Alin Vonica

FGF-20 and DKK1 are transcriptional targets of β-catenin and FGF-20 is implicated in cancer and development

β‐catenin is the major effector of the canonical Wnt signaling pathway. Mutations in components of the pathway that stabilize β‐catenin result in augmented gene transcription and play a major role in many human cancers. We employed microarrays to identify transcriptional targets of deregulated β‐catenin in a human epithelial cell line (293) engineered to produce mutant β‐catenin and in ovarian endometrioid adenocarcinomas characterized with respect to mutations affecting the Wnt/β‐catenin pathway. Two genes strongly induced in both systems—FGF20 and DKK1—were studied in detail. Elevated levels of FGF20 RNA were also observed in adenomas from mice carrying the ApcMinallele. Both XFGF20 and Xdkk‐1 are expressed early in Xenopus embryogenesis under the control of the Wnt signaling pathway. Furthermore, FGF20 and DKK1 appear to be direct targets for β‐catenin/TCF transcriptional regulation via LEF/TCF‐binding sites. Finally, by using small inhibitory RNAs specific for FGF20, we show that continued expression of FGF20 is necessary for maintenance of the anchorage‐independent growth state in RK3E cells transformed by β‐catenin, implying that FGF‐20 may be a critical element in oncogenesis induced by the Wnt signaling pathway.

The BMP Inhibitor Coco Reactivates Breast Cancer Cells at Lung Metastatic Sites

The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-β ligands, induces dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco induces a discrete gene expression signature, which is strongly associated with metastatic relapse to the lung, but not to the bone or brain in patients. Experiments in mouse models suggest that these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-initiating cells need to overcome organ-specific antimetastatic signals in order to undergo reactivation.

APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex

Hereditary hypotrichosis simplex is a rare autosomal dominant form of hair loss characterized by hair follicle miniaturization. Using genetic linkage analysis, we mapped a new locus for the disease to chromosome 18p11.22, and identified a mutation (Leu9Arg) in the adenomatosis polyposis down-regulated 1 (APCDD1) gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human hair follicles, and can interact in vitro with WNT3A and LRP5—two essential components of Wnt signalling. Functional studies show that APCDD1 inhibits Wnt signalling in a cell-autonomous manner and functions upstream of β-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signalling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus laevis embryos. The mutation Leu9Arg is located in the signal peptide of APCDD1, and perturbs its translational processing from the endoplasmic reticulum to the plasma membrane. APCDD1(L9R) probably functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signalling pathway with an essential role in human hair growth. As APCDD1 is expressed in a broad repertoire of cell types, our findings indicate that APCDD1 may regulate a diversity of biological processes controlled by Wnt signalling.

Cell fate specification and competence by Coco, a maternal BMP, TGFbeta and Wnt inhibitor.

Patterning of the pre-gastrula embryo and subsequent neural induction post-gastrulation are very complex and intricate processes of which little, until recently, has been understood. The earliest decision in neural development, the choice between epidermal or neural fates, is regulated by bone morphogenetic protein (BMP) signaling within the ectoderm. Inhibition of BMP signaling is sufficient for neural induction. Many secreted BMP inhibitors are expressed exclusively within the organizer of the Xenopus gastrula embryo and therefore are predicted to act as bona fide endogenous neural inducers. Other cell-autonomous inhibitors of the BMP pathway are more widely expressed, such as the inhibitory Smads, Smad6 and Smad7. In this report we describe the biological and biochemical characterization of 51-B6, a novel member of Cerberus/Dan family of secreted BMP inhibitors, which we identified in a screen for Smad7-induced genes. This gene is expressed maternally in an animal to vegetal gradient, and its expression levels decline rapidly following gastrulation. In contrast to known BMP inhibitors, 51-B6 is broadly expressed in the ectoderm until the end of gastrulation. The timing, pattern of expression, and activities of this gene makes it unique when compared to other BMP/TGFβ/Wnt secreted inhibitors which are expressed only zygotically and maintained post-gastrulation. We propose that a function of 51-B6 is to block BMP and TGFβ signals in the ectoderm in order to regulate cell fate specification and competence prior to the onset of neural induction. In addition, we demonstrate that 51-B6 can act as a neural inducer and induce ectopic head-like structures in neurula staged embryos. Because of this embryological activity, we have renamed this clone Coco, after the Spanish word meaning head.

Gene profiling during neural induction in Xenopus laevis: regulation of BMP signaling by post-transcriptional mechanisms and TAB3, a novel TAK1-binding protein

The earliest decision in vertebrate neural development is the acquisition of a neural identity by embryonic ectodermal cells. The default model for neural induction postulates that neural fate specification in the vertebrate embryo occurs by inhibition of epidermal inducing signals in the gastrula ectoderm. Bone morphogenetic proteins (BMPs) act as epidermal inducers, and all identified direct neural inducers block BMP signaling either intra- or extracellularly. Although the mechanism of action of the secreted neural inducers has been elucidated, the relevance of intracellular BMP inhibitors in neural induction is not clear. In order to address this issue and to identify downstream targets after BMP inhibition, we have monitored the transcriptional changes in ectodermal explants neuralized by Smad7 using a Xenopus laevis 5000-clone gastrula-stage cDNA microarray. We report the identification and initial characterization of 142 genes whose transcriptional profiles change in the neuralized explants. In order to address the potential involvement during neural induction of genes identified in the array, we performed gain-of-function studies in ectodermal explants. This approach lead to the identification of four genes that can function as neural inducers in Xenopus and three others that can synergize with known neural inducers in promoting neural fates. Based on these studies, we propose a role for post-transcriptional control of gene expression during neural induction in vertebrates and present a model whereby sustained BMP inhibition is promoted partly through the regulation of TGFβ activated kinase (TAK1) activity by a novel TAK1-binding protein (TAB3).

Dynamics of TGF-β signaling reveal adaptive and pulsatile behaviors reflected in the nuclear localization of transcription factor Smad4

The TGF-β pathway plays a vital role in development and disease and regulates transcription through a complex composed of receptor-regulated Smads (R-Smads) and Smad4. Extensive biochemical and genetic studies argue that the pathway is activated through R-Smad phosphorylation; however, the dynamics of signaling remain largely unexplored. We monitored signaling and transcriptional dynamics and found that although R-Smads stably translocate to the nucleus under continuous pathway stimulation, transcription of direct targets is transient. Surprisingly, Smad4 nuclear localization is confined to short pulses that coincide with transcriptional activity. Upon perturbation, the dynamics of transcription correlate with Smad4 nuclear localization rather than with R-Smad activity. In Xenopus embryos, Smad4 shows stereotyped, uncorrelated bursts of nuclear localization, but activated R-Smads are uniform. Thus, R-Smads relay graded information about ligand levels that is integrated with intrinsic temporal control reflected in Smad4 into the active signaling complex.

APOBEC2, a selective inhibitor of TGFβ signaling, regulates left–right axis specification during early embryogenesis

The specification of left-right asymmetry is an evolutionarily conserved developmental process in vertebrates. The interplay between two TGFβ ligands, Derrière/GDF1 and Xnr1/Nodal, together with inhibitors such as Lefty and Coco/Cerl2, have been shown to provide the signals that lead to the establishment of laterality. However, molecular events leading to and following these signals remain mostly unknown. We find that APOBEC2, a member of the cytidine deaminase family of DNA/RNA editing enzymes, is induced by TGFβ signaling, and that its activity is necessary to specify the left-right axis in Xenopus and zebrafish embryos. Surprisingly, we find that APOBEC2 selectively inhibits Derrière, but not Xnr1, signaling. The inhibitory effect is conserved, as APOBEC2 blocks TGFβ signaling, and promotes muscle differentiation, in a mammalian myoblastic cell line. This demonstrates for the first time that a putative RNA/DNA editing enzyme regulates TGFβ signaling and plays a major role in development.

Scaling of BMP gradients in Xenopus embryos.

Arising from: D. Ben-Zvi et al. 453, 1205–1211 (2008)10.1038/nature07059; Francois et al. replyMetazoan organisms can ‘scale’, that is, maintain similar proportions regardless of size. Ben-Zvi et al. use experiments in Xenopus to support a quantitative model that explains morphological scaling as the result of scaling of a gradient of bone morphogenetic protein (BMP) signals. We believe that the evidence for scaling in Xenopus is misinterpreted, and that their model for embryonic patterning disagrees with prior data. The experiments they present supporting their model admit alternative interpretations.

Coco regulates dorsoventral specification of germ layers via inhibition of TGFβ signalling.

One of the earliest steps in embryonic development is the specification of the germ layers, the subdivision of the blastula embryo into endoderm, mesoderm and ectoderm. Maternally expressed members of the Transforming Growth Factor β (TGFβ) family influence all three germ layers; the ligands are required to induce endoderm and mesoderm, whereas inhibitors are required for formation of the ectoderm. Here, we demonstrate a vital role for maternal Coco, a secreted antagonist of TGFβ signalling, in this process. We show that Coco is required to prevent Activin and Nodal signals in the dorsal marginal side of the embryo from invading the prospective ectoderm, thereby restricting endoderm- and mesoderm-inducing signals to the vegetal and marginal zones of the pre-gastrula Xenopus laevis embryo.