The transmembrane Semaphorin Sema I is required in Drosophila for embryonic motor and CNS axon guidance

Parasite-derived antioxidant proteins have been implicated in playing an important role in protection against the oxygen radicals that are generated during aerobic metabolism and in defense against host immune cell attack. Here we report that filarial nematodes include the thioredoxin peroxidase/thiol-specific antioxidant (TPx/TSA) family of antioxidant proteins as part of their complex defense against radical-mediated damage. At the protein level, the TPx/TSA from Brugia malayi (Bm-TPx-1) was approximately 50% identical and approximately 60% similar to TPx/TSAs from mammals, amphibians and yeast. Bm-TPx-1 was also approximately 60% identical to putative TPx proteins from a related filarial nematode, Onchocerca volvulus, and from the free-living nematode Caenorhabditis elegans. That B. malayi may express multiple forms of molecules with TPx/TSA activity was indicated by the identification of a B. malayi gene encoding a second, distinct member of the TPx/TSA family (Bm-tpx-2). Bm-tpx-1 was found to be transcribed in all stages of the parasite present in the mammalian host and the 25 kDa translation product was present in all of the developmental stages studied. The results of immunohistochemical, immunofluorescent and immunoprecipitation studies showed Bm-TPx-1 to be localized in the cells of the hypodermis/lateral chord in adult parasites and not to be present at the surface or in excretory/secretory products. The distribution in the parasite suggests that Bm-TPx-1 may play its major role in countering radicals produced within cells. A recombinant form of Bm-TPx-1 was biologically active and capable of protecting DNA from oxygen radical-mediated damage. Thioredoxin peroxidases may prove to be a critical component in the parasite's defense against injury caused by oxygen radicals derived from endogenous and exogenous sources.     

The trithorax group gene moira encodes a brahma-associated putative chromatin-remodeling factor in Drosophila melanogaster

The genes of the trithorax group (trxG) in Drosophila melanogaster are required to maintain the pattern of homeotic gene expression that is established early in embryogenesis by the transient expression of the segmentation genes. The precise role of each of the diverse trxG members and the functional relationships among them are not well understood. Here, we report on the isolation of the trxG gene moira (mor) and its molecular characterization. mor encodes a fruit fly homolog of the human and yeast chromatin-remodeling factors BAF170, BAF155, and SWI3. mor is widely expressed throughout development, and its 170-kDa protein product is present in many embryonic tissues. In vitro, MOR can bind to itself and it interacts with Brahma (BRM), an SWI2-SNF2 homolog, with which it is associated in embryonic nuclear extracts. The leucine zipper motif of MOR is likely to participate in self-oligomerization; the equally conserved SANT domain, for which no function is known, may be required for optimal binding to BRM. MOR thus joins BRM and Snf5-related 1 (SNR1), two known Drosophila SWI-SNF subunits that act as positive regulators of the homeotic genes. These observations provide a molecular explanation for the phenotypic and genetic relationships among several of the trxG genes by suggesting that they encode evolutionarily conserved components of a chromatin-remodeling complex.     

The Drosophila peanut gene is required for cytokinesis and encodes a protein similar to yeast putative bud neck filament proteins

We have identified a Drosophila gene, peanut (pnut), that is related in sequence to the CDC3, CDC10, CDC11, and CDC12 genes of S. cerevisiae. These genes are required for cytokinesis, and their products are present at the bud neck during cell division. We find that pnut is also required for cytokinesis: in pnut mutants, imaginal tissues fail to proliferate and instead develop clusters of large, multinucleate cells. Pnut protein is localized to the cleavage furrow of dividing cells during cytokinesis and to the intercellular bridge connecting postmitotic daughter cells. In addition to its role in cytokinesis, pnut displays genetic interactions with seven in absentia, a gene required for neuronal fate determination in the compound eye, suggesting that pnut may have pleiotropic functions. Our results suggest that this class of proteins is involved in aspects of cytokinesis that have been conserved between flies and yeast.     

The PHANTASTICA gene encodes a MYB transcription factor involved in growth and dorsoventrality of lateral organs in Antirrhinum

To study the impact of yeast populations on wine flavour and to better understand yeast growth dynamics, wines were produced by the (i) indigenous microflora, (ii) vigorous yeast starter EC1118 and (iii) slowly fermenting yeast Assmannshausen. Sensory analysis revealed that wines differed depending on the fermentation type. However, these yeast-related differences did not exceed the varietal character. Both added starter cultures clearly dominated the Saccharomyces population from the middle of fermentation onwards. The starter cultures differed in their repression of indigenous non-Saccharomyces yeast. EC1118 limited growth of non-Saccharomyces yeasts more strongly than Assmannshausen. Sulphite addition further repressed growth of non-Saccharomyces yeasts. On completion, more than one Saccharomyces strain was present in each fermentation, with the largest variety in the non-inoculated and the smallest in the EC1118-inoculated fermentation. Results from the two genetic assays, karyotyping, and PCR using delta-primers were not fully equivalent, limiting the usefulness of delta-PCR in studies of native Saccharomyces yeasts.     

The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal

Smoothened (smo) is a segment polarity gene required for correct patterning of every segment in Drosophila. The earliest defect in smo mutant embryos is loss of expression of the Hedgehog-responsive gene wingless between 1 and 2 hr after gastrulation. Since smo mutant embryos cannot respond to exogenous Hedgehog (Hh) but can respond to exogenous Wingless, the smo product functions in Hh signaling. Smo acts downstream of or in parallel to Patched, an antagonist of the Hh signal. The smo gene encodes an integral membrane protein with characteristics of G protein-coupled receptors and shows homology to the Drosophila Frizzled protein. Based on its predicted physical characteristics and on its position in the Hh signaling pathway, we suggest that smo encodes a receptor for the Hh signal.     

Genetic analysis on the role of integrin during axon guidance in Drosophila

Heterodimeric cell surface receptor integrin is widely expressed in the nervous system, but its specific role during axon development has not been directly tested in vivo. We show that the Drosophila nervous system expresses low levels of positron-specific (PS) integrin subunits alphaPS1, alphaPS2, and betaPS during embryonic axogenesis. Furthermore, certain subsets of neurons express higher levels of integrin mRNAs than do the rest. Null mutations in either the alphaPS1 or alphaPS2 subunit gene cause widespread axon pathfinding errors that can be rescued by supplying the wild-type integrin subunit to the mutant nervous system. In contrast, misexpressing either the alphaPS1 or alphaPS2 integrin subunit in all neurons leads to no obvious axon pathfinding errors. We propose that integrin does not itself serve as either a "clutch" constituting molecule or a specific growth cone "receptor," as proposed previously, but rather as part of a molecular network that cooperatively guarantees accurate axon guidance.     

The Drosophila maternal effect locus deadhead encodes a thioredoxin homolog required for female meiosis and early embryonic development

This study describes the identification, function and molecular characterization of deadhead, a Drosophila thioredoxin homolog. Although in vitro studies have shown that thioredoxin can post-translationally regulate the activity of many different proteins, we find that this homolog is not essential for viability. The phenotypic analysis of two different mutations which eliminate function suggests that dhd is essential for female meiosis. The majority of eggs laid by females homozygous for null mutations are fertilized but fail to complete meiosis. A small number of escaper embryos initiate development and display a range of phenotypes suggesting functions in both preblastoderm mitosis and head development. Our analysis of deadhead's RNA expression pattern is consistent with its maternal effect function: the RNA is predominately expressed in the nurse cells of the ovary, is maternally deposited into the egg, but does not appear to be zygotically expressed during embryogenesis. Thus both our genetic and molecular data are consistent with a function during meiosis and preblastoderm mitosis. Whether the head defect indicates an additional function or is an indirect consequence of earlier defects remains to be determined.     

muscleblind, a gene required for photoreceptor differentiation in Drosophila, encodes novel nuclear Cys3His-type zinc-finger-containing proteins

We have isolated the embryonic lethal gene muscleblind (mbl) as a suppressor of the sev-svp2 eye phenotype. Analysis of clones mutant for mbl during eye development shows that it is autonomously required for photoreceptor differentiation. Mutant cells are recruited into developing ommatidia and initiate neural differentiation, but they fail to properly differentiate as photoreceptors. Molecular analysis reveals that the mbl locus is large and complex, giving rise to multiple different proteins with common 5' sequences but different carboxy termini. Mbl proteins are nuclear and share a Cys3His zinc-finger motif which is also found in the TIS11/NUP475/TTP family of proteins and is highly conserved in vertebrates and invertebrates. Functional analysis of mbl, the observation that it also dominantly suppresses the sE-Jun(Asp) gain-of-function phenotype and the phenotypic similarity to mutants in the photoreceptor-specific glass gene suggest that mbl is a general factor required for photoreceptor differentiation.     

Clonal analysis of cmp44E, which encodes a conserved putative transmembrane protein, indicates a requirement for cell viability in Drosophila

We have identified the cmp44E gene which encodes a putative multi-pass transmembrane protein that is conserved from yeast to humans. The expression of cmp44E during embryogenesis is ubiquitous with notably higher levels in the CNS and brain. It is also expressed in the germline during the germarial stages as well as several later stages of oogenesis. Utilizing a P-element insertion at the 5' end of cmp44E we have isolated several deletions, created by imprecise excision events which eliminate most or all of its coding region. Analysis of these deficiencies has revealed that cmp44E is an essential gene required for embryogenesis. Results obtained from germline clone analysis indicate that cmp44E is not only required in the germline slem cells early in oogenesis, but is also required in other tissues probably due to it being required for cell viability. Finally, using germline transformation, we have identified a minimal genomic fragment capable of fully rescuing a null allele of cmp44E.     

crooked legs encodes a family of zinc finger proteins required for leg morphogenesis and ecdysone-regulated gene expression during Drosophila metamorphosis

Drosophila imaginal discs undergo extensive pattern formation during larval development, resulting in each cell acquiring a specific adult fate. The final manifestation of this pattern into adult structures is dependent on pulses of the steroid hormone ecdysone during metamorphosis, which trigger disc eversion, elongation and differentiation. We have defined genetic criteria that allow us to screen for ecdysone-inducible regulatory genes that are required for this transformation from patterned disc to adult structure. We describe here the first genetic locus isolated using these criteria: crooked legs (crol). crol mutants die during pupal development with defects in adult head eversion and leg morphogenesis. The crol gene is induced by ecdysone during the onset of metamorphosis and encodes at least three protein isoforms that contain 12-18 C2H2 zinc fingers. Consistent with this sequence motif, crol mutations have stage-specific effects on ecdysone-regulated gene expression. The EcR ecdysone receptor, and the BR-C, E74 and E75 early regulatory genes, are submaximally induced in crol mutants in response to the prepupal ecdysone pulse. These changes in gene activity are consistent with the crol lethal phenotypes and provide a basis for understanding the molecular mechanisms of crol action. The genetic criteria described here provide a new direction for identifying regulators of adult tissue development during insect metamorphosis.     

The Drosophila Medea gene is required downstream of dpp and encodes a functional homolog of human Smad4

The Transforming Growth Factor-beta superfamily member decapentaplegic (dpp) acts as an extracellular morphogen to pattern the embryonic ectoderm of the Drosophila embryo. To identify components of the dpp signaling pathway, we screened for mutations that act as dominant maternal enhancers of a weak allele of the dpp target gene zerkn?llt. In this screen, we recovered new alleles of the Mothers against dpp (Mad) and Medea genes. Phenotypic analysis of the new Medea mutations indicates that Medea, like Mad, is required for both embryonic and imaginal disc patterning. Genetic analysis suggests that Medea may have two independently mutable functions in patterning the embryonic ectoderm. Complete elimination of maternal and zygotic Medea activity in the early embryo results in a ventralized phenotype identical to that of null dpp mutants, indicating that Medea is required for all dpp-dependent signaling in embryonic dorsal-ventral patterning. Injection of mRNAs encoding DPP or a constitutively activated form of the DPP receptor, Thick veins, into embryos lacking all Medea activity failed to induce formation of any dorsal cell fates, demonstrating that Medea acts downstream of the thick veins receptor. We cloned Medea and found that it encodes a protein with striking sequence similarity to human SMAD4. Moreover, injection of human SMAD4 mRNA into embryos lacking all Medea activity conferred phenotypic rescue of the dorsal-ventral pattern, demonstrating conservation of function between the two gene products.     

Signalling by the Drosophila epidermal growth factor receptor is required for the specification and diversification of embryonic muscle progenitors

Muscle development initiates in the Drosophila embryo with the segregation of single progenitor cells, from which a complete set of myofibres arises. Each progenitor is assigned a unique fate, characterized by the expression of particular identity genes. We now demonstrate that the Drosophila epidermal growth factor receptor provides an inductive signal for the specification of a large subset of muscle progenitors. In the absence of the receptor or its ligand, SPITZ, specific progenitors fail to segregate. The resulting unspecified mesodermal cells undergo programmed cell death. In contrast, receptor hyperactivation generates supernumerary progenitors, as well as the duplication of at least one SPITZ-dependent myofibre. The development of individual muscles is differentially sensitive to variations in the level of signalling by the epidermal growth factor receptor. Such graded myogenic effects can be influenced by alterations in the functions of Star and rhomboid. In addition, muscle patterning is dependent on the generation of a spatially restricted, activating SPITZ signal, a process that may rely on the localized mesodermal expression of RHOMBOID. Thus, the epidermal growth factor receptor contributes both to muscle progenitor specification and to the diversification of muscle identities.     

windbeutel, a gene required for dorsoventral patterning in Drosophila, encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum

The formation of the dorsoventral axis of the Drosophila embryo depends on cell-cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The gene windbeutel functions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis of windbeutel. We have found that windbeutel encodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.