Gert O. Pflugfelder

Control of the Gene optomotor-blind in Drosophila Wing Development by decapentaplegic and wingless

Diffusible factors of several protein families control appendage outgrowth and patterning in both insects and vertebrates. In Drosophila wing development, the gene decapentaplegic (dpp) is expressed along the anteroposterior compartment boundary. Early wingless (wg) expression is involved in setting up the dorsoventral boundary. Interaction between dpp- and wg-expressing cells promotes appendage outgrowth. Here, it is shown that optomotor-blind (omb) expression is required for distal wing development and is controlled by both dpp and wg. Ectopic omb expression can lead to the growth of additional wings. Thus, omb is essential for wing development and is controlled by two signaling pathways.

A Cysteine-String Protein is Expressed in Retina and Brain of Drosophila

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.

The DrosDel Deletion Collection: A Drosophila Genomewide Chromosomal Deficiency Resource

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering ∼77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.

A homology domain shared between Drosophila optomotor-blind and mouse Brachyury is involved in DNA binding

The distribution of sequence elements divides the optomotor-blind protein into three regions and is suggestive of a transcriptional regulatory role of this protein. The central region of Omb is homologous to the N-terminal half of the Brachyury protein. The conserved domain of Omb is here shown to possess general DNA binding affinity but has no significant similarity to recognized DNA binding motifs.

A fluorescent core-shell dendritic macromolecule specifically stains the extracellular matrix

The extracellular cell matrix (ECM) surrounds cells and plays important roles in many aspects of cellular fate, including cell migration, stem cell differentiation, and cancer progression. So far, there is no fluorescent dye to directly visualize the ECM network. Here we present a positively charged fluorescent core-shell dendritic macromolecule containing multiple -NH2 groups which specifically binds to highly negatively charged ECM components. Due to its advantageous optical properties and biological specificity, the dye is useful as a routine tool to label the ECM in life science research.

Fluorescent Core/Shell Nanoparticles for Specific Cell-Nucleus Staining†

The highly fluorescent perylene-3,4,9,10-tetracarboxdiimide (PDI) chromophore is a popular dye and pigment because of its excellent chemical, thermal, and photochemical stability. Due to these outstanding properties, there have been several successful applications of PDI chromophores in various fields. Water-soluble and fluorescent PDI dyes have been used in biological applications such as the in vitro staining of cells and proteins. The combination of water solubility and high fluorescence quantum yield still represents a challenging goal since PDI dyes have a strong tendency to form aggregates in aqueous solution even at very low concentrations. Water solubility and high fluorescence quantum yields of ionic PDIs were obtained by introducing positively or negatively charged substituents into the bay region of the chromophore. Although these ionic PDIs penetrate the cytoplasmic membrane of living cells, the synthesis of PDIs with specific binding properties to subcellular compartments has not yet been achieved. Recently, the preparation of core/shell nanoparticles with high structural perfection, water-solubility, and biocompatibility has been reported. However, the synthesis and biological characterization of fluorescent core/shell nanoparticles with specific biological applications have not been demonstrated. Herein we describe a novel water-soluble, negatively charged PDI derivative that specifically labels the cell nucleus by strong binding to positively charged nuclear proteins, thus allowing their application as a fluorescent dye in pathological and histochemical studies. The core/shell nanoparticle (Figure 1, P1) consists of a central PDI chromophore, a rigid first-generation polyphenylene dendrimer scaffold for suppressing aggregation of the central PDI chromophore in aqueous media and a polymer shell with multiple carboxylic acid groups for inducing water solubility and biological specificity. The synthetic strategy towards P1 is shown in Scheme 1, starting from the previously

Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila

Patients with polyglutamine expansion diseases, like Huntingtons disease or several spinocerebellar ataxias, first present with neurological symptoms that can occur in the absence of neurodegeneration. Behavioral symptoms thus appear to be caused by neuronal dysfunction, rather than cell death. Pathogenesis in polyglutamine expansion diseases is largely viewed as a cell‐autonomous process in neurons. It is likely, however, that this process is influenced by changes in glial physiology and, at least in the case of DRPLA glial inclusions and glial cell death, seems to be an important part in the pathogenesis. To investigate these aspects in a Drosophila model system, we expressed polyglutamine proteins in the adult nervous system. Glial‐specific expression of a polyglutamine (Q)‐expanded (n = 78) and also a nonexpanded (n = 27) truncated version of human ataxin‐3 led to the formation of protein aggregates and glial cell death. Behavioral changes were observed prior to cell death. This reveals that glia is susceptible to the toxic action of polyglutamine proteins. Neuronal expression of the same constructs resulted in behavioral changes similar to those resulting from glial expression but did not cause neurodegeneration. Behavioral deficits were selective and affected two analyzed fly behaviors differently. Both glial and neuronal aggregates of Q78 and Q27 appeared early in pathogenesis and, at the electron microscopic resolution, had a fibrillary substructure. This shows that a nonexpanded stretch can cause similar histological and behavioral symptoms as the expanded stretch, however, with a significant delay.

Optomotor-blind of Drosophila melanogaster: a neurogenetic approach to optic lobe development and optomotor behaviour

The gene optomotor-blind (omb) plays a crucial role in Drosophila optic lobe development. Various mutations in omb lead to different structural defects in the adult optic lobes with correlated behavioural phenotypes. Molecular analysis of omb allows one to trace back behavioural defects to the spatio-temporal misexpression of the gene in mutant development.

Wide distribution of the cysteine string proteins in Drosophila tissues revealed by targeted mutagenesis.

Abstract The “cysteine string protein” (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a “J” domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the “tall cells” of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.

Isolation of a Drosophila T-box gene closely related to human TBX1

T-box genes, in all metazoans studied from nematode to man, exist in small gene families. They encode transcription factors with a novel, large, and highly conserved DNA binding domain termed the T-domain. In all cases studied, T-box genes have important developmental roles. Two familial diseases, Holt-Oram syndrome and ulnar-mammary syndrome, were recently shown to be caused by mutations in the human T-box genes TBX5 and TBX3, respectively. T-box genes were first identified in Drosophila and mouse. Two of the three known Drosophila T-box genes show a close sequence homology to mammalian genes. Similarities in the phenotypes of fly and mammalian mutants can be taken as evidence of functional conservation. We report here the isolation of a fourth Drosophila T-box gene, optomotor-blind-related gene-1 (org-1), closely related to mouse and human TBX1. We localized TBX1 to chromosomal band 22q11, confirming a recent report, and discuss TBX1 as a candidate gene for DiGeorge and related syndromes.