Markus Affolter

Structural basis of BMP signalling inhibition by the cystine knot protein Noggin.

The interplay between bone morphogenetic proteins (BMPs) and their antagonists governs developmental and cellular processes as diverse as establishment of the embryonic dorsal–ventral axis, induction of neural tissue, formation of joints in the skeletal system and neurogenesis in the adult brain. So far, the three-dimensional structures of BMP antagonists and the structural basis for inactivation have remained unknown. Here we report the crystal structure of the antagonist Noggin bound to BMP-7, which shows that Noggin inhibits BMP signalling by blocking the molecular interfaces of the binding epitopes for both type I and type II receptors. The BMP-7-binding affinity of site-specific variants of Noggin is correlated with alterations in bone formation and apoptosis in chick limb development, showing that Noggin functions by sequestering its ligand in an inactive complex. The scaffold of Noggin contains a cystine (the oxidized form of cysteine) knot topology similar to that of BMPs; thus, ligand and antagonist seem to have evolved from a common ancestral gene.

Hox proteins meet more partners

The Hox genes are clustered sets of homeobox-containing genes that play a central role in animal development. Recent genetic and molecular data suggest that Hox proteins interact with pre-existing homeodomain protein complexes. These complexes may help to regulate Hox activity and Hox specificity, and help cells to interpret signaling cascades during development.

Protein - DNA contacts in the structure of a homeodomain - DNA complex determined by nuclear magnetic resonance spectroscopy in solution

The 1:1 complex of the mutant Antp(C39––S) homeodomain with a 14 bp DNA fragment corresponding to the BS2 binding site was studied by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. The complex has a molecular weight of 17,800 and its lifetime is long compared with the NMR chemical shift time scale. Investigations of the three‐dimensional structure were based on the use of the fully 15N‐labelled protein, two‐dimensional homonuclear proton NOESY with 15N(omega 2) half‐filter, and heteronuclear three‐dimensional NMR experiments. Based on nearly complete sequence‐specific resonance assignments, both the protein and the DNA were found to have similar conformations in the free form and in the complex. A sufficient number of intermolecular 1H‐1H Overhauser effects (NOE) could be identified to enable a unique docking of the protein on the DNA, which was achieved with the use of an ellipsoid algorithm. In the complex there are intermolecular NOEs between the elongated second helix in the helix‐turn‐helix motif of the homeodomain and the major groove of the DNA. Additional NOE contacts with the DNA involve the polypeptide loop immediately preceding the helix‐turn‐helix segment, and Arg5. This latter contact is of special interest, both because Arg5 reaches into the minor groove and because in the free Antp(C39––S) homeodomain no defined spatial structure could be found for the apparently flexible N‐terminal segment comprising residues 0‐6.

The Decapentaplegic morphogen gradient: from pattern formation to growth regulation

Morphogens have been linked to numerous developmental processes, including organ patterning and the control of organ size. Here we review how different experimental approaches have led to an unprecedented level of molecular knowledge about the patterning role of the Drosophila melanogaster morphogen Decapentaplegic (DPP, the homologue of vertebrate bone morphogenetic protein, or BMP), the first validated secreted morphogen. In addition, we discuss how little is known about the role of the DPP morphogen in the control of organ growth and organ size. Continued efforts to elucidate the role of DPP in D. melanogaster is likely to shed light on this fundamental question in the near future.

An absolute requirement for both the type II and type I receptors, punt and thick veins, for Dpp signaling in vivo

TGF beta elicits diverse cellular responses by signaling through receptor complexes formed by two distantly related transmembrane serine/threonine kinases called type II and type I receptors. Previous studies have indicated that the product of the Drosophila thick veins (tkv) gene is a type I receptor for decapentaplegic (dpp). Here, we show that the Drosophila gene punt encodes a homolog of a vertebrate type II receptor, and we demonstrate that punt, like tkv, is essential in vivo for dpp-dependent patterning processes. Because no dpp-related signalling is apparent in the absence of either the punt or tkv receptor, we infer that both receptors act in concert to transduce the dpp signal and that their functions cannot be replaced by the other extant type II and I receptors.

Receptor serine/threonine kinases implicated in the control of Drosophila body pattern by decapentaplegic

Members of the TGF beta superfamily of secreted signaling molecules regulate growth and cellular patterning during development and interact with specific type I and type II membrane receptors possessing a cytoplasmic serine/threonine kinase domain. We describe two members of the type I receptor family in Drosophila and demonstrate that they are encoded by the genes saxophone (sax) and thick veins (tkv). Further, we show that mutations that abolish sax or tkv activity cause phenotypes similar to partial or complete loss of activity, respectively, of the TGF beta homolog decapentaplegic (dpp). We propose that specification of distinct cell fates in response to different concentrations of dpp may be achieved combinatorially by the sax and tkv receptors.

Tube or not tube : remodeling epithelial tissues by branching morphogenesis

Branching morphogenesis involves the restructuring of epithelial tissues into complex and organized ramified tubular networks. Early rounds of branching are controlled genetically in a hardwired fashion in many organs, whereas later, branching is stochastic, responding to environmental cues. We discuss this sequential process from formation of an organ anlage and invagination of the epithelium to branch initiation and outgrowth in several model systems including Drosophila trachea and mammalian lung, mammary gland, and kidney.

Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo.

The formation of intersegmental blood vessels (ISVs) in the zebrafish embryo serves as a paradigm to study angiogenesis in vivo. ISV formation is thought to occur in discrete steps. First, endothelial cells of the dorsal aorta migrate out and align along the dorsoventral axis. The dorsal-most cell, also called tip cell, then joins with its anterior and posterior neighbours, thus establishing a simple vascular network. The vascular lumen is then established via formation of vacuoles, which eventually fuse with those of adjacent endothelial cells to generate a seamless tube with an intracellular lumen. To investigate the cellular architecture and the development of ISVs in detail, we have analysed the arrangement of endothelial cell junctions and have performed single cell live imaging. In contrast to previous reports, we find that endothelial cells are not arranged in a linear head-to-tail configuration but overlap extensively and form a multicellular tube, which contains an extracellular lumen. Our studies demonstrate that a number of cellular behaviours, such as cell divisions, cell rearrangements and dynamic alterations in cell-cell contacts, have to be considered when studying the morphological and molecular processes involved in ISV and endothelial lumen formation in vivo.

schnurri is required for drosophila Dpp signaling and encodes a zinc finger protein similar to the mammalian transcription factor PRDII-BF1

Cytokines of the TGF beta superfamily regulate many aspects of cellular function by activating receptor complexes consisting of two distantly related serine/threonine kinases. Previous studies have indicated that Drosophila dpp uses similar signaling complexes and strictly requires the punt and thick veins receptors to transduce the signal across the membrane. Here, we show that the schnurri (shn) gene is required for many aspects of dpp signaling. Genetic epistasis experiments indicate that shn functions downstream of the dpp signal and its receptors. The shn gene encodes a large protein similar to a family of mammalian zinc finger transcription factors. The shn protein might therefore act as a nuclear target in the dpp signaling pathway directly regulating the expression of dpp-responsive genes.

Fluorescent fusion protein knockout mediated by anti-GFP nanobody

The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio.