Drosophila abelson interacting protein (dAbi) is a positive regulator of abelson tyrosine kinase activity.

Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. We report a new member of this gene family, a Drosophila Abi (dAbi) that is a substrate for Abl kinase and that co-immunoprecipitates with Abl if the Abi SH3 domain is intact. We have identified a new function for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell.

Dominant effects of the bcr-abl oncogene on Drosophila morphogenesis.

We targeted expression of human/fly chimeric Bcr-Abl proteins to the developing central nervous system (CNS) and eye imaginal disc of Drosophila melanogaster. Neural expression of human/fly chimeric P210 Bcr-Abl or P185 Bcr-Abl rescued abl mutant flies from pupal lethality, indicating that P210 and P185 Bcr-Abl can substitute functionally for Drosophila Abl during axonogenesis. However, increased levels of neurally expressed P210 or P185 Bcr-Abl but not Drosophila Abl produced CNS defects and lethality. Expression of P210 or P185 in the eye imaginal disc produced a dominant rough eye phenotype that was dependent on dosage of the transgene. Drosophila Enabled, previously identified as a suppressor of the abl mutant phenotype and substrate for Drosophila Abl kinase, had markedly increased phosphotyrosine levels in Bcr-Abl expressing Drosophila, indicating that it is a substrate for Bcr-Abl as well. Drosophila, therefore, is a suitable model system to identify Bcr-Abl interactions important for signal transduction and oncogenesis.

A genetic screen for modifiers of Drosophila decapentaplegic signaling identifies mutations in punt, Mothers against dpp and the BMP-7 homologue, 60A.

decapentaplegic (dpp) is a Transforming Growth Factor beta (TGF-beta)-related growth factor that controls multiple developmental processes in Drosophila. To identify components involved in dpp signaling, we carried out a genetic screen for dominant enhancer mutations of a hypomorphic allele of thick veins (tkv), a type I receptor for dpp. We recovered new alleles of tkv, punt, Mothers against dpp (Mad) and Medea (Med), all of which are known to mediate dpp signaling. We also recovered mutations in the 60A gene which encodes another TGF-beta-related factor in Drosophila. DNA sequence analysis established that all three 60A alleles were nonsense mutations in the prodomain of the 60A polypeptide. These mutations in 60A caused defects in midgut morphogenesis and fat body differentiation. We present evidence that when dpp signaling is compromised, lowering the level of 60A impairs several dpp-dependent developmental processes examined, including the patterning of the visceral mesoderm, the embryonic ectoderm and the imaginal discs. These results provide the first in vivo evidence for the involvement of 60A in the dpp pathway. We propose that 60A activity is required to maintain optimal signaling capacity of the dpp pathway, possibly by forming biologically active heterodimers with Dpp proteins.

Phosphorylation of Enabled by the Drosophila Abelson tyrosine kinase regulates the in vivo function and protein-protein interactions of Enabled.

Drosophila Enabled (Ena) is a member of a family of cytoskeleton-associated proteins including mammalian vasodilator-stimulated phosphoprotein and murine Enabled that regulate actin cytoskeleton assembly. Mutations in Drosophila ena were discovered as dominant genetic suppressors of mutations in the Abelson tyrosine kinase (Abl), suggesting that Ena and Abl function in the same pathway or process. We have identified six tyrosine residues on Ena that are phosphorylated by Abl in vitro and in vivo. Mutation of these phosphorylation sites to phenylalanine partially impaired the ability of Ena to restore viability to ena mutant animals, indicating that phosphorylation is required for optimal Ena function. Phosphorylation of Ena by Abl inhibited the binding of Ena to SH3 domains in vitro, suggesting that one effect of Ena phosphorylation may be to modulate its association with other proteins.

decapentaplegic overexpression affects Drosophila wing and leg imaginal disc development and wingless expression.

We have used the GAL4-UAS expression system to increase the level of expression of the Drosophila gene decapentaplegic (dpp) in a pattern approximating its normal pattern in leg and wing imaginal discs. Intermediate increases of dpp expression have little effect in wing discs but high levels of dpp overexpression lead to reduction of the scutellum and duplication of posterior wing structures. In leg discs intermediate increases cause supernumerary outgrowths of ventral leg structures in the anterior-ventral region. Greater increases of dpp expression cause the loss of ventral leg structures with the concomitant fusion of left and right dorsal forelegs. The defects observed in both legs and wings appear to arise through dose-dependent effects of dpp on wingless (wg) expression. A high level of dpp overexpression in the wing disc causes reduction of wg expression in the presumptive scutellar region, consistent with the subsequent reduction of the scutellum. An intermediate increase of dpp expression in leg discs induces the expansion of wg expression into the ventral outgrowths. At higher dpp expression levels, ventral wg expression in leg discs is eliminated, consistent with the loss of ventral leg cuticle. In the leg disc end knob and in the wing margin primordium, where wg and dpp cooperate in producing distal outgrowth, dpp overexpression has no detectable effect either on patterning or on wg expression. We propose that a critical role for dpp in other regions of the leg and wing discs is to reduce or block the expression of wg. This role of dpp is supported by the observation that ectopic wg expression is detected in imaginal discs where dpp signaling is compromised by lowering the activity of one of its receptors, tkv. This antagonism between dpp and wg expression may be critical to assigning only one disc region as the distal organizer.

Decapentaplegic restricts the domain of wingless during Drosophila limb patterning.

Signalling proteins in the BMP-decapentaplegic (dpp), WNT-wingless (wg) and Shh-hedgehog (hh) families have been implicated in limb and appendage development in both invertebrates and vertebrates. In Drosophila, dpp protein (Dpp) induces distal outgrowth and patterning of legs and wings, but the molecular responses to Dpp are not well characterized. Analysis of clones mutant for the Dpp receptors encoded by punt or thickveins (tkv) reveals that repression of wg expression is one critical function of Dpp signalling in leg and wing discs. Distal clones that lie on the anterior edge of the anterior-posterior compartment boundary ectopically express wg and cause pattern abnormalities, suggesting that Dpp represses Hh activation of wg in the distal primordia of the leg and wing. By repressing wg expression in the leg, Dpp signalling limits the region that responds to high levels of Wg and Dpp to the site of distal outgrowth. Such negative regulatory feedback loops between signalling molecules are likely to be critical for limb patterning in other species.

Genetic interactions between the Drosophila Abelson (Abl) tyrosine kinase and failed axon connections (fax), a novel protein in axon bundles.

Mutations in the failed axon connections (fax) gene have been identified as dominant genetic enhancers of the Abl mutant phenotype. These mutations in fax all result in defective or absent protein product. In a genetic background with wild-type Abl function, the fax loss-of-function alleles are homozygous viable, demonstrating that fax is not an essential gene unless the animal is also mutant for Abl. The fax gene encodes a novel 47-kD protein expressed in a developmental pattern similar to that of Abl in the embryonic mesoderm and axons of the central nervous system. The conditional, extragenic noncomplementation between fax and another Abl modifier gene, disabled, reveal that the two proteins are likely to function together in a process downstream or parallel to the Abl protein tyrosine kinase.

Can clues to the molecular defects in chronic myelogenous leukemia come from genetic studies on the Abelson tyrosine kinase in fruit flies?

Translocations affecting the structure of the c-abl proto-oncogene are involved in the development or progression of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL). Leukemic cells from patients with CML show alterations in adhesive properties that may play a part in the pathology of these diseases. Mutations in the Drosophila Abl homolog are lethal and indicate that Abl may mediate processes involving differential cell adhesion. These observations suggest that Abl may regulate similar adhesive processes in human beings and Drosophila. Genetic analysis of Abl function in Drosophila has identified novel proteins that function in Abl-related processes. Analysis of the functions of these new molecules may provide insight into mechanisms by which oncogenic abl proteins participate in the etiology of CML and ALL.

enabled, a dosage-sensitive suppressor of mutations in the Drosophila Abl tyrosine kinase, encodes an Abl substrate with SH3 domain-binding properties.

Genetic screens for dominant second-site mutations that suppress the lethality of Abl mutations in Drosophila identified alleles of only one gene, enabled (ena). We report that the ena protein contains proline-rich motifs and binds to Abl and Src SH3 domains, ena is also a substrate for the Abl kinase; tyrosine phosphorylation of ena is increased when it is coexpressed in cells with human or Drosophila Abl and endogenous ena tyrosine phosphorylation is reduced in Abl mutant animals. Like Abl, ena is expressed at highest levels in the axons of the embryonic nervous system and ena mutant embryos have defects in axonal architecture. We conclude that a critical function of Drosophila Abl is to phosphorylate and negatively regulate ena protein during neural development.

Ectopic decapentaplegic in the Drosophila midgut alters the expression of five homeotic genes, dpp, and wingless, causing specific morphological defects.

The patterns of homeotic gene expression in the Drosophila midgut visceral mesoderm are instrumental in several morphogenetic events, including the formation of the gastric caeca and the positioning of the three midgut constrictions. We demonstrate that a potent regulator of homeotic gene expression in the visceral mesoderm is the secreted growth factor-like molecule encoded by the decapentaplegic (dpp) gene. Ectopic dpp in the visceral mesoderm caused changes in the gene expression of Sex combs reduced, Antennapedia, Ultrabithorax (Ubx), and abdominal-A (abd-A) and disrupted the formation of the gastric caeca and the first and third midgut constrictions. Ectopic dpp also induced expression of teashirt, wingless (wg) and the endogenous dpp gene in the visceral mesoderm and enhanced labial expression in the adjacent endoderm. The patterns of gene expression and the formation of the second midgut constriction in the presence of ectopic dpp are most consistent with a dpp-induced transformation of virtually the entire midgut to cell fates normally seen only in the parasegment (ps)7 and ps8 regions of the midgut. We conclude that dpp is a primary signal in maintaining Ubx expression in the visceral mesoderm in a pattern different from Ubx expression in the embryonic ectoderm and in providing a cell-cell communication mechanism by which Ubx expression influences gene expression across germlayers and across the ps7 to ps8 parasegment boundary in the visceral mesoderm.

Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A.

Reported assays of the bone morphogenetic proteins (BMPs) have not in general revealed specific functions for the different proteins, belying the specificity implied by the evolutionary conservation and distinct expression patterns of the genes encoding BMPs. We have used assays of developmental function to show that the two Drosophila homologues of the BMPs, decapentaplegic (dpp) and 60A, that both induce ectopic bone formation in mammalian assay systems, have distinct effects in Drosophila development. A binary expression system using the yeast transcriptional activator GAL4 directed identical patterns of tissue and temporally specific dpp and 60A expression. When dpp enhancer elements drove GAL4 expression, GAL4-responsive dpp transgenes rescued dpp mutant phenotypes, but GAL4-responsive 60A transgenes did not. Ectopic ectodermal expression of dpp during gastrulation respecified the dorsal/ventral pattern of the embryo. In contrast, ectopic 60A expression had no detectable effects on embryonic development but led to defects in adult structures or lethality during metamorphosis. Expression of 60A in cells expressing dpp did not interfere with dpp functions, indicating that dysfunctional heterodimers did not form at sufficient levels to inhibit dpp. These specific developmental responses in Drosophila indicate that in vivo functions of BMP-like factors can be more specific than indicated by the ectopic bone formation assays and that the Drosophila embryo provides an assay system sensitive to the structural differences that contribute to BMP specificity in vivo.

Dosage-sensitive modifiers of Drosophila abl tyrosine kinase function: prospero, a regulator of axonal outgrowth, and disabled, a novel tyrosine kinase substrate.

In the absence of the Drosophila abl protein-tyrosine kinase (PTK), loss-of-function mutations in either disabled or prospero have dominant phenotypic effects on embryonic development. Molecular and genetic characterizations indicate that the products of these genes interact with the abl PTK by different mechanisms. The interaction between abl and prospero, which encodes a nuclear protein required for correct axonal outgrowth, is likely to be indirect. In contrast, the product of disabled may be a substrate for the abl PTK. The disabled protein is colocalized with abl in axons, its predicted amino acid sequence contains 10 motifs similar to the major autophosphorylation site of abl, and the protein is recognized by antibodies to phosphotyrosine.

Increased levels of the Drosophila Abelson tyrosine kinase in nerves and muscles: subcellular localization and mutant phenotypes imply a role in cell-cell interactions.

Mutations in the Drosophila Abelson tyrosine kinase have pleiotropic effects late in development that lead to pupal lethality or adults with a reduced life span, reduced fecundity and rough eyes. We have examined the expression of the abl protein throughout embryonic and pupal development and analyzed mutant phenotypes in some of the tissues expressing abl. abl protein, present in all cells of the early embryo as the product of maternally contributed mRNA, transiently localizes to the region below the plasma membrane cleavage furrows as cellularization initiates. The function of this expression is not yet known. Zygotic expression of abl is first detected in the post-mitotic cells of the developing muscles and nervous system midway through embryogenesis. In later larval and pupal stages, abl protein levels are also highest in differentiating muscle and neural tissue including the photoreceptor cells of the eye. abl protein is localized subcellularly to the axons of the central nervous system, the embryonic somatic muscle attachment sites and the apical cell junctions of the imaginal disk epithelium. Evidence for abl function was obtained by analysis of mutant phenotypes in the embryonic somatic muscles and the eye imaginal disk. The expression patterns and mutant phenotypes indicate a role for abl in establishing and maintaining cell-cell interactions.

TGF-beta family factors in Drosophila morphogenesis.

Many Drosophila genes have now been identified with substantial sequence similarity to vertebrate protooncogenes and growth factors. Some of these have been isolated directly by cross-hybridization with vertebrate probes and some have been recognized in the sequences of genes cloned because of their intiguing mutant phenotypes. An example of a gene isolated for its interesting development functions but with homology to a vertebrate growth factor is the Drosophila decapentaplegic gene (dpp). An example of a Drosophila gene isolated by virtue of its sequence conservation is the vgr/60A gene. Both dpp and vgr/60A are members of the transforming growth factor-beta family and are most similar to the human bone morphogenetic proteins. The regulation of the dpp gene by several different groups of pattern formation genes including the dorsal/ventral group, the terminal group, the segment polarity genes, and the homeotic genes indicates that many events in embryogenesis require the cell to cell communication mediated by the secreted dpp protein. The temporal and spatial pattern of vgr/60A expression differs from that of dpp indicating that it may be regulated by different pattern information genes. The experimental advantages of the Drosophila system should permit a better understanding of the importance of growth factor homologs in specific developmental events, aid in establishing the functional interactions between these regulatory molecules, and identify new genes that are important for the biological functions of growth factors. It is likely that some of the newly identified genes will have vertebrate homologs and the analysis of these may be helpful in studies on vertebrate development and tumor biology.

Sequence, biochemical characterization, and developmental expression of a new member of the TGF-beta superfamily in Drosophila melanogaster.

More than 20 members of the transforming growth factor-beta (TGF-beta) superfamily of growth and differentiation factors have been implicated in development. One member of the TGF-beta family has been previously reported from Drosophila, the decapentaplegic (dpp) gene which is involved in embryonic dorsal/ventral polarity, embryonic gut formation, and imaginal disk development. Using PCR methods, we have identified a second Drosophila gene in the TGF-beta family. It encodes a protein product that is more similar to the TGF-beta-related human bone morphogenetic proteins (BMPs) 5, 6, and 7 than it is to the Drosophila dpp gene product. Because of its localization on the polytene chromosome map, we refer to this gene as 60A. Expression of a 60A cDNA in Drosophila S2 cells was used to determine that 60A encodes a preproprotein that is processed to yield secreted amino- and carboxy-terminal polypeptides. The carboxy-terminal peptides are recovered as disulfide-linked homodimers. The 60A transcripts and protein are first detected at the onset of gastrulation, primarily in the mesoderm of the extending germ band. As the germ band retracts, and throughout later stages of embryonic development, the 60A transcript and protein are most readily detected in cells of the developing foregut and hindgut.

Learning about cancer genes through invertebrate genetics.

Genetic studies in yeast, nematodes and Drosophila are revealing the signal transduction pathways that regulate differentiation and cell proliferation. Some of the critical molecules involved are homologous to proto-oncogenes and others are likely to be analogous to the products of tumor suppressor genes.

Drosophila abl and genetic redundancy in signal transduction.

Genetic studies on Drosophila Abl and, more recently, on mouse c-Abl and c-Src indicate that the functions of these non-receptor tyrosine kinases may duplicate activities of other molecules within signal transduction pathways. In Drosophila, second-site mutations have been recovered that disrupt the redundant functions so that the Abl tyrosine kinase is essential to the formation of axonal connections in the embryonic central nervous system and for attachment of embryonic muscles to the body wall. Molecular isolation and analysis of the genes identified by these second-site mutations should define the molecular basis for the genetic redundancy.

Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos.

During Drosophila embryogenesis homeotic genes control the developmental diversification of body structures. The genes probably coordinate the expression of as yet unidentified target genes that carry out cell differentiation processes. At least four homeotic genes expressed in the visceral mesoderm are required for midgut morphogenesis. In addition, two growth factor homologs are expressed in specific regions of the visceral mesoderm surrounding the midgut epithelium. One of these, decapentaplegic (dpp), is a member of the transforming growth factor beta (TGF-beta) family; the other, wingless (wg), is a relative of the mammalian proto-oncogene int-1. Here we show that the spatially restricted expression of dpp in the visceral mesoderm is regulated by the homeotic genes Ubx and abd-A. Ubx is required for the expression of dpp while abd-A represses dpp. One consequence of dpp expression is the induction of labial (lab) in the underlying endoderm cells. In addition, abd-A function is required for the expression of wg in the visceral mesoderm posterior to the dpp-expressing cells. The two growth factor genes therefore are excellent candidates for target genes that are directly regulated by the homeotic genes.

Molecular organization of the decapentaplegic gene in Drosophila melanogaster.

The decapentaplegic (dpp) locus of Drosophila melanogaster is a greater than 55 kb genetic unit required for proper pattern formation during the embryonic and imaginal development of the organism. We have proposed that these morphogenetic functions result from the action of a secreted transforming growth factor-beta (TGF-beta)-related protein product encoded by dpp. In this paper we localize 60 mutations on the molecular map of dpp. The positions of these mutations cluster according to phenotypic class, identifying the locations of specific dpp functions. By Northern and cDNA analysis, we characterize five overlapping dpp transcripts. On the basis of the locations of the overlaps relative to a previously sequenced cDNA, it is likely that these transcripts all encode similar or identical polypeptides. We propose that the bulk of dpp DNA consists of extensive arrays of cis-regulatory information. The large (greater than 25-kb) 3' cis-regulatory region represents a novel feature of dpp gene organization