Amanda Simcox

minidiscs encodes a putative amino acid transporter subunit required non-autonomously for imaginal cell proliferation.

Drosophila minidiscs mutant larvae have smaller imaginal discs than wild-type larvae. However, transplantation experiments have revealed that minidiscs mutant imaginal discs can grow if cultured in non-mutant hosts. These data suggest that minidiscs is required in one or more non-imaginal tissues for synthesis and/or secretion of a diffusible factor that stimulates imaginal cell proliferation. The 2. 3 kb minidiscs transcript accumulates in the larval fat body and encodes a protein containing 12 putative membrane spanning domains that is similar in sequence to amino acid transporter subunits from other eukaryotes, including humans. We propose that in response to amino acid uptake by the transporter encoded by minidiscs, the fat body secretes a diffusible factor required for imaginal disc proliferation.

Genome-wide activities of Polycomb complexes control pervasive transcription

Polycomb group (PcG) complexes PRC1 and PRC2 are well known for silencing specific developmental genes. PRC2 is a methyltransferase targeting histone H3K27 and producing H3K27me3, essential for stable silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates ∼70% of total H3K27. We show that H3K27me2 occurs in inverse proportion to transcriptional activity in most non-PcG target genes and intergenic regions and is governed by opposing roaming activities of PRC2 and complexes containing the H3K27 demethylase UTX. Surprisingly, loss of H3K27me2 results in global transcriptional derepression proportionally greatest in silent or weakly transcribed intergenic and genic regions and accompanied by an increase of H3K27ac and H3K4me1. H3K27me2 therefore sets a threshold that prevents random, unscheduled transcription all over the genome and even limits the activity of highly transcribed genes. PRC1-type complexes also have global roles. Unexpectedly, we find a pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub) outside of canonical PcG target regions, dependent on the RING/Sce subunit of PRC1-type complexes. We show, however, that H2AK118ub does not mediate the global PRC2 activity or the global repression and is predominantly produced by a new complex involving L(3)73Ah, a homolog of mammalian PCGF3.

Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling

Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.

Differential requirement for EGF-like ligands in Drosophila wing development

Signaling through the Drosophila EGF receptor (DER) is important for the growth and differentiation of the wing. These processes may be mediated by different DER ligands including Spitz (Spi) and Vein (Vn). Here I investigate the roles of these ligands and other DER pathway components in wing disc development using in vivo culture to produce mutant discs from genotypes which are normally embryonic lethal. I find no role for spi in wing disc growth, whereas vn is essential. spi mutant wing discs are morphologically normal as judged by expression of the vein marker rhomboid (rho) and analysis of the differentiated wing tissue. rho, Star (S) and argos (aos) which are known to be involved in Spi/DER signaling are likewise not required for wing growth, whereas pointed (pnt), which acts at the end of the intracellular pathway, is required. The results suggest different ligands and molecular mechanisms control DER signaling in wing growth and differentiation.

Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype.

Changes in myofibrillar structure and function produced by N-terminal deletion of the regulatory light chain in Drosophila

The similarity of amino acid sequence and motifs of the N-terminal extensions of certain class II myosin light chains, found throughout the animal kingdom, suggest a common functional role. One possible role of the N-terminal extension is to enhance oscillatory work and power production in striated muscles that normally operate in an oscillatory mode. We conducted small-angle X-ray diffraction experiments and small-length-perturbation analysis to examine the structural and functional consequences of deleting the N-terminal extension of the myosin regulatory light chain (RLC) in Drosophila flight muscle. The in vivo lattice spacing of dorsal longitudinal muscle (DLM) of flies lacking the RLC N-terminal extension (Dmlc2Δ2–46) was ∼1 nm less than that of wild type (48.56 ± 0.02 nm). The myofilament lattice of detergent-treated, demembranated DLM swelled, with the Dmlc2Δ2–46 lattice expanding more than wild type and requiring roughly twice the concentration of Dextran T500 to restore its lattice to in vivo spacing (9–10% vs. 4% w/v). The calcium sensitivity and maximum amplitude of net oscillatory work near the in vivo lattice spacing was significantly lower in Dmlc2Δ2–46 compared to wild type (pCa50 shifted by approximately one-third of a pCa unit; amplitude reduced by approximately one-half). These changes were in contrast to the lack of effect reported in a previous study carried out in the absence of Dextran T500. The results are consistent with the N-terminal extension interacting with actin to increase the probability that crossbridges form during stretch-activated oscillatory work and power production, especially at submaximal levels of calcium activation.

Dpp-induced Egfr signaling triggers postembryonic wing development in Drosophila

The acquisition of flight contributed to the success of insects and winged forms are present in most orders. Key to understanding the origin of wings will be knowledge of the earliest postembryonic events promoting wing outgrowth. The Drosophila melanogaster wing is intensely studied as a model appendage, and yet little is known about the beginning of wing outgrowth. Vein (Vn) is a neuregulin-like ligand for the EGF receptor (Egfr), which is necessary for global development of the early Drosophila wing disc. vn is not expressed in the embryonic wing primordium and thus has to be induced de novo in the nascent larval wing disc. We find that Decapentaplegic (Dpp), a Bone Morphogenetic Protein (BMP) family member, provides the instructive signal for initiating vn expression. The signaling involves paracrine communication between two epithelia in the early disc. Once initiated, vn expression is amplified and maintained by autocrine signaling mediated by the E-twenty six (ETS)-factor PointedP2 (PntP2). This interplay of paracrine and autocrine signaling underlies the spatial and temporal pattern of induction of Vn/Egfr target genes and explains both body wall development and wing outgrowth. It is possible this gene regulatory network governing expression of an EGF ligand is conserved and reflects a common origin of insect wings.

New Negative Feedback Regulators of Egfr Signaling in Drosophila

The highly conserved epidermal growth factor receptor (Egfr) pathway is required in all animals for normal development and homeostasis; consequently, aberrant Egfr signaling is implicated in a number of diseases. Genetic analysis of Drosophila melanogaster Egfr has contributed significantly to understanding this conserved pathway and led to the discovery of new components and targets. Here we used microarray analysis of third instar wing discs, in which Egfr signaling was perturbed, to identify new Egfr-responsive genes. Upregulated transcripts included five known targets, suggesting the approach was valid. We investigated the function of 29 previously uncharacterized genes, which had pronounced responses. The Egfr pathway is important for wing-vein patterning and using reverse genetic analysis we identified five genes that showed venation defects. Three of these genes are expressed in vein primordia and all showed transcriptional changes in response to altered Egfr activity consistent with being targets of the pathway. Genetic interactions with Egfr further linked two of the genes, Sulfated (Sulf1), an endosulfatase gene, and CG4096, an A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS) gene, to the pathway. Sulf1 showed a strong genetic interaction with the neuregulin-like ligand vein (vn) and may influence binding of Vn to heparan-sulfated proteoglycans (HSPGs). How Drosophila Egfr activity is modulated by CG4096 is unknown, but interestingly vertebrate EGF ligands are regulated by a related ADAMTS protein. We suggest Sulf1 and CG4096 are negative feedback regulators of Egfr signaling that function in the extracellular space to influence ligand activity.

Analysis of cDNAs encoding Drosophila melanogaster myosin light chain kinase

Myosin light chain kinase regulates the activity of myosin by phosphorylating the myosin regulatory light chain. Here we describe the cloning and characterization of cDNAs encoding Drosophila melanogaster myosin light chain kinase. We amplified a fragment of the Drosophila mlck gene using degenerate primers homologous to a highly conserved region in myosin light chain kinase proteins of vertebrate species. We used the gene fragment to isolate corresponding Drosophila mlck cDNAs. The deduced protein sequence of the cDNAs shows high homology to the catalytic and regulatory domains of vertebrate nonmuscle, smooth muscle and skeletal muscle myosin light chain kinase. Protein motifs I and II, which are present in vertebrate nonmuscle and smooth muscle myosin light chain kinase, but not in skeletal muscle myosin light chain kinase, are also present in Drosophila myosin light chain kinase. Transcript and cDNA analysis shows the gene encodes multiple messages and is expressed in non muscle and muscle cells, including the adult indirect flight muscle. Genomic Southern analysis and chromosome hybridization suggest mlck is a single copy gene which maps to chromosome band 52D, and is not haplo-insufficient for flight

Functional Analysis of Genes Differentially Expressed in the Drosophila Wing Disc: Role of Transcripts Enriched in the Wing Region

Differential gene expression is the major mechanism underlying the development of specific body regions. Here we assessed the role of genes differentially expressed in the Drosophila wing imaginal disc, which gives rise to two distinct adult structures: the body wall and the wing. Reverse genetics was used to test the function of uncharacterized genes first identified in a microarray screen as having high levels of expression in the presumptive wing. Such genes could participate in elaborating the specific morphological characteristics of the wing. The activity of the genes was modulated using misexpression and RNAi-mediated silencing. Misexpression of eight of nine genes tested caused phenotypes. Of 12 genes tested, 10 showed effective silencing with RNAi transgenes, but only 3 of these had resulting phenotypes. The wing phenotypes resulting from RNAi suggest that CG8780 is involved in patterning the veins in the proximal region of the wing blade and that CG17278 and CG30069 are required for adhesion of wing surfaces. Venation and apposition of the wing surfaces are processes specific to wing development providing a correlation between the expression and function of these genes. The results show that a combination of expression profiling and tissue-specific gene silencing has the potential to identify new genes involved in wing development and hence to contribute to our understanding of this process. However, there are both technical and biological limitations to this approach, including the efficacy of RNAi and the role that gene redundancy may play in masking phenotypes.