Redundant Mechanisms for Regulation of Midline Crossing in Drosophila

During development, all neurons have to decide on whether to cross the longitudinal midline to project on the contralateral side of the body. In vertebrates and invertebrates regulation of crossing is achieved by interfering with Robo signalling either through sorting and degradation of the receptor, in flies, or through silencing of its repulsive activity, in vertebrates. Here I show that in Drosophila a second mechanism of regulation exists that is independent from sorting. Using in vitro and in vivo assays I mapped the region of Robo that is sufficient and required for its interaction with Comm, its sorting receptor. By modifying that region, I generated new forms of Robo that are insensitive to Comm sorting in vitro and in vivo, yet still able to normally translate repulsive activity in vivo. Using gene targeting by homologous recombination I created new conditional alleles of robo that are sorting defective (roboSD). Surprisingly, expression of these modified proteins results in phenotypically normal flies, unveiling a sorting independent mechanism of regulation

Less than 5 Netrin-1 molecules initiate attraction but 200 Sema3A molecules are necessary for repulsion

Guidance molecules, such as Sema3A or Netrin-1, induce growth cone (GC) repulsion or attraction. In order to determine the speed of action and efficiency of these guidance cues we developed an experimental procedure to deliver controlled amounts of these molecules. Lipid vesicles encapsulating 10-10 4 molecules of Sema3A or Netrin-1 were manipulated with high spatial and temporal resolution by optical tweezers and their photolysis triggered by laser pulses. Guidance molecules released from the vesicles diffused and reached the GC membrane in a few seconds. Following their arrival, GCs retracted or grew in 20-120 s. By determining the number of guidance molecules trapped inside vesicles and estimating the fraction of guidance molecules reaching the GC, we show that the arrival of less than 5 Netrin-1 molecules on the GC membrane is sufficient to induce growth. In contrast, the arrival of about 200 Sema3A molecules is necessary to induce filopodia repulsion

Sex and the Single Cell. II. There Is a Time and Place for Sex

In both male and female Drosophila, only a subset of cells have the potential to sexually differentiate, making both males and females mosaics of sexually differentiated and sexually undifferentiated cells

A Functional Misexpression Screen Uncovers a Role for Enabled in Progressive Neurodegeneration

Drosophila is a well-established model to study the molecular basis of neurodegenerative diseases. We carried out a misexpression screen to identify genes involved in neurodegeneration examining locomotor behavior in young and aged flies. We hypothesized that a progressive loss of rhythmic activity could reveal novel genes involved in neurodegenerative mechanisms. One of the interesting candidates showing progressive arrhythmicity has reduced enabled (ena) levels. ena down-regulation gave rise to progressive vacuolization in specific regions of the adult brain. Abnormal staining of pre-synaptic markers such as cystein string protein (CSP) suggest that axonal transport could underlie the neurodegeneration observed in the mutant. Reduced ena levels correlated with increased apoptosis, which could be rescued in the presence of p35, a general Caspase inhibitor. Thus, this mutant recapitulates two important features of human neurodegenerative diseases, i.e., vulnerability of certain neuronal populations and progressive degeneration, offering a unique scenario in which to unravel the specific mechanisms in an easily tractable organism

The Caenorhabditis elegans Eph Receptor Activates NCK and N-WASP, and Inhibits Ena/VASP to Regulate Growth Cone Dynamics during Axon Guidance

The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity

Can hippocampal neurites and growth cones climb over obstacles?

Guidance molecules, such as Sema3A or Netrin-1, can induce growth cone (GC) repulsion or attraction in the presence of a flat surface, but very little is known of the action of guidance molecules in the presence of obstacles. Therefore we combined chemical and mechanical cues by applying a steady Netrin-1 stream to the GCs of dissociated hippocampal neurons plated on polydimethylsiloxane (PDMS) surfaces patterned with lines 2 \ub5m wide, with 4 \ub5m period and with a height varying from 100 to 600 nm. GC turning experiments performed 24 hours after plating showed that filopodia crawl over these lines within minutes. These filopodia do not show staining for the adhesion marker Paxillin. GCs and neurites crawl over lines 100 nm high, but less frequently and on a longer time scale over lines higher than 300 nm; neurites never crawl over lines 600 nm high. When neurons are grown for 3 days over patterned surfaces, also neurites can cross lines 300 nm and 600 nm high, grow parallel to and on top of these lines and express Paxillin. Axons - selectively stained with SMI 312 - do not differ from dendrites in their ability to cross these lines. Our results show that highly motile structures such as filopodia climb over high obstacle in response to chemical cues, but larger neuronal structures are less prompt and require hours or days to climb similar obstacles

Midline Signalling Systems Direct the Formation of a Neural Map by Dendritic Targeting in the Drosophila Motor System

During embryonic development of the motor system of Drosophila, motorneurons target their dendrites to different regions along the body axis in response to midline guidance cues

A new strategy for isolating genes controlling dosage compensation in Drosophila using a simple epigenetic mosaic eye phenotype

<p>Abstract</p> <p>Background</p> <p>The <it>Drosophila </it>Male Specific Lethal (MSL) complex contains chromatin modifying enzymes and non-coding <it>roX </it>RNA. It paints the male X at hundreds of bands where it acetylates histone H4 at lysine 16. This epigenetic mark increases expression from the single male X chromosome approximately twofold above what gene-specific factors produce from each female X chromosome. This equalises X-linked gene expression between the sexes. Previous screens for components of dosage compensation relied on a distinctive male-specific lethal phenotype.</p> <p>Results</p> <p>Here, we report a new strategy relying upon an unusual male-specific mosaic eye pigmentation phenotype produced when the MSL complex acts upon autosomal <it>roX1 </it>transgenes. Screening the second chromosome identified at least five loci, two of which are previously described components of the MSL complex. We focused our analysis on the modifier alleles of MSL1 and MLE (for 'maleless'). The MSL1 lesions are not simple nulls, but rather alter the PEHE domain that recruits the MSL3 chromodomain and MOF ('males absent on first') histone acetyltransferase subunits to the complex. These mutants are compromised in their ability to recruit MSL3 and MOF, dosage compensate the X, and support long distance spreading from <it>roX1 </it>transgenes. Yet, paradoxically, they were isolated because they somehow increase MSL complex activity immediately around <it>roX1 </it>transgenes in combination with wild-type MSL1 subunits.</p> <p>Conclusions</p> <p>We propose that these diverse phenotypes arise from perturbations in assembly of MSL subunits onto nascent <it>roX </it>transcripts. This strategy is a promising alternative route for identifying previously unknown components of the dosage compensation pathway and novel alleles of known MSL proteins.</p

Sex-biased transcription enhancement by a 5' tethered Gal4-MOF histone acetyltransferase fusion protein in Drosophila

<p>Abstract</p> <p>Background</p> <p>In male <it>Drosophila melanogaster</it>, the male specific lethal (MSL) complex is somehow responsible for a two-fold increase in transcription of most X-linked genes, which are enriched for histone H4 acetylated at lysine 16 (H4K16ac). This acetylation requires MOF, a histone acetyltransferase that is a component of the MSL complex. MOF also associates with the non-specific lethal or NSL complex. The MSL complex is bound within active genes on the male X chromosome with a 3' bias. In contrast, the NSL complex is enriched at promoter regions of many autosomal and X-linked genes in both sexes. In this study we have investigated the role of MOF as a transcriptional activator.</p> <p>Results</p> <p>MOF was fused to the DNA binding domain of Gal4 and targeted to the promoter region of UAS-reporter genes in <it>Drosophila</it>. We found that expression of a UAS-red fluorescent protein (DsRed) reporter gene was strongly induced by Gal4-MOF. However, DsRed RNA levels were about seven times higher in female than male larvae. Immunostaining of polytene chromosomes showed that Gal4-MOF co-localized with MSL1 to many sites on the X chromosome in male but not female nuclei. However, in female nuclei that express MSL2, Gal4-MOF co-localized with MSL1 to many sites on polytene chromosomes but DsRed expression was reduced. Mutation of conserved active site residues in MOF (Glu714 and Cys680) reduced HAT activity <it>in vitro </it>and UAS-DsRed activation in <it>Drosophila</it>. In the presence of Gal4-MOF, H4K16ac levels were enriched over UAS-<it>lacZ </it>and UAS-<it>arm-lacZ </it>reporter genes. The latter utilizes the constitutive promoter from the <it>arm </it>gene to drive <it>lacZ </it>expression. In contrast to the strong induction of UAS-DsRed expression, UAS-<it>arm-lacZ </it>expression increased by about 2-fold in both sexes.</p> <p>Conclusions</p> <p>Targeting MOF to reporter genes led to transcription enhancement and acetylation of histone H4 at lysine 16. Histone acetyltransferase activity was required for the full transcriptional response. Incorporation of Gal4-MOF into the MSL complex in males led to a lower transcription enhancement of UAS-<it>DsRed </it>but not UAS-<it>arm-lacZ </it>genes. We discuss how association of Gal4-MOF with the MSL or NSL proteins could explain our results.</p

Mena deficiency delays tumor progression and decreases metastasis in polyoma middle-T transgenic mouse mammary tumors

Introduction The actin binding protein Mammalian enabled (Mena), has been implicated in the metastatic progression of solid tumors in humans. Mena expression level in primary tumors is correlated with metastasis in breast, cervical, colorectal and pancreatic cancers. Cells expressing high Mena levels are part of the tumor microenvironment for metastasis (TMEM), an anatomical structure that is predictive for risk of breast cancer metastasis. Previously we have shown that forced expression of Mena adenocarcinoma cells enhances invasion and metastasis in xenograft mice. Whether Mena is required for tumor progression is still unknown. Here we report the effects of Mena deficiency on tumor progression, metastasis and on normal mammary gland development. Methods To investigate the role of Mena in tumor progression and metastasis, Mena deficient mice were intercrossed with mice carrying a transgene expressing the polyoma middle T oncoprotein, driven by the mouse mammary tumor virus. The progeny were investigated for the effects of Mena deficiency on tumor progression via staging of primary mammary tumors and by evaluation of morbidity. Stages of metastatic progression were investigated using an in vivo invasion assay, intravital multiphoton microscopy, circulating tumor cell burden, and lung metastases. Mammary gland development was studied in whole mount mammary glands of wild type and Mena deficient mice. Results Mena deficiency decreased morbidity and metastatic dissemination. Loss of Mena increased mammary tumor latency but had no affect on mammary tumor burden or histologic progression to carcinoma. Elimination of Mena also significantly decreased epidermal growth factor (EGF) induced in vivo invasion, in vivo motility, intravasation and metastasis. Non-tumor bearing mice deficient for Mena also showed defects in mammary gland terminal end bud formation and branching. Conclusions Deficiency of Mena decreases metastasis by slowing tumor progression and reducing tumor cell invasion and intravasation. Mena deficiency during development causes defects in invasive processes involved in mammary gland development. These findings suggest that functional intervention targeting Mena in breast cancer patients may provide a valuable treatment option to delay tumor progression and decrease invasion and metastatic spread leading to an improved prognostic outcome.National Cancer Institute (U.S.). Integrative Cancer Biology Program (grant U54 CA112967)Virginia and D.K. Ludwig Fund for Cancer Researc