Compartment specific mitochondrial dysfunction in Drosophila knock-in model of ALS reversed by altered gene expression of OXPHOS subunits and pro-fission factor Drp1.

Amyotrophic Lateral Sclerosis (ALS) is a fatal multisystem neurodegenerative disease, characterized by a loss in motor function. ALS is genetically diverse, with mutations in genes ranging from those regulating RNA metabolism, like TAR DNA-binding protein (TDP-43) and Fused in sarcoma (FUS), to those that act to maintain cellular redox homeostasis, like superoxide dismutase 1 (SOD1). Although varied in genetic origin, pathogenic and clinical commonalities are clearly evident between cases of ALS. Defects in mitochondria is one such common pathology, thought to occur prior to, rather than as a consequence of symptom onset, making these organelles a promising therapeutic target for ALS, as well as other neurodegenerative diseases. Depending on the homeostatic needs of neurons throughout life, mitochondria are normally shuttled to different subcellular compartments to regulate metabolite and energy production, lipid metabolism, and buffer calcium. While originally considered a motor neuron disease due to the dramatic loss in motor function accompanied by motor neuron cell death in ALS patients, many studies have now implicated non-motor neurons and glial cells alike. Defects in non-motor neuron cell types often preceed motor neuron death suggesting their dysfunction may initiate and/or facilitate the decline in motor neuron health. Here, we investigate mitochondria in a Drosophila Sod1 knock-in model of ALS. In depth, in vivo, examination reveals mitochondrial dysfunction evident prior to onset of motor neuron degeneration. Genetically encoded redox biosensors identify a general disruption in the electron transport chain (ETC). Compartment specific abnormalities in mitochondrial morphology is observed in diseased sensory neurons, accompanied by no apparent defects in the axonal transport machinery, but instead an increase in mitophagy in synaptic regions. The decrease in networked mitochondria at the synapse is reversed upon downregulation of the pro-fission factor Drp1. Furthermore, altered expression of specific OXPHOS subunits reverses ALS-associated defects in mitochondrial morphology and function.

The MuSK-BMP pathway regulates synaptic Nav1.4 localization and muscle excitability.

The neuromuscular junction (NMJ) is the linchpin of nerve-evoked muscle contraction. Broadly considered, the function of the NMJ is to transduce a nerve action potential into a muscle fiber action potential (MFAP). Efficient information transfer requires both cholinergic signaling, responsible for the generation of endplate potentials (EPPs), and excitation, the activation of postsynaptic voltage-gated sodium channels (Nav1.4) to trigger MFAPs. In contrast to the cholinergic apparatus, the signaling pathways that organize Nav1.4 and muscle fiber excitability are poorly characterized. Muscle-specific kinase (MuSK), in addition to its Ig1 domain-dependent role as an agrin-LRP4 receptor, is also a BMP co-receptor that binds BMPs via its Ig3 domain and shapes BMP-induced signaling and transcriptional output. Here we probed the function of the MuSK-BMP pathway at the NMJ using mice lacking the MuSK Ig3 domain ('ΔIg3-MuSK'). Synapses formed normally in ΔIg3-MuSK animals, but the postsynaptic apparatus was fragmented from the first weeks of life. Anatomical denervation was not observed at any age examined. Moreover, spontaneous and nerve-evoked acetylcholine release, AChR density, and endplate currents were comparable to WT. However, trains of nerve-evoked MFAPs in ΔIg3-MuSK muscle were abnormal as revealed by increased jitter and blocking in single fiber electromyography. Further, nerve-evoked compound muscle action potentials (CMAPs), as well as twitch and tetanic muscle torque force production, were also diminished. Finally, Nav1.4 levels were reduced at ΔIg3-MuSK synapses but not at the extrajunctional sarcolemma, indicating that the observed excitability defects are the result of impaired localization of this voltage-gated ion channel at the NMJ. We propose that MuSK plays two distinct roles at the NMJ: as an agrin-LRP4 receptor necessary for establishing and maintaining cholinergic signaling, and as a BMP co-receptor required for maintaining proper Nav1.4 density, nerve-evoked muscle excitability and force production. The MuSK-BMP pathway thus emerges as a target for modulating excitability and functional innervation, which are defective in conditions such as congenital myasthenic syndromes and aging.

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells.

The bundle of filaments within microvilli of intestinal epithelial cells contains five major proteins including actin, calmodulin, and subunits of 105-, 95-, and 70-kdaltons. It has been previously shown (Howe, C. L., M. S. Mooseker, and T. A. Graves. 1980. Brush-border calmodulin: a major component of the isolated microvillus core. J. Cell Biol. 85: 916-923) that the addition of Ca++ (> 10(-6) M) to microvillus cores causes a rapid, drastic, but at least partially reversible disruption of this actin filament bundle. High-speed centrifugation of microvillus cores treated with Ca++ indicates that several core proteins are solubilized, including 30-50% of the actin and calmodulin, along with much of the 95- and 70-kdalton subunits. Gel filtration of such Ca++ extracts in the presence and absence of Ca++ indicates that microvillar actin "solated" by Ca++ is in an oligomeric state probably complexed with the 95-kdalton subunit. Removal of Ca++ results in the reassembly of F-actin, probably still complexed with 95-kdalton subunit, as determined by gel filtration, cosedimentation, viscometry, and electron microscopy. The 95-kdalton subunit (95K) was purified from Ca++ extracts by DEAE-Sephadex chromatography and its interaction with actin characterized by viscometry, cosedimentation, and EM in the presence and absence of Ca++. In the presence, but not absence, of Ca++, 95K inhibits actin assembly (50% inhibition at 1:50-60 95K to actin) and also reduces the viscosity of F-actin solutions. Similarly, sedimentation of actin is inhibited by 95K, but a small, presumably oligomeric actin- 95K complex formed in the presence of Ca++ is pelletable after long-term centrifugation. In the absence of Ca++, 95K cosediments with F-actin. EM of 95K-actin mixtures reveals that 95K "breaks" actin into small, filamentous fragments in the presence of Ca++. Reassembly of filaments occurs once Ca++ is removed. In the absence of Ca++, 95K has no effect on filament structure and, at relatively high ratios (1:2-6) of 95K to actin, this core protein will aggregate actin filaments into bundles.

Physical and functional definition of the Drosophila Notch locus by P element transformation.

Notch is a developmentally regulated locus which controls the differentiation of various Drosophila tissues, among them the embryonic nervous system. Molecular analysis has suggested that Notch is defined by an approximately 40-kb transcription unit which is spliced into a 10.2-kb mRNA composed of nine exonic regions and coding for a 2703-amino acid long transmembrane protein that shows homology to the mammalian epidermal growth factor. Here, we define the 5' end of the transcription unit and determine the sequences deleted in a Notch mutation involving the 5' nontranscribed region. Using a Notch cosmid vector we demonstrate by P element-mediated transformation that all sequences necessary for Notch function are confined in an approximately 40-kb long genomic region. cDNA sequences are used to construct a 15-kb "minigene" which lacks most, but not all, introns and its functionality is also tested by P element transformation. We show that, unlike the cosmid vector which is capable of rescuing completely all Notch mutations, only certain phenotypes can be rescued by the "minigene." The functional implications of our findings are discussed.

Genetic analysis of the bone morphogenetic protein-related gene, gbb, identifies multiple requirements during Drosophila development.

We have isolated mutations in the Drosophila melanogaster gene glass bottom boat (gbb), which encodes a TGF-beta signaling molecule (formerly referred to as 60A) with highest sequence similarity to members of the bone morphogenetic protein (BMP) subgroup including vertebrate BMPs 5-8. Genetic analysis of both null and hypomorphic gbb alleles indicates that the gene is required in many developmental processes, including embryonic midgut morphogenesis, patterning of the larval cuticle, fat body morphology, and development and patterning of the imaginal discs. In the embryonic midgut, we show that gbb is required for the formation of the anterior constriction and for maintenance of the homeotic gene Antennapedia in the visceral mesoderm. In addition, we show a requirement for gbb in the anterior and posterior cells of the underlying endoderm and in the formation and extension of the gastric caecae. gbb is required in all the imaginal discs for proper disc growth and for specification of veins in the wing and of macrochaete in the notum. Significantly, some of these tissues have been shown to also require the Drosophila BMP2/4 homolog decapentaplegic (dpp), while others do not. These results indicate that signaling by both gbb and dpp may contribute to the development of some tissues, while in others, gbb may signal independently of dpp.

CNS midline enhancers of the Drosophila slit and Toll genes.

The Drosophila CNS midline cells comprise a small, well-characterized group of neurons and glia in which the transcriptional control of CNS development can be studied. Using germ-line transformation of lacZ fusion constructs, we have dissected putative regulatory regions of the slit and Toll genes to identify CNS midline-restricted transcriptional enhancers. This analysis has uncovered DNA regions able to drive lacZ expression in most tissues in which embryonic slit and Toll are expressed, including three separable CNS midline-conferring regions: one in the Toll gene which is expressed early in all of the CNS midline precursors, and two in the slit gene which are expressed later in the midline glia (MG).

Thromboxane (Tx) A2 receptor blockade and TxA2 synthase inhibition alone and in combination: comparison of anti-aggregatory efficacy in human platelets.

1. The present study has compared the relative anti-aggregatory effect of various compounds which interfere with thromboxane (Tx) A2-dependent aggregation of human platelets in whole blood in vitro. These included the cyclo-oxygenase inhibitor aspirin, the TxA2 synthase inhibitor dazoxiben, the TxA2 (TP-) receptor blocking drug GR32191 and two compounds, R.68070 ((E)-5-[[[(3-pyridinyl) [3-(trifluoromethyl)phenyl]-methylen] amino]oxy] pentanoic acid) and CV-4151 [E)-7-phenyl-7-(3-pyridyl)-6-heptenoic acid), which possess both TP-receptor blocking and TxA2 synthase inhibitory activities in the same molecule. 2. GR32191, R.68070 and CV-4151 all antagonized aggregation to the TxA2 mimetic U-46619, with pA2 values of approximately 8.2, 5.4 and 4.8 respectively. This effect was specific, platelet aggregation induced by adenosine 5'-diphosphate (ADP) being unaffected by concentrations up to 10, 1000 and 300 microM respectively. In contrast, neither aspirin nor dazoxiben exhibited any measurable TP-receptor blocking activity. 3. The rank order of potency (pIC50) for inhibition of TxA2 formation in serum was R.68070 (7.4) greater than CV-4151 (6.9) greater than dazoxiben (5.7) greater than aspirin (5.3). In addition, all four drugs abolished collagen-induced platelet TxA2 formation. In contrast, GR32191 produced no consistent inhibition of TxA2 formation in either system up to concentrations of 10-30 microM. 4. The specificity of R.68070, CV-4151 and dazoxiben for TxA2 synthase was indicated by their ability to increase serum levels of prostaglandin E2 (PGE2) and PGD2 in parallel with decreases in TxA2 formation. This profile was not observed with aspirin or GR32191. However, high concentrations of R.68070 (100,microM) and CV-4151 (1000 microM) necessary for maximum TP-receptor blocking activity, produced substantially smaller increases in PGE2 and PGD2, consistent with an aspirin-like effect of these compounds upon cyclo-oxygenase. With dazoxiben (1000 microM), PGE2 and PGD2 levels remained elevated. 5. Aspirin inhibited collagen-induced platelet aggregation, the effect correlating with inhibition of TxA2 formation. Dazoxiben, whilst also achieving maximal inhibition of TxA2 formation, produced significantly less inhibition of aggregation than aspirin. In contrast, GR32191 (0.1-1O microM), at concentrations specific for TP-receptor blockade, produced a significantly greater antagonism of collagen-induced platelet aggregation than aspirin. This additional effect of GR32191 was absent in platelets pretreated with aspirin, indicating the probable involvement of an endogenous anti-aggregatory cyclo-oxygenase product in response to collagen stimulation. 6. R.68070 and CV-4151 also inhibited collagen-induced aggregation, with very high concentrations of R.68070 (100 microM) producing an effect equivalent to that of GR32191. 7. In contrast, the combination of GR32191 with either dazoxiben, R.68070 or CV-4151, at concentrations specific for TxA2 synthase, produced a synergistic inhibitory effect upon collagen-induced platelet aggregation which was greater than that achieved with either aspirin or any of the compounds used alone. Pretreatment of platelets with aspirin reversed this synergistic effect, consistent with it being dependent upon the formation and action of anti-aggregatory prostaglandins. 8. In conclusion, the present study has confirmed the superior platelet inhibitory profile of a combination of a TP-receptor blocking drug and a TxA2 synthase inhibitor to that of either activity alone. However, the maximum inhibitory effect of the currently available compounds, R.68070 and CV4151, which possess both activities in the same molecule, appears to be no greater in vitro than that obtained with the potent TP-receptor blocking drug, GR32191. This most probably reflects the inhibition by R.68070 and CV-4151 of platelet cyclo-oxygenase at the concentrations required for effective TP-receptor blockade which results in a reduction in the formation of anti-aggregatory prostanoids.

How many receptors does it take?

Three recent reports identify two genes, thick veins (tkv) and saxophone (sax), which encode serine/threonine transmembrane proteins that act as receptors for mediating different aspects of the Drosophila TGF-beta-related signal, dpp. tkv is required for patterning the entire embryonic dorsal region, while sax is required for patterning only amnioserosa, the dorsalmost cell fates. dpp signaling in other developmental processes again requires both tkv and sax, but to differing degrees. tkv and sax encode type I receptors, which appear to directly bind ligand, unlike what has been observed for other type I receptors.

Context-dependent relationships between the BMPs gbb and dpp during development of the Drosophila wing imaginal disk.

The Drosophila BMP5/6/7/8 homolog, glass bottom boat (gbb), has been shown to be involved in proliferation and vein patterning in the wing disk. To better understand the roles for gbb in wing development, as well as its relationship with the Drosophila BMP2/4 homolog decapentaplegic (dpp), we have used clonal analysis to define the functional foci of gbb during wing development. Our results show that gbb has both local and long-range functions in the disk that coincide both spatially and functionally with the established functions of dpp, suggesting that both BMPs contribute to the same processes during wing development. Indeed, comparison of the mutant phenotypes of dpp and gbb hypomorphs and null clones shows that both BMPs act locally along the longitudinal and cross veins to affect the process of vein promotion during pupal development, and long-range from a single focus along the A/P compartment boundary to affect the processes of disk proliferation and vein specification during larval development. Moreover, we show that duplications of dpp are able to rescue many of the phenotypes associated with gbb mutants and clones, indicating that the functions of gbb are at least partially redundant with those of dpp. While this relationship is similar to that described for dpp and the BMP screw (scw) in the embryo, we show that the mechanisms underlying both local and long-range functions of gbb and dpp in the wing are different. For the local foci, gbb function is confined to the regions of the veins that require the highest levels of dpp signaling, suggesting that gbb acts to augment dpp signaling in the same way as scw is proposed to do in the embryo. However, unlike scw-dependent signals in the embryo, these gbb signals are not transduced by the Type I receptor saxophone (sax), thus, the cooperativity between gbb and dpp is not achieved by signaling through distinct receptor complexes. For the long-range focus along the A/P compartment boundary, gbb function does not appear to affect the high point of the dpp gradient, but, rather, appears to be required for low points, which is the reciprocal of the relationship between dpp and scw in the embryo. Moreover, these functions of gbb also do not require the Type I receptor sax. Given these results, we conclude that the relationships between gbb and dpp in the wing disk represent novel paradigms for how multiple BMP ligands signal during development, and that signaling by multiple BMPs involves a variety of different inter-ligand relationships that depend on the developmental context in which they act.

An activity gradient of decapentaplegic is necessary for the specification of dorsal pattern elements in the Drosophila embryo.

decapentaplegic (dpp) is a zygotically expressed gene encoding a TGF-beta-related ligand that is necessary for dorsal-ventral patterning in the Drosophila embryo. We show here that dpp is an integral part of a gradient that specifies many different cell fates via intercellular signalling. There is a graded requirement for dpp activity in the early embryo: high levels of dpp activity specify the amnioserosa, while progressively lower levels specify dorsal and lateral ectoderm. This potential for dpp to specify cell fate is highly dosage sensitive. In the wild-type embryo, increasing the gene dosage of dpp can shift cell fates along the dorsal-ventral axis. Furthermore, in mutant embryos, in which only a subset of the dorsal-ventral pattern elements are represented, increasing the gene dosage of dpp can specifically transform those pattern elements into more dorsal ones. We present evidence that the zygotic dpp gradient and the maternal dorsal gradient specify distinct, non-overlapping domains of the dorsal-ventral pattern.

Drosophila 60A gene, another transforming growth factor beta family member, is closely related to human bone morphogenetic proteins.

The 60A gene, a member of the transforming growth factor beta superfamily of signaling proteins, has been identified in Drosophila melanogaster. From its inferred protein sequence we predict the precursor is secreted and processed to release a growth factor-like molecule. The 60A gene is expressed throughout development with peaks of transcription during early embryogenesis, in pupae, and in adult males. The putative 60A protein shows greater sequence similarity to three vertebrate family members (human bone morphogenetic proteins 5, 6, and 7) than to its only Drosophila relative, the protein product of the decapentaplegic (dpp) gene. This observation suggests that the duplication event that gave rise to the two transforming growth factor beta-like proteins in Drosophila predates the divergence of chordates and arthropods.

The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development.

Development of the Drosophila CNS midline cells is dependent upon the function of the single-minded (sim) gene. Sequence analysis shows that sim is a member of the basic-helix-loop-helix class of transcription factors. Cell fate experiments establish that sim is required for early events in midline cell development, including a synchronized cell division, proper formation of nerve cell precursors, and positive auto-regulation of its midline expression. Induction of ectopic sim protein under the control of the hsp70 promoter shows that sim can direct cells of the lateral CNS to exhibit midline cell morphology and patterns of gene expression. We propose that sim functions as a master developmental regulator of the CNS midline lineage.

Nucleotide sequence from the neurogenic locus notch implies a gene product that shares homology with proteins containing EGF-like repeats.

The primary structure of the major embryonic Notch transcript is presented, as determined by sequence analysis of overlapping cDNA clones. The 10,148 bp sequence corresponding to this transcript possesses an 8109 bp open reading frame that potentially codes for a 2703 amino acid protein. We show that this polypeptide contains a repeated structure composed of 36 tandemly arranged 40 amino acid long repeats, which show homology to the epidermal growth factor and other proteins containing EGF-like repeats. Hydropathy plots suggest that the putative Notch protein may span the membrane. We relate these findings to the developmental action of Notch and speculate that the locus may be involved in a cell-cell interaction mechanism that is essential for the differentiation of the ectoderm into neural and epidermal precursors.

Control of CNS midline transcription by asymmetric E-box-like elements: similarity to xenobiotic responsive regulation.

Central nervous system midline cells constitute a discrete group of Drosophila embryonic cells with numerous functional and developmental roles. Corresponding to their separate identity, the midline cells display patterns of gene expression distinct from the lateral central nervous system. A conserved 5 base pair sequence (ACGTG) was identified in central nervous system midline transcriptional enhancers of three genes. Germ-line transformation experiments indicate that this motif forms the core of an element required for central nervous system midline transcription. The central nervous system midline element is related to the mammalian xenobiotic response element, which regulates transcription of genes that metabolize aromatic hydrocarbons. These data suggest a model whereby related basic-helix-loop-helix-PAS proteins interact with asymmetric E-box-like target sequences to control these disparate processes.

Vertebrate proteins related to Drosophila Naked Cuticle bind Dishevelled and antagonize Wnt signaling.

Wnt signals control cell fate decisions and orchestrate cell behavior in metazoan animals. In the fruit fly Drosophila, embryos defective in signaling mediated by the Wnt protein Wingless (Wg) exhibit severe segmentation defects. The Drosophila segment polarity gene naked cuticle (nkd) encodes an EF hand protein that regulates early Wg activity by acting as an inducible antagonist. Nkd antagonizes Wg via a direct interaction with the Wnt signaling component Dishevelled (Dsh). Here we describe two mouse and human proteins, Nkd1 and Nkd2, related to fly Nkd. The most conserved region among the fly and vertebrate proteins, the EFX domain, includes the putative EF hand and flanking sequences. EFX corresponds to a minimal domain required for fly or vertebrate Nkd to interact with the basic/PDZ domains of fly Dsh or vertebrate Dvl proteins in the yeast two-hybrid assay. During mouse development, nkd1 and nkd2 are expressed in multiple tissues in partially overlapping, gradient-like patterns, some of which correlate with known patterns of Wnt activity. Mouse Nkd1 can block Wnt1-mediated, but not beta-catenin-mediated, activation of a Wnt-dependent reporter construct in mammalian cell culture. Misexpression of mouse nkd1 in Drosophila antagonizes Wg function. The data suggest that the vertebrate Nkd-related proteins, similar to their fly counterpart, may act as inducible antagonists of Wnt signals.

Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila.

In Drosophila wing discs, a morphogen gradient of DPP has been proposed to determine the transcriptional response thresholds of the downstream genes sal and omb. We present evidence that the concentration of the type I receptor TKV must be low to allow long-range DPP diffusion. Low TKV receptor concentrations result, however, in low signaling activity. To enhance signaling at low DPP concentrations, we find that a second ligand, GBB, augments DPP/TKV activity. GBB signals primarily through the type I receptor SAX, which synergistically enhances TKV signaling and is required for proper OMB expression. We show that OMB expression in wing discs requires synergistic signaling by multiple ligands and receptors to overcome the limitations imposed on DPP morphogen function by receptor concentration levels.

opa: a novel family of transcribed repeats shared by the Notch locus and other developmentally regulated loci in D. melanogaster.

The principal transcription product of Notch, a locus involved in the neurogenesis of D. melanogaster, is a developmentally regulated poly(A)+ RNA approximately 10.5 kb in length. Analysis of the structure of this RNA has revealed a 93 bp repeated sequence that is shared by many other developmentally regulated transcription units. Nucleotide sequence analysis of the repeat shows an unusual structure consisting predominantly of the triplets CAG and CAA, both of which can code for the amino acid Gln. We present evidence indicating that the Notch repeat is a member of a novel family of repetitive elements, which we term the opa family. Our data suggest that some of these elements may be not only transcribed but also translated. We compare opa with other known transcribed repeats and speculate on its functional significance.

TGF-beta/BMP superfamily members, Gbb-60A and Dpp, cooperate to provide pattern information and establish cell identity in the Drosophila wing.

Within a developing organism, cells receive many signals which control their proliferation, fate specification and differentiation. One group of such proteins is the TGF-beta/BMP class of related signaling molecules. Based on expression studies, multiple members of this class of ligands must impinge upon the same cells of a developing tissue; however, the role that multiple TGF-beta/BMP ligands may play in directing the development of such a tissue is not understood. Here we provide evidence that multiple BMPs are required for growth and patterning of the Drosophila wing. The Drosophila BMP gene, gbb-60A, exhibits a requirement in wing morphogenesis distinct from that shown previously for dpp, a well-characterized Drosophila BMP member. gbb-60A mutants exhibit a loss of pattern elements from the wing, particularly those derived from cells in the posterior compartment, consistent with the gbb-60A RNA and protein expression pattern. Based on genetic analysis and expression studies, we conclude that Gbb-60A must signal primarily as a homodimer to provide patterning information in the wing imaginal disc. We demonstrate that gbb-60A and dpp genetically interact and that specific aspects of this interaction are synergistic while others are antagonistic. We propose that the positional information received by a cell at a particular location within the wing imaginal disc depends on the balance of Dpp to Gbb-60A signaling. Furthermore, the critical ratio of Gbb-60A to Dpp signaling appears to be mediated by both Tkv and Sax type I receptors.