is discussed. Given its neuralizing activity, the secreted protein Cer-l is a candidate for mediating inductive activities of anterior visceral endoderm.

Cleavage of Chordin by Xolloid Metalloprotease Suggests a Role for Proteolytic Processing in the Regulation of Spemann Organizer Activity

The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites in biochemical experiments Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein was unable to antagonize BMP activity. Furthermore, Xolloid digestion released biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicated that the in vivo function of Xolloid is to limit the extent of Spemanns organizer field. We propose that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis: [formula in text].

Gastrulation in the Mouse: The Role of the Homeobox Gene goosecoid

Mouse goosecoid is a homeobox gene expressed briefly during early gastrulation. Its mRNA accumulates as a patch on the side of the epiblast at the site where the primitive streak is first formed. goosecoid-expressing cells are then found at the anterior end of the developing primitive streak, and finally in the anteriormost mesoderm at the tip of the early mouse gastrula, a region that gives rise to the head process. Treatment of early mouse embryos with activin results in goosecoid mRNA accumulation in the entire epiblast, suggesting that a localized signal induces goosecoid expression during development. Transplantation experiments indicate that the tip of the murine early gastrula is the equivalent of the organizer of the amphibian gastrula.

The evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling

Dorsal–ventral patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional information gradient. The components involved include bone morphogenetic proteins (BMP/Dpp), a BMP antagonist (Chordin/Short gastrulation; Chd/Sog) and a secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. Here we describe Xenopus Twisted gastrulation (xTsg), another member of this signalling pathway. xTsg is expressed ventrally as part of the BMP-4 synexpression group and encodes a secreted BMP-binding protein that is a BMP signalling agonist. The data suggest a molecular mechanism by which xTsg dislodges latent BMPs bound to Chordin BMP-binding fragments generated by Xolloid cleavage, providing a permissive signal that allows high BMP signalling in the embryo. Drosophila Tsg also binds BMPs and is expressed dorsally, supporting the proposal that the dorsal–ventral axis was inverted in the course of animal evolution.

The homeobox gene goosecoid and the origin of organizer cells in the early chick blastoderm

The chick homeobox gene goosecoid (gsc) is first expressed in a barely noticeable cell population near the posterior margin (Kollers sickle) of the unincubated egg. Then it is detected in Hensens node, traditionally considered the chick organizer. Later, gsc-expressing cells leave the node with the prechordal plate. Fate mapping indicates that these three regions are related by cell lineage, and transplantation experiments suggest that they all have inducing activity. Quail posterior margin and anterior primitive streak grafts (gsc expressing) induce gsc transcription in neighboring chick host cells. We propose that development of the chick organizer starts earlier than previously thought and that gsc marks this changing cell population.

The Xenopus Dorsalizing Factor noggin Ventralizes Drosophila Embryos by Preventing DPP from Activating Its Receptor

noggin is expressed in the Spemann organizer region of the Xenopus embryo and can promote dorsal cell fates within the mesoderm and neural development within the overlying ectoderm. Here, we show that noggin promotes ventral development in Drosophila, specifying ventral ectoderm and CNS in the absence of all endogenous ventral-specific zygotic gene expression. We utilize constitutively active forms of the dpp receptors to demonstrate that noggin blocks dpp signaling upstream of dpp receptor activation. These results suggest a mechanistic basis for the action of Spemanns organizer during Xenopus development and provide further support for the conservation of dorsal-ventral patterning mechanisms between arthropods and chordates.