 |
Neural Induction and Self-Regulating Morphogenetic Fields in Vertebrate Embryos
Summary: Edward De Robertis studies how cell-cell signals induce the formation of the brain in the vertebrate embryo. These studies have led to the discovery of how secreted growth factor antagonists—such as Chordin, Cerberus, Crescent, and Frzb-1—regulate cell differentiation and embryonic dorsal-ventral patterning.
The amphibian embryo provides an excellent system for studying how the central nervous system (CNS) is formed in a vertebrate embryo. A rich heritage of experimental embryology exists, and classical transplantation techniques can now be combined with knockdowns of individual or multiple genes in development. A foundation for understanding CNS formation was provided by an experiment carried out by Spemann and Mangold 80 years ago involving grafting of the dorsal lip region of the amphibian embryo. They found that a small group of cells, called the organizer, is able to induce twinning and neural tissue in neighboring cells. Isolating the molecules involved in these cell-cell inductions has been the Holy Grail of amphibian embryology.
Through the work of several laboratories, extensive screens for organizer-specific genes have been performed in embryos of the frog Xenopus. We have isolated many genes that encode secreted proteins expressed specifically in Spemann's organizer. Studies on Chordin, Cerberus, Crescent, Frzb-1, and Sizzled have led to the realization that growth factor antagonists secreted into the extracellular space mediate embryonic patterning.
Chordin Is a BMP Antagonist
Chordin is one of the most abundant proteins secreted by organizer tissue at the gastrula stage, reaching concentrations of 6–10 nanomolar in the extracellular space. When microinjected into Xenopus embryos, chordin mRNA is able to induce twinning and CNS induction, recapitulating Spemann's experiment. When translation of Chordin is knocked down using antisense morpholino oligos, the CNS-inducing activity of Spemann organizer grafts is lost.
Biochemical studies on Chordin have provided a new paradigm for the regulation of growth factor signaling in the extracellular space. Chordin is a secreted protein with four cysteine-rich domains (CRs) of about 70 amino acids each. We have shown that each CR constitutes a bone morphogenetic protein (BMP)-binding module. The Chordin loss-of-function phenotype can be rescued by knockdown of BMPs, underscoring that Chordin is a dedicated BMP antagonist. BMP4 bound to Chordin is unable to bind BMP receptors on the cell surface.
Chordin has a cofactor, called Twisted gastrulation (Tsg), which we have shown functions as both a BMP- and Chordin-binding protein. The ternary complex of Chordin/BMP/Tsg is subjected to another layer of regulation by the secreted zinc metalloprotease Tolloid/Xolloid. In Xenopus, the Xolloid-related (Xlr) protease is expressed in the ventral side of the embryo and cleaves Chordin at two conserved sites. Once this proteolytic cleavage takes place, BMPs regain the ability to signal through BMP receptors. The Xolloid metalloprotease provides a critical switch between BMP-signaling inhibition and activation in the presence of Tsg and Chordin that helps establish the dorsal-ventral pattern of the embryo.
Sizzled Is an Inhibitor of Tolloid Proteases
The sizzled gene, which was isolated by Marc Kirschner (Harvard Medical School), is expressed at the ventral pole of the gastrula. We have discovered that Sizzled antagonizes BMP signaling by an indirect molecular mechanism: it is a competitive inhibitor of the Xolloid-related metalloproteinase. Although Sizzled has the structure of a secreted Frizzled-related protein (sFRP, a type of Wnt inhibitor) and was expected to be a Wnt antagonist, its loss-of-function phenotype in X. laevis and in zebrafish embryos is very similar to that of Chordin. The Xlr enzyme has a similar chemical affinity (dissociation constant, KD, of ~20 nM) for its substrate Chordin and for its inhibitor Sizzled, to which it can bind but not cleave.
Remarkably, embryonic dorsal-ventral pattern is controlled by modulating the relative levels of Chordin and Sizzled, which are abundant proteins in the extracellular space. The protease inhibitory activity of Sizzled is mediated by its Frizzled cysteine-rich domain (Fz-CRD). We are investigating whether the Fz-CRD domains present in many other extracellular proteins might also function as zinc metalloproteinase-binding domains. In addition, Sizzled is an inhibitor of BMP1/procollagen-C peptidase, a Tolloid metalloproteinase required for collagen fiber deposition, which is of considerable medical interest.
Establishment of a Self-Regulating Morphogenetic Field
Vertebrate embryos have the remarkable property of self-regulating toward the whole after experimental perturbation. This self-organizing property of "morphogenetic fields" was first described in 1918 by Ross Harrison, yet we do not know how self-regulation works. The Xenopus embryo has not only a dorsal signaling center but also a ventral one at the opposite pole. Both centers express molecules of similar molecular structure. Thus, ventral cells express BMP2/4/7, whereas another BMP called ADMP (antidorsalizing morphogenetic protein) is expressed dorsally. Similarly, Sizzled and Crossveinless-2 on the ventral side serve as counterparts for Crescent and Chordin, respectively. This provided the key for understanding how both poles of the embryo compensate for each other.
When BMP2, BMP4, and BMP7 were knocked down simultaneously, Xenopus embryos displayed increased dorsal structures but still retained a significant amount of dorsal-ventral pattern. Surprisingly, in embryos that lacked Spemann's organizer, the effects were much greater, with the depletion of BMP2, BMP4, and BMP7 causing massive brain differentiations. This suggested that the Spemann's organizer itself is a source of signals that compensate for the loss of BMP. A main component of this signal was found to be the dorsal BMP molecule ADMP.
Indeed, the quadruple knockdown of ADMP, BMP2, BMP4, and BMP7 caused a spectacular transformation: the entire ectoderm became CNS tissue, and epidermal differentiation was eliminated. Thus, when dorsal and ventral BMP signals are depleted, the self-regulating morphogenetic field collapses and ubiquitous CNS induction takes place. Moreover, by transplanting wild-type tissue into these BMP-depleted embryos, we were able to demonstrate that both the ventral and dorsal centers can serve as sources of BMPs that diffuse over considerable distances in the embryo and trigger changes in cell differentiation. We propose that this double gradient of BMP signals that emanate from opposite poles of the embryo assures the robustness of pattern formation in the vertebrate embryo.
Cell-Cell Communication over Long Distances
Spemann organizer (dorsal) genes are transcribed where BMP levels are low, whereas expression of ventral genes requires high BMP levels. Embryonic field self-regulation can be explained by a "seesaw" model of reciprocal transcriptional regulation. As BMP levels are lowered, transcription of ADMP increases dorsally, which leads to compensation, since ADMP has BMP-signaling activity. As BMP-signaling levels increase, the BMP antagonist Sizzled is transcribed in the ventral center, where it functions as an inhibitor of the enzyme that inactivates Chordin. Although ADMP is produced dorsally, it is unable to signal in this location because it binds to Chordin. ADMP only signals in the ventral side once Chordin is cleaved by the Xolloid-related zinc metalloproteinase that is produced by the ventral center. These findings provide a framework for understanding how self-organizing morphogenetic fields are established. Interestingly, self-regulation may also help us understand the behavior of cultured human embryonic stem cells, which are notoriously difficult to differentiate into homogeneous cell types.
In conclusion, efforts to uncover the molecular basis of an embryological experiment carried out 80 years ago have shown that the differentiation of embryonic tissue types is regulated by secreted inhibitory proteins originating from dorsal and ventral organizing centers. These studies in Xenopus embryos have led to new insights on how the CNS is formed and how a network of extracellular proteins controlled by the proteolysis of Chordin mediates pattern self-regulation.
A grant from the National Institutes of Health provided support for some of these projects.
Last updated: July 2, 2007
|
|
|
|
