Retrograde Gbb signaling through the Bmp type 2 receptor wishful thinking regulates systemic FMRFa expression in Drosophila

Amidated neuropeptides of the FMRFamide class regulate numerous physiological processes including synaptic efficacy at the Drosophila neuromuscular junction (NMJ). We demonstrate here that mutations in wishful thinking (wit) a gene encoding a Drosophila Bmp type 2 receptor that is required for proper neurotransmitter release at the neuromuscular junction, also eliminates expression of FMRFa in that subset of neuroendocrine cells (Tv neurons) which provide the systemic supply of FMRFa peptides. We show that Gbb, a Bmp ligand expressed in the neurohemal organ provides a retrograde signal that helps specify the peptidergic phenotype of the Tv neurons. Finally, we show that supplying FMRFa in neurosecretory cells partially rescues the wit lethal phenotype without rescuing the primary morphological or electrophysiological defects of wit mutants. We propose that Wit and Gbb globally regulate NMJ function by controlling both the growth and transmitter release properties of the synapse as well as the expression of systemic modulators of NMJ synaptic activity.     

The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function

Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. Here, we describe the phenotypic consequences of mutations in the wishful thinking (wit) gene, the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. These results reveal a novel role for BMP signaling in regulating Drosophila neuromuscular junction synapse assembly and activity and may indicate that similar pathways could govern vertebrate synapse development.     

Highwire regulates synaptic growth in Drosophila

The formation, stabilization, and growth of synaptic connections are dynamic and highly regulated processes. The glutamatergic neuromuscular junction (NMJ) in Drosophila grows new boutons and branches throughout larval development. A primary walking behavior screen followed by a secondary anatomical screen led to the identification of the highwire (hiw) gene. In hiw mutants, the specificity of motor axon pathfinding and synapse formation appears normal. However, NMJ synapses grow exuberantly and are greatly expanded in both the number of boutons and the extent and length of branches. These synapses appear normal ultrastructurally but have reduced quantal content physiologically. hiw encodes a large protein found at presynaptic terminals. Within presynaptic terminals, HIW is localized to the periactive zone surrounding active zones; Fasciclin II (Fas II), which also controls synaptic growth, is found at the same location.     

LIM Kinase1 controls synaptic stability downstream of the type II BMP receptor

Here, we demonstrate that the BMP receptor Wishful Thinking (Wit) is required for synapse stabilization. In the absence of BMP signaling, synapse disassembly and retraction ensue. Remarkably, downstream Smad-mediated signaling cannot fully account for the stabilizing activity of the BMP receptor. We identify LIM Kinase1 (DLIMK1)-dependent signaling as a second, parallel pathway that confers the added synapse-stabilizing activity of the BMP receptor. We show that DLIMK1 binds a region of the Wit receptor that is necessary for synaptic stability but is dispensable for Smad-mediated synaptic growth. A genetic analysis demonstrates that DLIMK1 is necessary, presynaptically, for synapse stabilization, but is not necessary for normal synaptic growth or function. Furthermore, presynaptic expression of DLIMK1 in a wit or mad mutant significantly rescues synaptic stability, growth, and function. DLIMK1 localizes near synaptic microtubules and functions independently of ADF/cofilin, highlighting a novel requirement for DLIMK1 during synapse stabilization rather than actin-dependent axon outgrowth.     

The Drosophila type II receptor, Wishful thinking, binds BMP and myoglianin to activate multiple TGFbeta family signaling pathways

Wishful thinking (Wit) is a Drosophila transforming growth factor-beta (TGFbeta) superfamily type II receptor most related to the mammalian bone morphogenetic protein (BMP) type II receptor, BMPRII. To better understand its function, we undertook a biochemical approach to establish the ligand binding repertoire and downstream signaling pathway. We observed that BMP4 and BMP7, bound to receptor complexes comprised of Wit and the type I receptor thickveins and saxophone to activate a BMP-like signaling pathway. Further we demonstrated that both myoglianin and its most closely related mammalian ligand, myostatin, interacted with a Wit and Baboon (Babo) type II-type I receptor complex to activate TGFbeta/activin-like signaling pathways. These results thereby demonstrate that Wit binds multiple ligands to activate both BMP and TGFbeta-like signaling pathways. Given that myoglianin is expressed in muscle and glial-derived cells, these results also suggest that Wit may mediate myoglianin-dependent signals in the nervous system.     

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. Here, we demonstrate that cholecystokinin-like receptor (CCKLR) and drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR or dsk produced severe NMJ undergrowth, whereas overexpression of CCKLR caused overgrowth. Presynaptic expression of CCKLR was necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduced synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. Our results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms.     

Retrograde neurotrophin signaling through Tollo regulates synaptic growth in Drosophila

Toll-like receptors (TLRs) are best characterized for their roles in mediating dorsoventral patterning and the innate immune response. However, recent studies indicate that TLRs are also involved in regulating neuronal growth and development. Here, we demonstrate that the TLR Tollo positively regulates growth of the Drosophila melanogaster larval neuromuscular junction (NMJ). Tollo mutants exhibited NMJ undergrowth, whereas increased expression of Tollo led to NMJ overgrowth. Tollo expression in the motoneuron was both necessary and sufficient for regulating NMJ growth. Dominant genetic interactions together with altered levels of phosphorylated c-Jun N-terminal kinase (JNK) and puc-lacZ expression revealed that Tollo signals through the JNK pathway at the NMJ. Genetic interactions also revealed that the neurotrophin Spätzle3 (Spz3) is a likely Tollo ligand. Spz3 expression in muscle and proteolytic activation via the Easter protease was necessary and sufficient to promote NMJ growth. These results demonstrate the existence of a novel neurotrophin signaling pathway that is required for synaptic development in Drosophila.     

lazaro encodes a lipid phosphate phosphohydrolase that regulates phosphatidylinositol turnover during Drosophila phototransduction

An essential step in Drosophila phototransduction is the hydrolysis of phosphatidylinositol 4,5 bisphosphate PI(4,5)P2 by phospholipase Cbeta (PLCbeta) to generate a second messenger that opens the light-activated channels TRP and TRPL. Although the identity of this messenger remains unknown, recent evidence has implicated diacylglycerol kinase (DGK), encoded by rdgA, as a key enzyme that regulates its levels, mediating both amplification and response termination. In this study, we demonstrate that lazaro (laza) encodes a lipid phosphate phosphohydrolase (LPP) that functions during phototransduction. We demonstrate that the synergistic activity of laza and rdgA regulates response termination during phototransduction. Analysis of retinal phospholipids revealed a reduction in phosphatidic acid (PA) levels and an associated reduction in phosphatidylinositol (PI) levels. Together our results demonstrate the contribution of PI depletion to the rdgA phenotype and provide evidence that depletion of PI and its metabolites might be a key signal for TRP channel activation in vivo.     

BMP type II receptor regulates positioning of outflow tract and remodeling of atrioventricular cushion during cardiogenesis

Signaling of bone morphogenetic protein (BMP) via type I and type II receptors is involved in multiple processes contributing to cardiogenesis. To investigate the role of the BMP type II receptor (BMPRII) in heart development, the BMPRII gene was deleted throughout the embryo during gastrulation using a Mox2-Cre transgene. BMPRII^flox/−;Mox2-Cre mice exhibited cardiac defects including double-outlet right ventricle, ventricular septal defect (VSD), atrioventricular (AV) cushion defects, and thickened valve leaflets. To characterize the tissue-specific functions of BMPRII in cardiogenesis, a series of Cre transgenes (αMHC-, Tie2-, Wnt1-, and SM22α-Cre) was employed. Interestingly, myocardial development was normal when the BMPRII gene was deleted in myocardial cells using Mox2-Cre, αMHC-Cre, or SM22α-Cre transgenes, suggesting that signaling by other BMP type II receptors may compensate for the absence of BMPRII in the myocardial cells. AV cushion defects including atrial septal defect, membranous VSD, and thickened valve leaflets were found in BMPRII^flox/−;Tie2-Cre mice. Abnormal positioning of the aorta was observed in BMPRII^flox/−;Wnt1-Cre and BMPRII^flox/−;SM22α-Cre mice. Taken together, these results demonstrate that endocardial BMPRII expression is required for septal formation and valvulogenesis. Moreover, mesenchymal BMPRII expression in the outflow tract cushion is required for proper positioning of the aorta.     

Hephaestus encodes a polypyrimidine tract binding protein that regulates Notch signalling during wing development in Drosophila melanogaster

We describe the role of the Drosophila melanogaster hephaestus gene in wing development. We have identified several hephaestus mutations that map to a gene encoding a predicted RNA-binding protein highly related to human polypyrimidine tract binding protein and Xenopus laevis 60 kDa Vg1 mRNA-binding protein. Polypyrimidine tract binding proteins play diverse roles in RNA processing including the subcellular localization of mRNAs, translational control, internal ribosome entry site use, and the regulation of alternate exon selection. The analysis of gene expression in imaginal discs and adult cuticle of genetic mosaic animals supports a role for hephaestus in Notch signalling. Somatic clones lacking hephaestus express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of DELTA: These results lead to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta. This is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in the Notch signalling pathway.     

The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye.

The argos gene encodes a protein that is required for viability and that regulates the determination of cells in the Drosophila eye. A developmental analysis of argos mutant eyes indicates that the mystery cells, which are usually nonneuronal, are transformed into extra photoreceptors, and that supernumerary cone cells and pigment cells are also recruited. Clonal analysis indicates that argos acts nonautonomously and can diffuse over the range of several cell diameters. Conceptual translation of the argos gene suggests that it encodes a secreted protein.     

Population density regulates Drosophila synaptic morphology in a Fasciclin-II-dependent manner

Genetic analysis of the Drosophila larval neuromuscular junction has identified some of the key molecules that regulate synaptic plasticity. Among these molecules, the expression level of Fasciclin II (FasII), a homophilic cell adhesion molecule, is critically important for determining the final form of the neuromuscular junction. Genetic reduction of FasII expression by 50% yields more elaborate nerve terminals, while a greater reduction in expression, to 10% of wild-type, yields a substantial reduction in the nerve terminal morphology. Importantly, regulation of FasII expression seems to be the final output for several genetic manipulations that transform NMJ morphology. In an effort to understand the importance of this regulatory pathway in the normal animal, we have undertaken studies to identify environmental cues that might be important for initiating FasII-dependent changes in synaptic plasticity. Here we report on the relationship between larval population density and synaptic morphology, synaptic strength, and FasII levels. We raised Drosophila larvae under conditions of increasing population density and found an inverse exponential relationship between population density and the number of synaptic boutons, the number of branches, and the length of branches. We also observed population-dependent alteration in FasII levels, with lower densities having less FasII at the synapse. The correlation between density and morphological change was abrogated in larvae constitutively expressing FasII, and in wild-type larvae grown on soft culture medium. Together these data show that environmental cues can induce regulation of FasII. Interestingly, however, the quantal content of synaptic transmission was not different among the different population densities, suggesting that other factors contribute to maintaining synaptic strength at a defined level.     

Lost-a-fin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation

TGFbeta signaling pathways of the bone morphogenetic protein (BMP) subclass are essential for dorsoventral pattern formation of both vertebrate and invertebrate embryos. Here we determine by chromosomal mapping, linkage analysis, cDNA sequencing and mRNA rescue that the dorsalized zebrafish mutant lost-a-fin (laf) is defective in the gene activin receptor-like kinase 8 (alk8), which encodes a novel type I TGFbeta receptor. The alk8 mRNA is expressed both maternally and zygotically. Embyros that lack zygotic, but retain maternal Laf/Alk8 activity, display a weak dorsalization restricted to the tail and die by 3 days postfertilization. We rescued the laf dorsalized mutant phenotype by alk8 mRNA injection and generated homozygous laf/alk8 mothers to investigate the maternal role of Laf/Alk8 activity. Adult fish lacking Laf/Alk8 activity are fertile, exhibit a growth defect and are significantly smaller than their siblings. Embryos derived from homozygous females, which lack both maternal and zygotic Laf/Alk8 activity, display a strongly dorsalized mutant phenotype, no longer limited to the tail. These mutant embryos lack almost all gastrula ventral cell fates, with a concomitant expansion of dorsal cell types. During later stages, most of the somitic mesoderm and neural tissue circumscribe the dorsoventral axis of the embryo. Zygotic laf/alk8 mutants can be rescued by overexpression of the BMP signal transducer Smad5, but not the Bmp2b or Bmp7 ligands, consistent with the Laf/Alk8 receptor acting within a BMP signaling pathway, downstream of a Bmp2b/Bmp7 signal. Antibodies specific for the phosphorylated, activated form of Smad1/5, show that BMP signaling is nearly absent in gastrula lacking both maternal and zygotic Laf/Alk8 activity, providing further evidence that Laf/Alk8 transduces a BMP signal. In total, our work strongly supports the role of Laf/Alk8 as a type I BMP receptor required for the specification of ventral cell fates.     

KEL-8 is a substrate receptor for CUL3-dependent ubiquitin ligase that regulates synaptic glutamate receptor turnover

The regulated localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors (AMPARs) to synapses is an important component of synaptic signaling and plasticity. Regulated ubiquitination and endocytosis determine the synaptic levels of AMPARs, but it is unclear which factors conduct these processes. To identify genes that regulate AMPAR synaptic abundance, we screened for mutants that accumulate high synaptic levels of the AMPAR subunit GLR-1 in Caenorhabditis elegans. GLR-1 is localized to postsynaptic clusters, and mutants for the BTB-Kelch protein KEL-8 have increased GLR-1 levels at clusters, whereas the levels and localization of other synaptic proteins seem normal. KEL-8 is a neuronal protein and is localized to sites adjacent to GLR-1 postsynaptic clusters along the ventral cord neurites. KEL-8 is required for the ubiquitin-mediated turnover of GLR-1 subunits, and kel-8 mutants show an increased frequency of spontaneous reversals in locomotion, suggesting increased levels of GLR-1 are present at synapses. KEL-8 binds to CUL-3, a Cullin 3 ubiquitin ligase subunit that we also find mediates GLR-1 turnover. Our findings indicate that KEL-8 is a substrate receptor for Cullin 3 ubiquitin ligases that is required for the proteolysis of GLR-1 receptors and suggest a novel postmitotic role in neurons for Kelch/CUL3 ubiquitin ligases.     

The Drosophila estrogen-related receptor directs a metabolic switch that supports developmental growth

Metabolism must be coordinated with development to provide the appropriate energetic needs for each stage in the life cycle. Little is known, however, about how this temporal control is achieved. Here, we show that the Drosophila ortholog of the estrogen-related receptor (ERR) family of nuclear receptors directs a critical metabolic transition during development. dERR mutants die as larvae with low ATP levels and elevated levels of circulating sugars. The expression of active dERR protein in mid-embryogenesis triggers a coordinate switch in gene expression that drives a metabolic program normally associated with proliferating cells, supporting the dramatic growth that occurs during larval development. This study shows that dERR plays a central role in carbohydrate metabolism, demonstrates that a proliferative metabolic program is used in normal developmental growth, and provides a molecular context to understand the close association between mammalian ERR family members and cancer.     

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.