Richard M. Harland

The Spemann Organizer Signal noggin Binds and Inactivates Bone Morphogenetic Protein 4

Signals released by the Spemann organizer of the amphibian gastrula can directly induce neural tissue from ectoderm and can dorsalize ventral mesoderm to form muscle. The secreted polypeptide noggin mimics these activities and is expressed at the appropriate time and place to participate in the organizer signal. Neural induction and mesoderm dorsalization are antagonized by bone morphogenetic proteins (BMPs), which induce epidermis and ventral mesoderm instead. Here we report that noggin protein binds BMP4 with high affinity and can abolish BMP4 activity by blocking binding to cognate cell-surface receptors. These data suggest that noggin secreted by the organizer patterns the embryo by interrupting BMP signaling.

Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos

We have cloned a cDNA encoding a novel polypeptide capable of inducing dorsal development in Xenopus embryos. RNA transcripts from this clone rescue normal development when injected into ventralized embryos and result in excessive head development at high doses. Therefore, we have named the cDNA noggin, noggin cDNA contains a single reading frame encoding a 26 kd protein with a hydrophobic amino-terminal sequence, suggesting that it is secreted. In Northern blot analysis this cDNA hybridizes to two mRNAs that are expressed both maternally and zygotically. Although noggin transcript is not localized in the oocyte and cleavage stage embryo, zygotic transcripts are initially restricted to the presumptive dorsal mesoderm and reach their highest levels at the gastrula stage in the dorsal lip of the blastopore (Spemann organizer). In the neurula, noggin is transcribed in the notochord and prechordal mesoderm. The activity of exogenous noggin RNA in embryonic axis induction and the localized expression of endogenous noggin transcripts suggest that noggin plays a role in normal dorsal development.

Dishevelled controls cell polarity during Xenopus gastrulation

Although cell movements are vital for establishing the normal architecture of embryos, it is unclear how these movements are regulated during development in vertebrates. Inhibition of Xenopus Dishevelled (Xdsh) function disrupts convergent extension movements of cells during gastrulation, but the mechanism of this effect is unclear, as cell fates are not affected. In Drosophila, Dishevelled controls both cell fate and cell polarity, but whether Dishevelled is involved in controlling cell polarity in vertebrate embryos has not been investigated. Here we show, using time-lapse confocal microscopy, that the failure of cells lacking Xdsh function to undergo convergent extension results from defects in cell polarity. Furthermore, Xdsh mutations that inhibit convergent extension correspond to mutations in Drosophila Dishevelled that selectively perturb planar cell polarity. Finally, the localization of Xdsh at the membrane of normal dorsal mesodermal cells is consistent with Xdsh controlling cell polarity. Our results show that polarized cell behaviour is essential for convergent extension and is controlled by vertebrate Dishevelled. Thus, a vertebrate equivalent of the Drosophila planar cell polarity signalling cascade may be required for normal gastrulation.

The Xenopus Dorsalizing Factor Gremlin Identifies a Novel Family of Secreted Proteins that Antagonize BMP Activities

Using a Xenopus expression-cloning screen, we have isolated Gremlin, a novel antagonist of bone morphogenetic protein (BMP) signaling that is expressed in the neural crest. Gremlin belongs to a novel gene family that includes the head-inducing factor Cerberus and the tumor suppressor DAN. We show that all family members are secreted proteins and that they act as BMP antagonists in embryonic explants. We also provide support for the model that Gremlin, Cerberus, and DAN block BMP signaling by binding BMPs, preventing them from interacting with their receptors. In addition, Cerberus alone blocks signaling by Activin- and Nodal-like members of the TGF beta superfamily. Therefore, we propose that Gremlin, Cerberus, and DAN control diverse processes in growth and development by selectively antagonizing the activities of different subsets of the TGF beta ligands.

Injected Xwnt-8 RNA acts early in Xenopus embryos to promote formation of a vegetal dorsalizing center

Expression cloning from a pool of gastrula cDNAs identified the Wnt family member Xwnt-8 as having dorsal axis-inducing activity in Xenopus embryos. Microinjected Xwnt-8 mRNA was able to rescue the development of a dorsally complete anterior-posterior axis in embryos ventralized by exposure to UV light. Axis induction was observed in embryos injected in either marginal or vegetal blastomeres at the 32-cell stage. Vegetal blastomeres receiving Xwnt-8 mRNA contributed progeny not to the induced dorsal axis, but to the endoderm, a result consistent with Xwnt-8 causing cells to act as a Nieuwkoop center (the vegetal-inducing component of normal dorsal axis formation), rather than as a Spemann organizer (the induced dorsal marginal zone component that directly forms the dorsal mesoderm). Xwnt-8, which is normally expressed ventrally in midgastrula and neurula embryos, appears to mimic, when injected, maternally encoded dorsal mesoderm-inducing factors that act early in development.

Convergent extension: The molecular control of polarized cell movement during embryonic development

During development, vertebrate embryos undergo dramatic changes in shape. The lengthening and narrowing of a field of cells, termed convergent extension, contributes to a variety of morphogenetic processes. Focusing on frogs and fish, we review the different cellular mechanisms and the well-conserved signaling pathways that underlie this process.

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.

The Genome of the Western Clawed Frog Xenopus tropicalis

Frog Genome The African clawed frog Xenopus tropicalis is the first amphibian to have its genome sequenced. Hellsten et al. (p. 633, see the cover) present an analysis of a draft assembly of the genome. The genome of the frog, which is an important model system for developmental biology, encodes over 20,000 protein-coding genes, of which more than 1700 genes have identified human disease associations. Detailed comparison of the content of protein-coding genes with other tetrapods—human and chicken—reveals extensive shared synteny, occasionally spanning entire chromosomes. Assembly, annotation, and analysis of the frog genome compares gene content and synteny with the human and chicken genomes. The western clawed frog Xenopus tropicalis is an important model for vertebrate development that combines experimental advantages of the African clawed frog Xenopus laevis with more tractable genetics. Here we present a draft genome sequence assembly of X. tropicalis. This genome encodes more than 20,000 protein-coding genes, including orthologs of at least 1700 human disease genes. Over 1 million expressed sequence tags validated the annotation. More than one-third of the genome consists of transposable elements, with unusually prevalent DNA transposons. Like that of other tetrapods, the genome of X. tropicalis contains gene deserts enriched for conserved noncoding elements. The genome exhibits substantial shared synteny with human and chicken over major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, mainly in the mammalian lineage.

A nodal-related gene defines a physical and functional domain within the Spemann organizer

A functional screen for gene products that rescue dorsal development in ventralized Xenopus embryos has yielded Xenopus nodal-related 3 (Xnr3), a diverged member of the TGF beta superfamily. Xnr3 is specifically expressed in the Spemann organizer and is only expressed in the epithelial layer of the organizer immediately preceding and extending through gastrulation. Like noggin, Xnr3 can induce muscle in ventral mesoderm explants, consistent with a role in patterning the gastrula. In other ways, the activity of Xnr3 is different from noggin. Embryos receiving injections of Xnr3, particularly in the animal pole, send out tube-like extensions of tissue from the site of injection. These protrusions usually contain no axial mesoderm and only occasionally are positive for neural markers. It has previously been proposed that the epithelial layer of the organizer initiates and coordinates the morphogenetic movements at gastrulation. The protrusions observed may reflect an activity of Xnr3 in promoting morphogenesis.

Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning

During limb outgrowth, signaling by bone morphogenetic proteins (BMPs) must be moderated to maintain the signaling loop between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Gremlin, an extracellular Bmp antagonist, has been proposed to fulfill this function and therefore be important in limb patterning. We tested this model directly by mutating the mouse gene encoding gremlin (Cktsf1b1, herein called gremlin). In the mutant limb, the feedback loop between the ZPA and the AER is interrupted, resulting in abnormal skeletal pattern. We also show that the gremlin mutation is allelic to the limb deformity mutation (ld). Although Bmps and their antagonists have multiple roles in limb development, these experiments show that gremlin is the principal BMP antagonist required for early limb outgrowth and patterning.