Negative modulation of bone morphogenetic protein signaling by Dullard during wing vein formation in Drosophila

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2/Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-β and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.     

Human Crossveinless-2 is a novel inhibitor of bone morphogenetic proteins

Drosophila Crossveinless-2 (dCV-2) is required for local activation of Mad phosphorylation in the fruit fly wing and has been postulated to be a positive regulator of BMP-mediated signaling. In contrast, the presence of 5 Chordin-like cysteine-rich domains in the CV-2 protein suggests that CV-2 belongs to a family of well-established inhibitors of BMP function that includes Chordin and Sog [Development 127 (2000) 3947]. We have identified a human homolog of Drosophila CV-2 (hCV-2). Here we show that purified recombinant hCV-2 protein inhibits BMP-2 and BMP-4 dependent osteogenic differentiation of W-20-17 cells, as well as BMP dependent chondrogenic differentiation of ATDC5 cells. Interestingly, hCV-2 messenger RNA is expressed at high levels in human primary chondrocytes, whereas expression in primary human osteoblasts is low. These results suggest that hCV-2 may regulate BMP responsiveness of osteoblasts and chondrocytes in vivo. Taken together we have shown that contrary to the function predicted from the fruit fly, Crossveinless-2 is a novel inhibitor of BMP function.     

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development.     

WRM-1 activates the LIT-1 protein kinase to transduce anterior/posterior polarity signals in C. elegans.

During C. elegans development, Wnt/WG signaling is required for differences in cell fate between sister cells born from anterior/posterior divisions. A beta-catenin-related gene, wrm-1, and the lit-1 gene are effectors of this signaling pathway and appear to downregulate the activity of POP-1, a TCF/LEF-related protein, in posterior daughter cells. We show here that lit-1 encodes a serine/threonine protein kinase homolog related to the Drosophila tissue polarity protein Nemo. We demonstrate that the WRM-1 protein binds to LIT-1 in vivo and that WRM-1 can activate the LIT-1 protein kinase when coexpressed in vertebrate tissue culture cells. This activation leads to phosphorylation of POP-1 and to apparent changes in its subcellular localization. Our findings provide evidence for novel regulatory avenues for an evolutionarily conserved Wnt/WG signaling pathway.     

R-Smad competition controls activin receptor output in Drosophila

Animals use TGF-β superfamily signal transduction pathways during development and tissue maintenance. The superfamily has traditionally been divided into TGF-β/Activin and BMP branches based on relationships between ligands, receptors, and R-Smads. Several previous reports have shown that, in cell culture systems, "BMP-specific" Smads can be phosphorylated in response to TGF-β/Activin pathway activation. Using Drosophila cell culture as well as in vivo assays, we find that Baboon, the Drosophila TGF-β/Activin-specific Type I receptor, can phosphorylate Mad, the BMP-specific R-Smad, in addition to its normal substrate, dSmad2. The Baboon-Mad activation appears direct because it occurs in the absence of canonical BMP Type I receptors. Wing phenotypes generated by Baboon gain-of-function require Mad, and are partially suppressed by over-expression of dSmad2. In the larval wing disc, activated Baboon cell-autonomously causes C-terminal Mad phosphorylation, but only when endogenous dSmad2 protein is depleted. The Baboon-Mad relationship is thus controlled by dSmad2 levels. Elevated P-Mad is seen in several tissues of dSmad2 protein-null mutant larvae, and these levels are normalized in dSmad2; baboon double mutants, indicating that the cross-talk reaction and Smad competition occur with endogenous levels of signaling components in vivo. In addition, we find that high levels of Activin signaling cause substantial turnover in dSmad2 protein, providing a potential cross-pathway signal-switching mechanism. We propose that the dual activity of TGF-β/Activin receptors is an ancient feature, and we discuss several ways this activity can modulate TGF-β signaling output.     

Ter94/VCP Is a Novel Component Involved in BMP Signaling

Bone morphogenetic proteins (BMPs), a subgroup of the transforming growth factor (TGF)-β family, transduce their signal through multiple components downstream of their receptors. Even though the components involved in the BMP signaling pathway have been intensely studied, many molecules mediating BMP signaling remain to be addressed. To identify novel components that participate in BMP signaling, RNA interference (RNAi)-based screening was established by detecting phosphorylated Mad (pMad) in Drosophila S2 cells. Ter94, a member of the family of AAA ATPases, was identified as a novel mediator of BMP signaling, which is required for the phosphorylation of Mad in Drosophila S2 cells. Moreover, the mammalian orthlog of Ter94 valosin-containing protein (VCP) plays a critical role in the BMP-Smad1/5/8 signaling pathway in mammalian cells. Genetic evidence suggests that Ter94 is involved in the dorsal-ventral patterning of the Drosophila early embryo through regulating decapentaplegic (Dpp)/BMP signals. Taken together, our data suggest that Ter94/VCP appears to be an evolutionarily conserved component that regulates BMP-Smad1/5/8 signaling.     

Characterization of a Smad motif similar to Drosophila mad in the mouse Msx 1 promoter

Mouse Msx 1 gene, orthologous of the Drosophila msh, is involved in several developmental processes. BMP family members are major proteins in the regulation of Msx 1 expression. BMP signaling activates Smad 1/5/8 proteins, which associate to Smad 4 before translocating to the nucleus. Analysis of Msx 1 promoter revealed the presence of three elements similar to the consensus established for Mad, the Smad 1 Drosophila counterpart. Notably, such an element was identified in an enhancer important for Msx 1 regulation. Gel shift analysis demonstrated that proteins from 13.5 dpc embryo associate to this enhancer. Remarkably, supershift assays showed that Smad proteins are present in the complex. Purified Smad 1 and 4 also bind to this fragment. We demonstrate that functional binding sites in this enhancer are confined to the Mad motif and flanking region. Our data suggest that this Mad motif may be functional in response to BMP signaling.     

Disruption of Axonal Transport Perturbs Bone Morphogenetic Protein (BMP) - Signaling and Contributes to Synaptic Abnormalities in Two Neurodegenerative Diseases

Formation of new synapses or maintenance of existing synapses requires the delivery of synaptic components from the soma to the nerve termini via axonal transport. One pathway that is important in synapse formation, maintenance and function of the Drosophila neuromuscular junction (NMJ) is the bone morphogenetic protein (BMP)-signaling pathway. Here we show that perturbations in axonal transport directly disrupt BMP signaling, as measured by its downstream signal, phospho Mad (p-Mad). We found that components of the BMP pathway genetically interact with both kinesin-1 and dynein motor proteins. Thick vein (TKV) vesicle motility was also perturbed by reductions in kinesin-1 or dynein motors. Interestingly, dynein mutations severely disrupted p-Mad signaling while kinesin-1 mutants showed a mild reduction in p-Mad signal intensity. Similar to mutants in components of the BMP pathway, both kinesin-1 and dynein motor protein mutants also showed synaptic morphological defects. Strikingly TKV motility and p-Mad signaling were disrupted in larvae expressing two human disease proteins; expansions of glutamine repeats (polyQ77) and human amyloid precursor protein (APP) with a familial Alzheimer''s disease (AD) mutation (APPswe). Consistent with axonal transport defects, larvae expressing these disease proteins showed accumulations of synaptic proteins along axons and synaptic abnormalities. Taken together our results suggest that similar to the NGF-TrkA signaling endosome, a BMP signaling endosome that directly interacts with molecular motors likely exist. Thus problems in axonal transport occurs early, perturbs BMP signaling, and likely contributes to the synaptic abnormalities observed in these two diseases.     

Nemo is an inducible antagonist of Wingless signaling during Drosophila wing development

The cellular events that govern patterning during animal development must be precisely regulated. This is achieved by extrinsic factors and through the action of both positive and negative feedback loops. Wnt/Wg signals are crucial across species in many developmental patterning events. We report that Drosophila nemo (nmo) acts as an intracellular feedback inhibitor of Wingless (Wg) and that it is a novel Wg target gene. Nemo antagonizes the activity of the Wg signal, as evidenced by the finding that reduction of nmo rescues the phenotypic defects induced by misexpression of various Wg pathway components. In addition, the activation of Wg-dependent gene expression is suppressed in wing discs ectopically expressing nmo and enhanced cell autonomously in nmo mutant clones. We find that nmo itself is a target of Wg signaling in the imaginal wing disc. nmo expression is induced upon high levels of Wg signaling and can be inhibited by interfering with Wg signaling. Finally, we observe alterations in Arm stabilization upon modulation of Nemo. These observations suggest that the patterning mechanism governed by Wg involves a negative feedback circuit in which Wg induces expression of its own antagonist Nemo.     

A positive role for Short gastrulation in modulating BMP signaling during dorsoventral patterning in the Drosophila embryo

Positional information in the dorsoventral axis of the Drosophila embryo is encoded by a BMP activity gradient formed by synergistic signaling between the BMP family members Decapentaplegic (DPP) and Screw (SCW). short gastrulation (sog), which is functionally homologous to Xenopus Chordin, is expressed in the ventrolateral regions of the embryo and has been shown to act as a local antagonist of BMP signaling. Here we demonstrate that SOG has a second function, which is to promote BMP signaling on the dorsal side of the embryo. We show that a weak, homozygous-viable sog mutant is enhanced to lethality by reduction in the activities of the Smad family members Mad or Medea, and that the lethality is caused by defects in the molecular specification and subsequent cellular differentiation of the dorsal-most cell type, the amnioserosa. While previous data had suggested that the negative function of SOG is directed against SCW, we present data that suggests that the positive activity of SOG is directed towards DPP. We demonstrate that Chordin shares the same apparent ligand specificity as does SOG, preferentially inhibiting SCW but not DPP activity. However, in Drosophila assays Chordin does not have the same capacity to elevate BMP signaling as does SOG, identifying a functional difference in the otherwise well conserved process of dorsoventral pattern formation in arthropods and chordates.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.     

Evolution of BMP signaling in Drosophila oogenesis: a receptor-based mechanism

The bone morphogenetic protein (BMP) signaling pathway is a conserved regulator of cellular and developmental processes in animals. The mechanisms underlying BMP signaling activation differ among tissues and mostly reflect changes in the expression of pathway components. BMP signaling is one of the major pathways responsible for the patterning of the Drosophila eggshell, a complex structure derived from a layer of follicle cells (FCs) surrounding the developing oocyte. Activation of BMP signaling in the FCs is dynamic. Initially, signaling is along the anterior-posterior (A/P) axis; later, signaling acquires dorsal-ventral (D/V) polarity. These dynamics are regulated by changes in the expression pattern of the type I BMP receptor thickveins (tkv). We recently found that signaling dynamics and TKV patterning are highly correlated in the FCs of multiple Drosophila species. In addition, we showed that signaling patterns are spatially different among species. Here, we use a mathematical model to simulate the dynamics and differences of BMP signaling in numerous species. This model predicts that qualitative and quantitative changes in receptor expression can lead to differences in the spatial pattern of BMP signaling. We tested these predications experimentally in three different Drosophila species and through genetic perturbations of BMP signaling in D. melanogaster. On the basis of our results, we concluded that changes in tkv patterning can account for the experimentally observed differences in the patterns of BMP signaling in multiple Drosophila species.     

Mouse Crossveinless-2 is the vertebrate homolog of a Drosophila extracellular regulator of BMP signaling

The Dpp/BMP signaling pathway is highly conserved between vertebrates and invertebrates. The recent molecular characterization of the Drosophila crossveinless-2 (cv-2) mutation by Conley and colleagues introduced a novel regulatory step in the Dpp/BMP pathway (Development 127 (2000) 3945). The CV-2 protein is secreted and contains five cysteine-rich (CR) domains similar to those observed in the BMP antagonist Short gastrulation (Sog) of Drosophila and Chordin (Chd) of vertebrates. The mutant phenotype in Drosophila suggests that CV-2 is required for the differentiation of crossvein structures in the wing which require high Dpp levels. Here we present the mouse and human homologs of the Drosophila cv-2 protein. The mouse gene is located on chromosome 9A3 while the human locus maps on chromosome 7p14. CV-2 is expressed dynamically during mouse development, in particular in regions of high BMP signaling such as the posterior primitive streak, ventral tail bud and prevertebral cartilages. We conclude that CV-2 is an evolutionarily conserved extracellular regulator of the Dpp/BMP signaling pathway.