Udo Häcker

Heparan sulphate proteoglycans : the sweet side of development

Pattern formation during development is controlled to a great extent by a small number of conserved signal transduction pathways that are activated by extracellular ligands such as Hedgehog, Wingless or Decapentaplegic. Genetic experiments have identified heparan sulphate proteoglycans (HSPGs) as important regulators of the tissue distribution of these extracellular signalling molecules. Several recent reports provide important new insights into the mechanisms by which HSPGs function during development.

Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner.

Members of the Rho/Rac/Cdc42 superfamily of GTPases and their upstream activators, guanine nucleotide exchange factors (GEFs) , have emerged as key regulators of actin and microtubule dynamics. In their GTP bound form, these proteins interact with downstream effector molecules that alter actin and microtubule behavior. During Drosophila embryogenesis, a Galpha subunit (Concertina) and a Rho-type guanine nucleotide exchange factor (DRhoGEF2) have been implicated in the dramatic epithelial-cell shape changes that occur during gastrulation and morphogenesis . Using Drosophila S2 cells as a model system, we show that DRhoGEF2 induces contractile cell shape changes by stimulating myosin II via the Rho1 pathway. Unexpectedly, we found that DRhoGEF2 travels to the cell cortex on the tips of growing microtubules by interaction with the microtubule plus-end tracking protein EB1. The upstream activator Concertina, in its GTP but not GDP bound form, dissociates DRhoGEF2 from microtubule tips and also causes cellular contraction. We propose that DRhoGEF2 uses microtubule dynamics to search for cortical subdomains of receptor-mediated Galpha activation, which in turn causes localized actomyosin contraction associated with morphogenetic movements during development.

piggyBac-based insertional mutagenesis in the presence of stably integrated P elements in Drosophila

P element-mediated mutagenesis has been used to disrupt an estimated 25% of genes essential for Drosophila adult viability. Mutation of all genes in the fly genome, however, poses a problem, because P elements show significant hotspots of integration. In addition, advanced screening scenarios often require the use of P element-based tools like the generation of germ-line mosaics using FLP recombinase-mediated recombination or gene misexpression using the UAS/Gal4 system. These techniques are P element-based and can therefore not be combined with the use of P elements as mutagenic agents. To circumvent these limitations, we have developed an insertional mutagenesis system using non-P element transposons. An enhanced yellow fluorescent protein-marked piggyBac-based mutator element was mobilized by a piggyBac specific transposase source expressed from a Hermes-based jump-starter transposon marked with enhanced cyan fluorescent protein. In a pilot screen, we have generated 798 piggyBac insertions on FRT bearing third chromosomes of which 9% have sustained a putatively piggyBac-related lethal hit. The FRTs present on the target chromosome remained stably integrated during the screen and could subsequently be used to generate germ-line clones associated with maternal and zygotic phenotypes. PCR-based analysis of insertion loci shows that 57% of the insertions are in genes for which no P element insertions have been reported. Our data demonstrate the potential of this technique to facilitate the quest for saturation mutagenesis of the Drosophila genome. The system is Drosophila nonspecific and potentially applicable in a broad spectrum of nonmodel organisms.

slalom encodes an adenosine 3′‐phosphate 5′‐phosphosulfate transporter essential for development in Drosophila

Sulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high‐energy sulfate donor adenosine 3′‐phosphate 5′‐phosphosulfate. Here we report the first molecular identification of a gene that encodes a transmembrane protein required to transport adenosine 3′‐phosphate 5′‐phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which we call slalom, display defects in Wg and Hh signaling, which are likely due to the lack of sulfation of glycos aminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal–ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal–ventral determinant pipe.

The mitotic arrest in response to hypoxia and of polar bodies during early embryogenesis requires Drosophila Mps1

Mps1 kinase plays an evolutionary conserved role in the mitotic spindle checkpoint. This system precludes anaphase onset until all chromosomes have successfully attached to spindle microtubules via their kinetochores. Mps1 overexpression in budding yeast is sufficient to trigger a mitotic arrest, which is dependent on the other mitotic checkpoint components, Bub1, Bub3, Mad1, Mad2, and Mad3. Therefore, Mps1 might act at the top of the mitotic checkpoint cascade. Moreover, in contrast to the other mitotic checkpoint components, Mps1 is essential for spindle pole body duplication in budding yeast. Centrosome duplication in mammalian cells might also be controlled by Mps1 , but the fission yeast homolog is not required for spindle pole body duplication. Our phenotypic characterizations of Mps1 mutant embryos in Drosophila do not reveal an involvement in centrosome duplication, while the mitotic spindle checkpoint is defective in these mutants. In addition, our analyses reveal novel functions. We demonstrate that Mps1 is also required for the arrest of cell cycle progression in response to hypoxia. Finally, we show that Mps1 and the mitotic spindle checkpoint are responsible for the developmental cell cycle arrest of the three haploid products of female meiosis that are not used as the female pronucleus.

DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo

Morphogenesis of the Drosophila melanogaster embryo is associated with a dynamic reorganization of the actin cytoskeleton that is mediated by small GTPases of the Rho family. Often, Rho1 controls different aspects of cytoskeletal function in parallel, requiring a complex level of regulation. We show that the guanine triphosphate (GTP) exchange factor DRhoGEF2 is apically localized in epithelial cells throughout embryogenesis. We demonstrate that DRhoGEF2, which has previously been shown to regulate cell shape changes during gastrulation, recruits Rho1 to actin rings and regulates actin distribution and actomyosin contractility during nuclear divisions, pole cell formation, and cellularization of syncytial blastoderm embryos. We propose that DRhoGEF2 activity coordinates contractile actomyosin forces throughout morphogenesis in Drosophila by regulating the association of myosin with actin to form contractile cables. Our results support the hypothesis that specific aspects of Rho1 function are regulated by specific GTP exchange factors.

Wingless, hedgehog and heparan sulfate Proteoglycans

Several recent papers published in Development challenge some of the previous views published by our group and others that: (1) the Drosophila glypican molecules Dally and Dally-like protein (Dlp) are involved in Wingless (Wg) signaling; and (2) Tout-velu/Ext/Ttv affects Hedgehog (Hh) but not Wg

DRhoGEF2 Regulates Cellular Tension and Cell Pulsations in the Amnioserosa during Drosophila Dorsal Closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.

Expression and regulation of Spätzle-processing enzyme in Drosophila

The Drosophila melanogaster Toll receptor controls embryonic dorsal‐ventral axis formation and is crucial for the innate immune response. In both cases, Toll is activated by the enzymatically cleaved form of its ligand Spätzle (Spz). During axis formation, Spz is cleaved by the maternally provided serine protease Easter while the Spätzle‐processing enzyme (SPE) activates Spz after infection. We confirm the role of SPE in immunity and show that it is a zygotic gene specifically expressed in immune tissues implying that the dual activation of Spz is achieved by differential spatiotemporal expression of two similar but distinct serine proteases.

Ingestion of gallium phosphide nanowires has no adverse effect on Drosophila tissue function

Engineered nanoparticles have been under increasing scrutiny in recent years. High aspect ratio nanoparticles such as carbon nanotubes and nanowires have raised safety concerns due to their geometrical similarity to asbestos fibers. III-V epitaxial semiconductor nanowires are expected to be utilized in devices such as LEDs and solar cells and will thus be available to the public. In addition, clean-room staff fabricating and characterizing the nanowires are at risk of exposure, emphasizing the importance of investigating their possible toxicity. Here we investigated the effects of gallium phosphide nanowires on the fruit fly Drosophila melanogaster. Drosophila larvae and/or adults were exposed to gallium phosphide nanowires by ingestion with food. The toxicity and tissue interaction of the nanowires was evaluated by investigating tissue distribution, activation of immune response, genome-wide gene expression, life span, fecundity and somatic mutation rates. Our results show that gallium phosphide nanowires applied through the diet are not taken up into Drosophila tissues, do not elicit a measurable immune response or changes in genome-wide gene expression and do not significantly affect life span or somatic mutation rate.