Apterous is a Drosophila LIM domain gene required for the development of a subset of embryonic muscles.

The recently discovered LIM motif is found in a set of homeodomain-containing proteins thought to mediate the generation of particular cell types. Of the four LIM domain family members described to date, mec-3 and lin-11 determine cell lineages in C. elegans. Isl-1 and Xlim-1 may play similar roles in vertebrates. We have identified a Drosophila member of this class, the product of the apterous (ap) gene. During embryogenesis, ap is expressed in a small subset of fusing mesodermal precursors that give rise to 6 muscles in each abdominal hemisegment and in 5 neurons within each corresponding CNS hemisegment. Lack of ap function results in loss of ap-expressing muscles, while misexpression of ap using a heterologous promoter produces ectopic muscles.     

Drosophila ERCC1 is required for a subset of MEI-9-dependent meiotic crossovers

Drosophila MEI-9 is the catalytic subunit of a DNA structure-specific endonuclease required for nucleotide excision repair (NER). The enzymatic activity of this endonuclease during NER requires the presence of a second, noncatalytic subunit called ERCC1. In addition to its role in NER, MEI-9 is required for the generation of most meiotic crossovers. To better understand the role of MEI-9 in crossover formation, we report here the characterization of the Drosophila Ercc1 gene. We created an Ercc1 mutant through homologous gene targeting. We find that Ercc1 mutants are identical to mei-9 mutants in sensitivity to DNA-damaging agents, but have a less severe reduction in the number of meiotic crossovers. MEI-9 protein levels are reduced in Ercc1 mutants; however, overexpression of MEI-9 is not sufficient to restore meiotic crossing over in Ercc1 mutants. We conclude that MEI-9 can generate some meiotic crossovers in an ERCC1-independent manner.     

Small wing PLCgamma is required for ER retention of cleaved Spitz during eye development in Drosophila

The Drosophila EGF receptor ligand Spitz is cleaved by Rhomboid to generate an active secreted molecule. Surprisingly, when a cleaved variant of Spitz (cSpi) was expressed, it accumulated in the ER, both in embryos and in cell culture. A cell-based RNAi screen for loss-of-function phenotypes that alleviate ER accumulation of cSpi identified several genes, including the small wing (sl) gene encoding a PLCgamma. sl mutants compromised ER accumulation of cSpi in embryos, yet they exhibit EGFR hyperactivation phenotypes predominantly in the eye. Spi processing in the eye is carried out primarily by Rhomboid-3/Roughoid, which cleaves Spi in the ER, en route to the Golgi. The sl mutant phenotype is consistent with decreased cSpi retention in the R8 cells. Retention of cSpi in the ER provides a novel mechanism for restricting active ligand levels and hence the range of EGFR activation in the developing eye.     

Calcineurin function is required for myofilament formation and troponin I isoform transition in Drosophila indirect flight muscle

Mutations in the Drosophila calcineurin B2 gene cause the collapse of indirect flight muscles during mid stages of pupal development. Examination of cell fate-specific markers indicates that unlike mutations in genes such as vestigial, calcineurin B2 does not cause a shift in cell fate from indirect flight muscle to direct flight muscle. Genetic and molecular analyses indicate a severe reduction of myosin heavy chain gene expression in calcineurin B2 mutants, which accounts at least in part for the muscle collapse. Myofibrils in calcineurin B2 mutants display a variety of phenotypes, ranging from normal to a lack of sarcomeric structure. Calcineurin B2 also plays a role in the transition to an adult-specific isoform of troponin I during the late pupal stages, although the incompleteness of this transition in calcineurin B2 mutants does not contribute to the phenotype of muscle collapse. Together, these findings suggest a molecular basis for the indirect flight muscle hypercontractility phenotype observed in flies mutant for Drosophila calcineurin B2.     

The EGFR ligands Spitz and Keren act cooperatively in the Drosophila eye

The EGFR signalling cascade is responsible for coordinating a wide variety of events during Drosophila eye development. It remains something of a mystery how it is that cells are able to interpret the signal so as to choose the appropriate response from the battery of possibilities: division, differentiation, cell shape change and so on. Since the cascade is essentially linear below the receptor, different cellular responses cannot be regulated by alternative signal transduction pathways. The main diversity lies upstream, in the multiple activating ligands. Spitz, Gurken and Vein have been long studied, but little is known about the physiological functions of the fourth ligand, Keren, although various roles have been predicted based on the differences between mutants in the known ligands and those of the receptor. Here, we have isolated a mutant in the keren gene, and demonstrate that Keren does indeed participate in EGFR signalling in the eye, where it acts redundantly with Spitz to control R8 spacing, cell clustering and survival. Thus, specificity cannot be determined by ligand choice, and must instead be a consequence of cell-intrinsic factors, although we speculate that there may be some quantitative differences in signalling elicited by the two ligands.     

The heterotrimeric protein Go is required for the formation of heart epithelium in Drosophila.

The gene encoding the alpha subunit of the Drosophila Go protein is expressed early in embryogenesis in the precursor cells of the heart tube, of the visceral muscles, and of the nervous system. This early expression coincides with the onset of the mesenchymal-epithelial transition to which are subjected the cardial cells and the precursor cells of the visceral musculature. This gene constitutes an appropriate marker to follow this transition. In addition, a detailed analysis of its expression suggests that the cardioblasts originate from two subpopulations of cells in each parasegment of the dorsal mesoderm that might depend on the wingless and hedgehog signaling pathways for both their determination and specification. In the nervous system, the expression of Goalpha shortly precedes the beginning of axonogenesis. Mutants produced in the Goalpha gene harbor abnormalities in the three tissues in which the gene is expressed. In particular, the heart does not form properly and interruptions in the heart epithelium are repeatedly observed, henceforth the brokenheart (bkh) name. Furthermore, in the bkh mutant embryos, the epithelial polarity of cardial cells was not acquired (or maintained) in various places of the cardiac tube. We predict that bkh might be involved in vesicular traffic of membrane proteins that is responsible for the acquisition of polarity.     

Feo, the Drosophila homolog of PRC1, is required for central-spindle formation and cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.     

The Drosophila SHC adaptor protein is required for signaling by a subset of receptor tyrosine kinases

Receptor tyrosine kinases (RTKs) transduce signals via cytoplasmic adaptor proteins to downstream signaling components. We have identified loss-of-function mutations in dshc, the Drosophila homolog of the mammalian adaptor protein SHC. A point mutation in the phosphotyrosine binding (PTB) domain completely abolishes DSHC function and provides in vivo evidence for the function of PTB domains. Unlike other adaptor proteins, DSHC is involved in signaling by only a subset of RTKs: dshc mutants show defects in Torso and DER but not Sevenless signaling, which is confirmed by epistasis experiments. We show by double-mutant analysis that the adaptors DOS, DRK, and DSHC act in parallel to transduce the Torso signal. Our results suggest that DSHC confers specificity to receptor signaling.     

The cyclophilin homolog ninaA is a tissue-specific integral membrane protein required for the proper synthesis of a subset of Drosophila rhodopsins.

Mutations in the Drosophila ninaA gene cause dramatic reductions in rhodopsin levels, leading to impaired visual function. The ninaA protein is a homolog of peptidyl-prolyl cis-trans isomerases. We find that ninaA is unique among this family of proteins in that it is an integral membrane protein, and it is expressed in a cell type-specific manner. We have used transgenic animals misexpressing different rhodopsins in the major class of photoreceptor cells to demonstrate that ninaA is required for normal function by two homologous rhodopsins, but not by a less conserved member of the Drosophila rhodopsin gene family. This demonstrates in vivo substrate specificity in a cyclophilin-like molecule. We also show that vertebrate retina contains a ninaA-related protein and that ninaA is a member of a gene family in Drosophila. These data offer insights into the in vivo role of this important family of proteins.     

Drosophila fasciclinII is required for the formation of odor memories and for normal sensitivity to alcohol.

Drosophila fasciclinII (fasII) mutants perform poorly after olfactory conditioning due to a defect in encoding, stabilizing, or retrieving short-term memories. Performance was rescued by inducing the expression of a normal transgene just before training and immediate testing. Induction after training but before testing failed to rescue performance, showing that Fas II does not have an exclusive role in memory retrieval processes. The stability of odor memories in fasII mutants are indistinguishable from control animals when initial performance is normalized. Like several other mutants deficient in odor learning, fasII mutants exhibit a heightened sensitivity to ethanol vapors. A combination of behavioral and genetic strategies have therefore revealed a role for Fas II in the molecular operations of encoding short-term odor memories and conferring alcohol sensitivity. The preferential expression of Fas II in the axons of mushroom body neurons furthermore suggests that short-term odor memories are formed in these neurites.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Star is required in a subset of photoreceptor cells in the developing Drosophila retina and displays dosage sensitive interactions with rough.

We report that mutations at the Star locus act as dominant enhancers of the eye phenotype displayed by flies carrying a null allele of rough. Our analysis of double mutants at different stages of eye development suggests that this phenotype results from defects in the early stages of photoreceptor cell differentiation in the eye imaginal disc. Complete loss of Star function during retinal development, analyzed in mosaic animals, results in cell death, visible as scars in the adult eye. The requirement for wild-type Star function, however, is confined to only a subset of photoreceptor cells, R8, R2, and R5, which are the first three cells to differentiate neurally in the developing retina. These results suggest an essential role for the Star gene in the initial events of ommatidial cluster formation during the development of the Drosophila compound eye.     

Drosophila mind bomb2 is required for maintaining muscle integrity and survival

We report that the Drosophila mind bomb2 (mib2) gene is a novel regulator of muscle development. Unlike its paralogue, mib1, zygotic expression of mib2 is restricted to somatic and visceral muscle progenitors, and their respective differentiated musculatures. We demonstrate that in embryos that lack functional Mib2, muscle detachment is observed beginning in mid stage 15 and progresses rapidly, culminating in catastrophic degeneration and loss of most somatic muscles by stage 17. Notably, the degenerating muscles are positive for apoptosis markers, and inhibition of apoptosis in muscles prevents to a significant degree the muscle defects. Rescue experiments with Mib1 and Neuralized show further that these E3 ubiquitin ligases are not capable of ameliorating the muscle mutant phenotype of mib2. Our data suggest strongly that mib2 is involved in a novel Notch- and integrin-independent pathway that maintains the integrity of fully differentiated muscles and prevents their apoptotic degeneration.     

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. Here, we report for the first time that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae.     

The dachsous gene, a member of the cadherin family, is required for Wg-dependent pattern formation in the Drosophila wing disc

The dachsous (ds) gene encodes a member of the cadherin family involved in the non-canonical Wnt signaling pathway that controls the establishment of planar cell polarity (PCP) in Drosophila. ds is the only known cadherin gene in Drosophila with a restricted spatial pattern of expression in imaginal discs from early stages of larval development. In the wing disc, ds is first expressed distally, and later is restricted to the hinge and lateral regions of the notum. Flies homozygous for strong ds hypomorphic alleles display previously uncharacterized phenotypes consisting of a reduction of the hinge territory and an ectopic notum. These phenotypes resemble those caused by reduction of the canonical Wnt signal Wingless (Wg) during early wing disc development. An increase in Wg activity can rescue these phenotypes, indicating that Ds is required for efficient Wg signaling. This is further supported by genetic interactions between ds and several components of the Wg pathway in another developmental context. Ds and Wg show a complementary pattern of expression in early wing discs, suggesting that Ds acts in Wg-receiving cells. These results thus provide the first evidence for a more general role of Ds in Wnt signaling during imaginal development, not only affecting cell polarization but also modulating the response to Wg during the subdivision of the wing disc along its proximodistal (PD) axis.     

Auxilin is required for formation of Golgi-derived clathrin-coated vesicles during Drosophila spermatogenesis

Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway.