E. M. De Robertis

The head inducer cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals

Embryological and genetic evidence indicates that the vertebrate head is induced by a different set of signals from those that organize trunk–tail development. The gene cerberus encodes a secreted protein that is expressed in anterior endoderm and has the unique property of inducing ectopic heads in the absence of trunk structures. Here we show that the cerberus protein functions as a multivalent growth-factor antagonist in the extracellular space: it binds to Nodal, BMP and Wnt proteins via independent sites. The expression of cerberus during gastrulation is activated by earlier nodal-related signals in endoderm and by Spemann-organizer factors that repress signalling by BMP and Wnt. In order for the head territory to form, we propose that signals involved in trunk development, such as those involving BMP, Wnt and Nodal proteins, must be inhibited in rostral regions.

Connective-tissue growth factor (CTGF) modulates cell signalling by BMP and TGF-β

Connective-tissue growth factor (CTGF) is a secreted protein implicated in multiple cellular events including angiogenesis, skeletogenesis and wound healing. It is a member of the CCN family of secreted proteins, named after CTGF, cysteine-rich 61 (CYR61), and nephroblastoma overexpressed (NOV) proteins. The molecular mechanism by which CTGF or other CCN proteins regulate cell signalling is not known. CTGF contains a cysteine-rich domain (CR) similar to those found in chordin and other secreted proteins, which in some cases have been reported to function as bone morphogenetic protein (BMP) and TGF-β binding domains. Here we show that CTGF directly binds BMP4 and TGF-β1 through its CR domain. CTGF can antagonize BMP4 activity by preventing its binding to BMP receptors and has the opposite effect, enhancement of receptor binding, on TGF-β1. These results show that CTGF inhibits BMP and activates TGF-β signals by direct binding in the extracellular space.

The organizer factors Chordin and Noggin are required for mouse forebrain development.

In mice, there is evidence suggesting that the development of head and trunk structures is organized by distinctly separated cell populations. The head organizer is located in the anterior visceral endoderm (AVE) and the trunk organizer in the node and anterior primitive streak. In amphibians, Spemanns organizer, which is homologous to the node, partially overlaps with anterior endoderm cells expressing homologues of the AVE markers cerberus, Hex and Hesx1 (refs 3,4,5,6). For mice, this raises the question of whether the AVE and node are independent of each other, as suggested by their anatomical separation, or functionally interdependent as is the case in amphibians. Chordin and Noggin are secreted bone morphogenetic protein (BMP) antagonists expressed in the mouse node, but not in the AVE. Here we show that mice double-homozygous mutants that are for chordin and noggin display severe defects in the development of the prosencephalon. The results show that BMP antagonists in the node and its derivatives are required for head development.

On the function of BMP-4 in patterning the marginal zone of the Xenopus embryo.

Bone morphogenetic protein 4 (BMP‐4) is expressed in the ventral marginal zone of the gastrulating embryo. At late gastrula stage this gene is expressed in the ventral‐most part of the slit blastopore and in tissues that derive from it. At tailbud stages BMP‐4 is expressed in the spinal cord roof plate, neural crest, eye and auditory vesicle. The interactions of BMP‐4 with dorsal genes such as goosecoid (gsc) and Xnot‐2 were studied in vivo. In embryos ventralized by UV irradiation and suramin treatment, BMP‐4 zygotic transcripts accumulate prematurely and the entire marginal zone expresses this gene. The patterning effect of BMP‐4 on ventro‐posterior development can be revealed by a sensitive assay involving the injection of BMP‐4 mRNA in the ventral marginal zone of embryos partially dorsalized with LiCl, which leads to the complete rescue of trunk and tail structures. The experiments presented here argue that BMP‐4 may act in vivo as a ventral signal for the proper patterning of the marginal zone, actively interacting with dorsal genes such as gsc and Xnot‐2. A model is proposed in which the timing of expression of various marginal zone‐specific genes plays a central role in patterning the mesoderm.

Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps.

Spemanns organizer has potent neural inducing and mesoderm dorsalizing activities in the Xenopus gastrula. A third activity, the organizers ability to induce a secondary gut, has been difficult to analyze experimentally due to the lack of early gene markers. Here we introduce endodermin, a pan‐endodermal gene marker, and use it to demonstrate that chordin (Chd), a protein secreted by the organizer region, is able to induce endodermal differentiation in Xenopus. The ability of chd, as well as that of noggin, to induce endoderm in animal cap explants is repressed by the ventralizing factor BMP‐4. When FGF signaling is blocked by a dominant‐negative FGF receptor in chd‐injected animal caps, neural induction is inhibited and most of the explant is induced to become endoderm. The results suggest that proteins secreted by the organizer, acting together with known peptide growth factors, regulate differentiation of the endodermal germ layer.

Evo-Devo: Variations on Ancestral Themes

Most animals evolved from a common ancestor, Urbilateria, which already had in place the developmental genetic networks for shaping body plans. Comparative genomics has revealed rather unexpectedly that many of the genes present in bilaterian animal ancestors were lost by individual phyla during evolution. Reconstruction of the archetypal developmental genomic tool-kit present in Urbilateria will help to elucidate the contribution of gene loss and developmental constraints to the evolution of animal body plans.Most animals evolved from a common ancestor, Urbilateria, which already had in place the developmental genetic networks for shaping body plans. Comparative genomics has revealed rather unexpectedly that many of the genes present in bilaterian animal ancestors were lost by individual phyla during evolution. Reconstruction of the archetypal developmental genomic tool-kit present in Urbilateria will help to elucidate the contribution of gene loss and developmental constraints to the evolution of animal body plans.

A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction

A homeobox sequence has been used to isolate a new Xenopus cDNA, named XIHbox6. A short probe from this gene serves as an early marker of posterior neural differentiation in the Xenopus nervous system. The gene recognized by this cDNA sequence is first transcribed at the late gastrula stage and solely in the posterior neural cells. The gene is expressed when ectodermal and mesodermal tissues of an early gastrula are placed in contact, but not by either tissue cultured on its own. However, gene expression is most easily inducible in ectoderm from the dorsal region, i.e., in ectoderm normally destined to form neural structures. This establishes the principle, in contrast to previous belief, that the induction of the embryonic nervous system involves a predisposition of the ectoderm and does not depend entirely on an interaction with inducing mesoderm.

Regulation of ADMP and BMP2/4/7 at opposite embryonic poles generates a self-regulating morphogenetic field.

Embryos have the ability to self-regulate and regenerate normal structures after being sectioned in half. How is such a morphogenetic field established? We discovered that quadruple knockdown of ADMP and BMP2/4/7 in Xenopus embryos eliminates self-regulation, causing ubiquitous neural induction throughout the ectoderm. ADMP transcription in the Spemann organizer is activated at low BMP levels. When ventral BMP2/4/7 signals are depleted, Admp expression increases, allowing for self-regulation. ADMP has BMP-like activity and signals via the ALK-2 receptor. It is unable to signal dorsally because of inhibition by Chordin. The ventral BMP antagonists Sizzled and Bambi further refine the pattern. By transplanting dorsal or ventral wild-type grafts into ADMP/BMP2/4/7-depleted hosts, we demonstrate that both poles serve as signaling centers that can induce histotypic differentiation over considerable distances. We conclude that dorsal and ventral BMP signals and their extracellular antagonists expressed under opposing transcriptional regulation provide a molecular mechanism for embryonic self-regulation.

Embryonic Dorsal-Ventral Signaling: Secreted Frizzled-Related Proteins as Inhibitors of Tolloid Proteinases

Here we report an unexpected role for the secreted Frizzled-related protein (sFRP) Sizzled/Ogon as an inhibitor of the extracellular proteolytic reaction that controls BMP signaling during Xenopus gastrulation. Microinjection experiments suggest that the Frizzled domain of Sizzled regulates the activity of Xolloid-related (Xlr), a metalloproteinase that degrades Chordin, through the following molecular pathway: Szl -| Xlr -| Chd -| BMP --> P-Smad1 --> Szl. In biochemical assays, the Xlr proteinase has similar affinities for its endogenous substrate Chordin and for its competitive inhibitor Sizzled, which is resistant to enzyme digestion. Extracellular levels of Sizzled and Chordin in the gastrula embryo and enzyme reaction constants were all in the 10(-8) M range, consistent with a physiological role in the regulation of dorsal-ventral patterning. Sizzled is also a natural inhibitor of BMP1, a Tolloid metalloproteinase of medical interest. Furthermore, mouse sFRP2 inhibited Xlr, suggesting a wider role for this molecular mechanism.

Neural and Head Induction by Insulin-like Growth Factor Signals

Evidence is presented for a new pathway participating in anterior neural development. It was found that IGF binding protein 5 (IGFBP-5), as well as three IGFs expressed in early embryos, promoted anterior development by increasing the head region at the expense of the trunk in mRNA-injected Xenopus embryos. A secreted dominant-negative type I IGF receptor (DN-IGFR) had the opposite effect. IGF mRNAs led to the induction of ectopic eyes and ectopic head-like structures containing brain tissue. In ectodermal explants, IGF signals induced anterior neural markers in the absence of mesoderm formation and DN-IGFR inhibited neural induction by the BMP antagonist Chordin. Thus, active IGF signals appear to be both required and sufficient for anterior neural induction in Xenopus.