Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.

An antibody against secretogranin I (chromogranin B) is packaged into secretory granules.

We have investigated the sorting and packaging of secretory proteins into secretory granules by an immunological approach. An mAb against secretogranin I (chromogranin B), a secretory protein costored with various peptide hormones and neuropeptides in secretory granules of many endocrine cells and neurons, was expressed by microinjection of its mRNA into the secretogranin I-producing cell line PC12. An mAb against the G protein of vesicular stomatitis virus--i.e., against an antigen not present in PC12 cells--was expressed as a control. The intracellular localization and the secretion of the antibodies was studied by double-labeling immunofluorescence using the conventional and the confocal microscope, as well as by pulse-chase experiments. The secretogranin I antibody, like the control antibody, was transported along the secretory pathway to the Golgi complex. However, in contrast to the control antibody, which was secreted via the constitutive pathway, the secretogranin I antibody formed an immunocomplex with secretogranin I, was packaged into secretory granules, and was released by regulated exocytosis. Our results show that a constitutive secretory protein, unaltered by genetic engineering, can be diverted to the regulated pathway of secretion by its protein-protein interaction with a regulated secretory protein. The data also provide the basis for immunologically studying the role of luminally exposed protein domains in the biogenesis and function of regulated secretory vesicles.

Mesodermal patterning by a gradient of the vertebrate homeobox gene goosecoid.

Amphibian mesoderm arises from the marginal zone of the early gastrula and generates various tissues such as notochord, muscle, kidney, and blood. Small changes (twofold) in the amount of microinjected messenger RNA encoding the goosecoid (gsc) homeodomain protein resulted in marked changes in the differentiation of mesoderm in Xenopus laevis. At least three thresholds were observed, which were sufficient to specify four mesodermal cell states. Endogenous gsc messenger RNA was expressed in the marginal zone in a graded fashion that is congruent with a role for this gene in dorso-ventral patterning of mesoderm at the early gastrula stage.

Vertebrate axis formation.

Molecular understanding of axis formation has recently taken a great leap forward with the identification and functional characterization of regulatory genes that appear to act at the top of the hierarchy leading to positional specification in the vertebrate. Analysis of these genes, which encode peptide growth factors and their receptors as well as transcription factors, is disclosing principles of early cell fate specification that are common to all vertebrates.

Function and biological roles of the Dickkopf family of Wnt modulators.

Dickkopf (Dkk) genes comprise an evolutionary conserved small gene family of four members (Dkk1-4) and a unique Dkk3-related gene, Dkkl1 (soggy). They encode secreted proteins that typically antagonize Wnt/beta-catenin signaling, by inhibiting the Wnt coreceptors Lrp5 and 6. Additionally, Dkks are high affinity ligands for the transmembrane proteins Kremen1 and 2, which also modulate Wnt signaling. Dkks play an important role in vertebrate development, where they locally inhibit Wnt regulated processes such as antero-posterior axial patterning, limb development, somitogenesis and eye formation. In the adult, Dkks are implicated in bone formation and bone disease, cancer and Alzheimer's disease.

Mutual antagonism between dickkopf1 and dickkopf2 regulates Wnt/beta-catenin signalling.

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family [1] to stabilise beta-catenin [2]. Wnts are antagonised by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown [3]. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established [3-6], the effect of other Dkks on the Wnt/beta-catenin pathway is unclear. Here, we report that a related family member, Dkk2, activates rather than inhibits the Wnt/beta-catenin signalling pathway in Xenopus embryos. Dkk2 strongly synergised with Wnt receptors of the Fz family to induce Wnt signalling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/beta-catenin signalling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signalling.

Head in the WNT: the molecular nature of Spemann's head organizer.

Formation of the head during vertebrate embryogenesis has been one of the most-studied topics in development, probably because we are such cephalized beings ourselves. Early experimenters found that the head is induced during gastrulation by Spemann's organizer. In 1999 we celebrate the 75th anniversary of the discovery of the organizer by Spemann and Mangold, a group of cells in amphibia that secretes powerful signalling molecules. Recently, advances have been made in identifying candidate head inducers. Not surprisingly, these inducers act in familiar molecular pathways, namely transforming growth factor beta (TGF-beta) and WNT signalling.

RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase.

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

The role of prechordal mesendoderm in neural patterning.

Prechordal mesendoderm is formed in response to Nodal and maternal beta-Catenin signaling and is regulated by signals from anterior endoderm and chordamesoderm. Prechordal mesendodermal cells are involved in neural induction and in anteroposterior and dorsoventral neural patterning. Inhibitors of Wnt and BMP growth factors secreted by prechordal mesendoderm mediate neural induction and anteroposterior and dorsoventral patterning, whereas SHH and TGF betas mediate dorsoventral patterning.

The comparative influence of substituted phenols (especially chlorophenols) on yeast cells assayed by electro-rotation and other methods.

The toxicity of 31 phenols was studied by electro-rotation of yeast cells. Control yeast cells show both anti-field and co-field rotation, depending upon the field frequency applied. After treatment with supra-threshold amounts of phenols the anti-field rotation is weakened or abolished and a stronger co-field rotation can be seen. The proportion of cells showing the co-field rotation was found to be a sensitive measure of toxicity. Doses of 2.2 mumol/l of pentachlorophenol, or of 0.3 mumol/l of pentabromophenol were detectable after 3 h incubation at pH 4.0. At a given pH, the toxicity of the chlorophenols correlated extremely well with their octanol:water partition coefficients (Pow). The complete set of phenols showed fair overall correlation with Pow, but less good correlation with their acidity constants (pKa). In particular the toxicity of a given phenol was less than predicted from its pKa if the incubation pH was higher than the pKa. Biochemical assays on 23 of the phenols showed that the rotational sensitivity runs closely parallel to the sensitivities of cell growth rate and of the plasmamembrane ATPase, but less closely to the inhibition of purine incorporation. It appears that the electro-rotation method provides a useful and rapid test for the presence of organic ecotoxins. The test enables us to distinguish differences between single cells, and is comparable in sensitivity to biochemical tests that use vesicles or homogenates derived from a cell population.

Requirement for anti-dorsalizing morphogenetic protein in organizer patterning.

The amphibian Spemann organizer is subdivided in trunk and head organizer and it is unclear how this division is regulated. The Xenopus trunk organizer expresses anti-dorsalizing morphogenetic protein (ADMP), a potent organizer antagonist. We show that ADMP represses head formation during gastrulation and that its expression is activated by BMP antagonists. A specifically acting dominant-negative ADMP anteriorizes embryos and its coexpression with BMP antagonists induces secondary embryonic axes with heads as well as expression of head inducers. Unlike other BMPs, ADMP is not inhibited by a dominant-negative BMP type I receptor, Noggin, Cerberus and Chordin but by Follistatin, suggesting that it utilizes a distinct TGF-beta receptor pathway and displays differential sensitivity to BMP antagonists. The results indicate that ADMP functions in the trunk organizer to antagonize head formation, thereby regulating organizer patterning.

Increased XRALDH2 activity has a posteriorizing effect on the central nervous system of Xenopus embryos.

Retinoic acid (RA) metabolizing enzymes play important roles in RA signaling during vertebrate embryogenesis. We have previously reported on a RA degrading enzyme, XCYP26, which appears to be critical for the anteroposterior patterning of the central nervous system (EMBO J. 17 (1998) 7361). Here, we report on the sequence, expression and function of its counterpart, XRALDH2, a RA generating enzyme in Xenopus. During gastrulation and neurulation, XRALDH2 and XCYP26 show non-overlapping, complementary expression domains. Upon misexpression, XRALDH2 is found to reduce the forebrain territory and to posteriorize the molecular identity of midbrain and individual hindbrain rhombomeres in Xenopus embryos. Furthermore, ectopic XRALDH2, in combination with its substrate, all-trans-retinal (ATR), can mimic the RA phenotype to result in microcephalic embryos. Taken together, our data support the notion that XRALDH2 plays an important role in RA homeostasis by the creation of a critical RA concentration gradient along the anteroposterior axis of early embryos, which is essential for proper patterning of the central nervous system in Xenopus.

Combinatorial signalling by Xwnt-11 and Xnr3 in the organizer epithelium.

The epithelium of the Spemann organizer plays an important role in embryonic axis formation and transplantation experiments have shown that epithelial organizer cells have potent axis-inducing potential. Known axis-inducing molecules like noggin and chordin are not expressed in the epithelium and cannot account for its inductive properties. Xwnt-11 is expressed in the epithelium but has only poor dorsalizing activity. In an expression screen for genes that are able to functionally cooperate with Xwnt-11 we have identified a cDNA encoding Xenopus nodal-related 3 (XNR3), a member of the TGF-beta family, coexpressed with Xwnt-11 in the organizer epithelium. Xwnt-11 and Xnr3 act highly cooperatively in inducing secondary embryonic axes and dorsalizing ventral mesoderm. Xwnt-11/Xnr3 interfere with BMP signalling without themselves inducing chordin or noggin. The results indicate that induction by the organizer epithelium may result from the combinatorial action of instructive Xnr3 and permissive Xwnt-11 signalling.

Dissecting GHRH- and pituitary adenylate cyclase activating polypeptide-mediated signalling in Xenopus.

The highly conserved neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in a broad variety of physiological processes. The PACAP precursor protein gives rise to three different peptides, the cryptic peptide, GHRH, and PACAP, respectively, and here we dissect their functional properties using Xenopus as model system. PACAP and GHRH but not the cryptic peptide directly neuralize animal caps. In contrast to GHRH, the neuralizing effect mediated by PACAP is independent of the PKA pathway. Moreover, PACAP but not GHRH behaves like a BMP-4 antagonist. Blastocoel injection of PACAP-38 but not of the closely related peptides PACAP-27 and VIP leads to strong anteriorization of the injected embryos suggesting the possible involvement of a novel PACAP-preferring receptor.

Essential role of CREB family proteins during Xenopus embryogenesis.

The leucine zipper transcription factors cAMP response element binding protein (CREB), cAMP response element modulatory protein (CREM) and activating transcription factor 1 (ATF1) bind to the cAMP response element (CRE) with the palindromic consensus sequence TGACGTCA. Their transcriptional activities are dependent on serine phosphorylation induced by various extracellular signals such as hormones, growth factors and neurotransmitters. Here we show that CREB is the predominant CRE-binding protein in Xenopus embryos and that it plays an essential role during early development. The importance of CREB for morphogenetic processes was assessed by injection of RNA encoding a dominant-negative form of CREB that is fused to a truncated progesterone receptor ligand binding domain. In this fusion protein, a dominant-negative function can be induced by application of the synthetic steroid RU486 at given developmental stages. The inhibition of CREB at blastula and early gastrula stages leads to severe posterior defects of the embryos reflected by strong spina bifida, whereas the inhibition of CREB at the beginning of neurulation resulted in stunted embryos with microcephaly. In these embryos, initial induction of neural and mesodermal tissues is not dependent on CREB function, as genes such as Otx2, Krox20, Shh and MyoD are still expressed in injected embryos. But the expression domains of Otx2 and MyoD were found to be distorted reflecting the abnormal development in both neural and somitic derivatives. In summary, our data show that CREB is essential during several developmental stages of Xenopus embryogenesis.

Phenotypic effects in Xenopus and zebrafish suggest that one-eyed pinhead functions as antagonist of BMP signalling.

Zebrafish one-eyed pinhead (oep) is essential for embryonic axis and dorsal midline formation by promoting Nodal signalling and is thought to act as a permissive factor. Here we describe that oep elicits profound phenotypic effects when overexpressed in Xenopus and zebrafish. In Xenopus, wild-type oep inhibits mesoderm induction, disrupts axis formation and neuralizes animal caps. A secreted Oep dorsoanteriorizes and neuralizes Xenopus embryos indicative of BMP inhibition. In zebrafish, misexpression of smad1 in oep mutant embryos also reveals an interaction of oep with BMP signalling. Furthermore, the phenotypic effect of nodal overexpression can be rescued by coexpression of oep both in Xenopus and zebrafish. Taken together, our results support an interaction between oep and nodal but they suggest also (1) that the role of oep in Nodal signalling may include negative as well as positive regulation, (2) that oep is able to function in an active fashion and (3) that oep exerts a regulatory effect on the BMP signalling pathway.

Gene expression screening in Xenopus identifies molecular pathways, predicts gene function and provides a global view of embryonic patterning.

In a large-scale gene expression screen 1765 randomly picked cDNAs were analyzed by whole-mount in situ hybridization in Xenopus embryos. Two hundred and seventy three unique, differentially expressed genes were identified, 204 of which are novel in Xenopus. Partial DNA sequences and expression patterns were documented and assembled into a database, 'AXelDB'. Approximately 30% of cDNAs analyzed represent differentially expressed genes and about 5% show highly regionalized expression. Novel marker genes and potential developmental regulators were found. Differential expression of mitochondrial genes was observed. Marker genes were used to study regionalization of the entire gastrula as well as the tail forming region and the epidermis of the tailbud embryo. Four 'synexpression' groups representing genes with shared, complex expression pattern that predict molecular pathways involved in patterning and differentiation were identified. According to their probable functional significance these groups are designated as Delta1, Bmp4, ER-import and Chromatin group. Within synexpression groups, a likely function of genes without sequence similarity can be predicted. The results indicate that synexpression groups have strong prognostic value. A cluster analysis was made by comparing gene expression patterns to derive a novel parameter, 'tissue relatedness'. In conclusion, this study describes a semi-functional approach to investigate genes expressed during early development and provides global insight into embryonic patterning.

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

The C-terminal domain of Mad-like signal transducers is sufficient for biological activity in the Xenopus embryo and transcriptional activation.

We report the characterization of two vertebrate homologs of Drosophila mothers against dpp (Mad) isolated from the mouse and the Xenopus embryo, named MusMLP (mad-like protein) and XenMLP, respectively, together with a summary of their expression patterns in the embryo. Overexpression of XenMLP causes ventralization of Xenopus embryos and we demonstrate that the C-terminal domain is necessary and sufficient to confer this biological effect. This domain also has the potential for transcriptional activation, as shown in one-hybrid assays in mammalian cells. We further demonstrate that MLPs are multidomain proteins by showing a cis-negative effect of the N-terminal domain on the transactivation by the C-terminal domain and that the proline-rich, middle domain maximizes the activity of the C-terminal domain. We also mapped the MusMLP gene to a region on mouse chromosome 13 that corresponds to a region on human chromosome 5q that contains cancer-related genes.

Dickkopf1 and the Spemann-Mangold head organizer.

Work in amphibians indicates that inhibition of Wnt and BMP signals is essential for head development and that head induction by the Spemann-Mangold organizer may be mediated by secreted Wnt antagonists. Wnts are potent posteriorizing factors and antagonize the Spemann-Mangold organizer. Dickkopf1 (dkk1) encodes a secreted effector expressed in head organizing centers of Xenopus, mouse and zebrafish. It acts as a Wnt inhibitor and is able together with BMP inhibitors to induce the formation of ectopic embryonic heads in Xenopus. It anteriorizes both mesendoderm and neuroectoderm, promoting prechordal plate and forebrain fates. Injection of inhibitory antibodies leads to microcephaly and cyclopia. Dkk1 thus is an essential mediator of the vertebrate head organizer.