Members of the transforming growth factor-beta (TGF-beta) superfamily, including TGF-beta, bone morphogenetic proteins (BMPs), activins and nodals, are vital for regulating growth and differentiation. These growth factors transduce their signals through pairs of transmembrane type I and type II receptor kinases. Here, we have cloned a transmembrane protein, BAMBI, which is related to TGF-beta-family type I receptors but lacks an intracellular kinase domain. We show that BAMBI is co-expressed with the ventralizing morphogen BMP4 (refs 5, 6) during Xenopus embryogenesis and that it requires BMP signalling for its expression. The protein stably associates with TGF-beta-family receptors and inhibits BMP and activin as well as TGF-beta signalling. Finally, we provide evidence that BAMBI's inhibitory effects are mediated by its intracellular domain, which resembles the homodimerization interface of a type I receptor and prevents the formation of receptor complexes. The results indicate that BAMBI negatively regulates TGF-beta-family signalling by a regulatory mechanism involving the interaction of signalling receptors with a pseudoreceptor.

The development of multicellular animals is initially controlled by maternal gene products deposited in the oocyte. During the maternal-to-zygotic transition, transcription of zygotic genes commences, and developmental control starts to be regulated by zygotic gene products. In Drosophila, the transcription factor Zelda specifically binds to promoters of the earliest zygotic genes and primes them for activation. It is unknown whether a similar regulation exists in other animals. We found that zebrafish Pou5f1, a homolog of the mammalian pluripotency transcription factor Oct4, occupies SOX-POU binding sites before the onset of zygotic transcription and activates the earliest zygotic genes. Our data position Pou5f1 and SOX-POU sites at the center of the zygotic gene activation network of vertebrates and provide a link between zygotic gene activation and pluripotency control.

The Spemann organizer of the amphibian embryo can be subdivided into two discrete activities, namely trunk organizer and head organizer. Several factors secreted from the organizer that are involved in trunk organization are thought to act by repressing Bmp signalling. With the exception of the secreted factor cerberus, little is known about head-organizer inducers. Here we show that co-expression of a dominant-negative Bmp receptor with inhibitors of the Wnt-signalling pathway in Xenopus leads to the induction of complete secondary axes, including a head. This induction does not require expression of the siamois marker of Nieuwkoop centre signalling, suggesting that cells are directly shifting to head-organizer fate. Furthermore, we find that cerberus is a potent inhibitor of Wnt signalling. Our results indicate that head-organizer activity results from the simultaneous repression of Bmp and Wnt signalling and they suggest a mechanism for region-specific induction by the organizer.

The phototoxic red fluorescent GFP-like protein KillerRed has recently been described. The phototoxicity of KillerRed exceeds that of EGFP by at least 1,000-fold, making it the first fully genetically encoded photosensitizer. KillerRed opens up new possibilities for precise light-induced cell killing and target protein inactivation. Because KillerRed is encoded by a gene, it can be expressed in a spatially and temporally regulated manner, under a chosen promoter, and fused with the desired protein of interest or localization signal. Here we provide a protocol for target protein inactivation in cell culture using KillerRed. As KillerRed is a new tool, the protocol focuses on aspects that will allow users to maximize the potential of this protein, guiding the design of chimeric constructs, recommended control experiments and preferred illumination parameters. The protocol, which describes target protein visualization and subsequent inactivation, is a 2- or 3-d procedure.

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Summary Initiation of motile cell behavior in embryonic development occurs during late blastula stages when gastrulation begins. At this stage, the strong adhesion of blastomeres has to be modulated to enable dynamic behavior, similar to epithelial-to-mesenchymal transitions. We show that in zebrafish MZspg embryos mutant for the stem cell transcription factor Pou5f1/Oct4, which are severely delayed in the epiboly gastrulation movement, all blastomeres are defective in E-cad endosomal trafficking and E-cad accumulates at the plasma membrane. We find that Pou5f1-dependent control of EGF expression regulates endosomal E-cad trafficking. EGFR may act via modulation of p120 activity. Loss of E-cad dynamics reduces cohesion of cells in reaggregation assays. Quantitative analysis of cell behavior indicates that dynamic E-cad endosomal trafficking is required for epiboly cell movements. We hypothesize that dynamic control of E-cad trafficking is essential to effectively generate new adhesion sites when cells move relative to each other.

Time-resolved transcriptome analysis of early pou5f1 mutant zebrafish embryos identified groups of developmental regulators, including SoxB1 genes, that depend on Pou5f1 activity, and a large cluster of differentiation genes which are prematurely expressed.Pou5f1 represses differentiation genes indirectly via activation of germlayer-specific transcriptional repressor genes, including her3, which may mediate in part Pou5f1-dependent repression of neural genes.A dynamic mathematical model is established for Pou5f1 and SoxB1 activity-dependent temporal behaviour of downstream transcriptional regulatory networks. The model predicts that Pou5f1-dependent increase in SoxB1 activity significantly contributes to developmental timing in the early gastrula.Comparison to mouse Pou5f1/Oct4 reveals evolutionary conserved targets. We show that Pou5f1 developmental function is also conserved by demonstrating rescue of Pou5f1 mutant zebrafish embryos by mouse POU5F1/OCT4.

The nucleus contains distinct nuclear bodies (NBs); nucleolus is the largest and the most studied NB, but its role in the functioning of the nucleus is far from being fully understood. The nucleolus is not surrounded by a membrane, yet it contains DNA, RNA and a set of proteins that can either be retained in the nucleolus or rapidly shuttle between the nucleoplasm, the nucleolus and the cytoplasm in response to various stimuli. The emerging evidence points to the central function of the nucleolus in organizing many nuclear functions besides RNA polymerase I transcription and ribosome biogenesis. Here we discuss the functions of the nucleolus related to the shuttling of proteins and nucleic acids between nucleolus and nucleoplasm. The functional processes affected by shuttling of nucleolar components include 3D organization of the genome, stress response, DNA repair and recombination, transcription regulation, telomere maintenance and other essential cellular functions. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Highlights  Nucleolus is a PolI transcription factory and a place of ribosome assembly, but is also performs many other functions that are commonly referred to as non-canonical functions of nucleolus  Nucleolus organizes the adjacent chromatin into a large-scale repressive hub underlying the spatial segregation of active and repressive chromatin compartments.  The interphase chromosomes are attached to the nucleolus via nucleolus-associated domains (NADs).  Protein shuttling between the nucleolus and the nucleoplasm regulates a multitude of nuclear processes including DNA repair, recombination, transcription and telomere maintenance.