Reduced cul-5 Activity Causes Aberrant Follicular Morphogenesis and Germ Cell Loss in Drosophila Oogenesis

Drosophila oogenesis is especially well suited for studying stem cell biology, cellular differentiation, and morphogenesis. The small modifier protein ubiquitin regulates many cellular pathways. Ubiquitin is conjugated to target proteins by a diverse class of enzymes called ubiquitin E3 ligases. Here we characterize the requirement of Cul-5, a key component of a subgroup of Cullin-RING-type ubiquitin E3 ligases, in Drosophila oogenesis. We find that reduced cul-5 activity causes the formation of aberrant follicles that are characterized by excess germ cells. We show that germ line cells overproliferate in cul-5 mutant females, causing the formation of abnormally large germ line cysts. Also, the follicular epithelium that normally encapsulates single germ line cysts develops aberrantly in cul-5 mutant, leading to defects in cyst formation. We additionally found that Cul-5 is required for germ cell maintenance, as germ cells are depleted in a substantial fraction of cul-5 mutant ovaries. All of these cul-5 phenotypes are strongly enhanced by reduced activity of gustavus (gus), which encodes a substrate receptor of Cul-5-based ubiquitin E3 ligases. Taken together, our results implicate Cul-5/Gus ubiquitin E3 ligases in ovarian tissue morphogenesis, germ cell proliferation and maintenance of the ovarian germ cell population

A Transient Luminal Chitinous Matrix Is Required to Model Epithelial Tube Diameter in the Drosophila Trachea

SummaryEpithelial tubes are found in many vital organs and require uniform and correct tube diameters for optimal function. Tube size depends on apical membrane growth and subapical cytoskeletal reorganization, but the cues that coordinate these events to ensure functional tube shape remain elusive. We find that epithelial tubes in the Drosophila trachea require luminal chitin polysaccharides to attain the correct diameter. Tracheal chitin forms a broad transient filament within the tubes during the restricted period of expansion. Loss of chitin causes tubular constrictions and cysts associated with irregular subapical cytoskeletal organization, without affecting epithelial integrity and polarity. Analysis of previously identified tube expansion mutants in genes encoding septate junction proteins further suggests that septate junction components may function in tubulogenesis through their role in luminal matrix assembly. We propose that the transient luminal protein/polysaccharide matrix is sensed by the epithelial cells and coordinates cytoskeletal organization to ensure uniform lumen diameter

The bHLH Transcription Factor, Hairy, Refines the Terminal Cell Fate in the Drosophila Embryonic Trachea

Background: The pair-rule gene, hairy, encodes a basic helix-loop-helix transcription factor and is required for patterning of the early Drosophila embryo and for morphogenesis of the embryonic salivary gland. Although hairy was shown to be expressed in the tracheal primordia and in surrounding mesoderm, whether hairy plays a role in tracheal development is not known. Principal Findings: Here, we report that hairy is required for refining the terminal cell fate in the embryonic trachea and that hairy’s tracheal function is distinct from its earlier role in embryonic patterning. In hairy mutant embryos where the repressive activity of hairy is lost due to lack of its co-repressor binding site, extra terminal cells are specified in the dorsal branches. We show that hairy functions in the muscle to refine the terminal cell fate to a single cell at the tip of the dorsal branch by limiting the expression domain of branchless (bnl), encoding the FGF ligand, in surrounding muscle cells. Abnormal activation of the Bnl signaling pathway in hairy mutant tracheal cells is exemplified by increased number of dorsal branch cells expressing Bnl receptor, Breathless (Btl) and Pointed, a downstream target of the Bnl/Btl signaling pathway. We also show that hairy genetically interacts with bnl in TC fate restriction and that overexpression of bnl in a subset of the muscle surrounding tracheal cells phenocopied the hairy mutant phenotype. Conclusions/Significance: Our studies demonstrate a novel role for Hairy in restriction of the terminal cell fate by limitin

The Drosophila LEM-domain protein MAN1 antagonizes BMP signaling at the neuromuscular junction and the wing crossveins

AbstractBMP signaling responses are refined by distinct secreted and intracellular antagonists in different cellular and temporal contexts. Here, we show that the nuclear LEM-domain protein MAN1 is a tissue-specific antagonist of BMP signaling in Drosophila. MAN1 contains two potential Mad-binding sites. We generated MAN1ΔC mutants, harbouring a MAN1 protein that lacks part of the C-terminus including the RNA recognition motif, a putative Mad-binding domain. MAN1ΔC mutants show wing crossvein (CV) patterning defects but no detectable alterations in nuclear morphology. MAN1ΔC pupal wings display expanded phospho-Mad (pMad) accumulation and ectopic expression of the BMP-responsive gene crossveinless-2 (cv-2) indicating that MAN1 restricts BMP signaling. Conversely, MAN1 overexpression in wing imaginal discs inhibited crossvein development and BMP signaling responses. MAN1 is expressed at high levels in pupal wing veins and can be activated in intervein regions by ectopic BMP signaling. The specific upregulation of MAN1 in pupal wing veins may thus represent a negative feedback circuit that limits BMP signaling during CV formation. MAN1ΔC flies also show reduced locomotor activity, and electrophysiology recordings in MAN1ΔC larvae uncover a new presynaptic role of MAN1 at the neuromuscular junction (NMJ). Genetic interaction experiments suggest that MAN1 is a BMP signaling antagonist both at the NMJ and during CV formation

Asrij Maintains the Stem Cell Niche and Controls Differentiation during Drosophila Lymph Gland Hematopoiesis

Several signaling pathways control blood cell (hemocyte) development in the Drosophila lymph gland. Mechanisms that modulate and integrate these signals are poorly understood. Here we report that mutation in a conserved endocytic protein Asrij affects signal transmission and causes aberrant lymph gland hematopoiesis. Mammalian Asrij (Ociad1) is expressed in stem cells of the blood vascular system and is implicated in several cancers. We found that Drosophila Asrij is a pan-hemocyte marker and localizes to a subset of endocytic vesicles. Loss of asrij causes hyperproliferation of lymph gland lobes coupled with increased hemocyte differentiation, thereby depleting the pool of quiescent hemocyte precursors. This co-relates with fewer Col+ cells in the hematopoietic stem cell niche of asrij mutants. Asrij null mutants also show excess specification of crystal cells that express the RUNX factor Lozenge (Lz), a target of Notch signaling. Asrij mutant lymph glands show increased N in sorting endosomes suggesting aberrant trafficking. In vitro assays also show impaired traffic of fluorescent probes in asrij null hemocytes. Taken together our data suggest a role for Asrij in causing increased Notch signaling thereby affecting hemocyte differentiation. Thus, conserved endocytic functions may control blood cell progenitor quiescence and differentiation

The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export

Many cellular responses rely on the control of nucleocytoplasmic transport of transcriptional regulators. The Drosophila nucleoporin Nup88 is selectively required for nuclear accumulation of Rel proteins and full activation of the innate immune response. Here, we investigate the mechanisms underlying its role in nucleocytoplasmic transport. Nuclear import of an nuclear localization signal-enhanced green fluorescent protein (NLS-EGFP) reporter is not affected in DNup88 (members only; mbo) mutants, whereas the level of CRM1-dependent EGFP-nuclear export signal (EGFP-NES) export is increased. We show that the nuclear accumulation of the Drosophila Rel protein Dorsal requires CRM1. DNup88 binds to DNup214 and DCRM1 in vitro, and both proteins become mislocalized from the nuclear rim into the nucleus of mbo mutants. Overexpression of DNup88 is sufficient to relocalize DNup214 and CRM1 on the nuclear envelope and revert the mutant phenotypes. We propose that a major function of DNup88 is to anchor DNup214 and CRM1 on the nuclear envelope and thereby attenuate NES-mediated nuclear export

Distinct functions of the Drosophila Nup153 and Nup214 FG domains in nuclear protein transport

The phenylanine-glycine (FG)–rich regions of several nucleoporins both bind to nuclear transport receptors and collectively provide a diffusion barrier to the nuclear pores. However, the in vivo roles of FG nucleoporins in transport remain unclear. We have inactivated 30 putative nucleoporins in cultured Drosophila melanogaster S2 cells by RNA interference and analyzed the phenotypes on importin α/β−mediated import and CRM1-dependent protein export. The fly homologues of FG nucleoporins Nup358, Nup153, and Nup54 are selectively required for import. The FG repeats of Nup153 are necessary for its function in transport, whereas the remainder of the protein maintains pore integrity. Inactivation of the CRM1 cofactor RanBP3 decreased the nuclear accumulation of CRM1 and protein export. We report a surprisingly antagonistic relationship between RanBP3 and the Nup214 FG region in determining CRM1 localization and its function in protein export. Our data suggest that peripheral metazoan FG nucleoporins have distinct functions in nuclear protein transport events

The Hrs/Stam Complex Acts as a Positive and Negative Regulator of RTK Signaling during Drosophila Development

BACKGROUND: Endocytosis is a key regulatory step of diverse signalling pathways, including receptor tyrosine kinase (RTK) signalling. Hrs and Stam constitute the ESCRT-0 complex that controls the initial selection of ubiquitinated proteins, which will subsequently be degraded in lysosomes. It has been well established ex vivo and during Drosophila embryogenesis that Hrs promotes EGFR down regulation. We have recently isolated the first mutations of stam in flies and shown that Stam is required for air sac morphogenesis, a larval respiratory structure whose formation critically depends on finely tuned levels of FGFR activity. This suggest that Stam, putatively within the ESCRT-0 complex, modulates FGF signalling, a possibility that has not been examined in Drosophila yet. PRINCIPAL FINDINGS: Here, we assessed the role of the Hrs/Stam complex in the regulation of signalling activity during Drosophila development. We show that stam and hrs are required for efficient FGFR signalling in the tracheal system, both during cell migration in the air sac primordium and during the formation of fine cytoplasmic extensions in terminal cells. We find that stam and hrs mutant cells display altered FGFR/Btl localisation, likely contributing to impaired signalling levels. Electron microscopy analyses indicate that endosome maturation is impaired at distinct steps by hrs and stam mutations. These somewhat unexpected results prompted us to further explore the function of stam and hrs in EGFR signalling. We show that while stam and hrs together downregulate EGFR signalling in the embryo, they are required for full activation of EGFR signalling during wing development. CONCLUSIONS/SIGNIFICANCE: Our study shows that the ESCRT-0 complex differentially regulates RTK signalling, either positively or negatively depending on tissues and developmental stages, further highlighting the importance of endocytosis in modulating signalling pathways during development

Yorkie controls tube length and apical barrier integrity during airway development

Epithelial organ size and shape depend on cell shape changes, cell-matrix communication, and apical membrane growth. The Drosophila melanogaster embryonic tracheal network is an excellent model to study these processes. Here, we show that the transcriptional coactivator of the Hippo pathway, Yorkie (YAP/TAZ in vertebrates), plays distinct roles in the developing Drosophila airways. Yorkie exerts a cytoplasmic function by binding Drosophila Twinstar, the orthologue of the vertebrate actin-severing protein Cofilin, to regulate F-actin levels and apical cell membrane size, which are required for proper tracheal tube elongation. Second, Yorkie controls water tightness of tracheal tubes by transcriptional regulation of the δ-aminolevulinate synthase gene (Alas). We conclude that Yorkie has a dual role in tracheal development to ensure proper tracheal growth and functionality

The Rap1 Guanine Nucleotide Exchange Factor C3G Is Required for Preservation of Larval Muscle Integrity in Drosophila melanogaster

C3G is a guanine nucleotide exchange factor (GEF) and modulator of small G-protein activity, which primarily acts on members of the Rap GTPase subfamily. Via promotion of the active GTP bound conformation of target GTPases, C3G has been implicated in the regulation of multiple cellular and developmental events including proliferation, differentiation and apoptosis. The Drosophila C3G orthologue exhibits a domain organization similar to that of vertebrate C3G. Through deletion of the C3G locus, we have observed that loss of C3G causes semi-lethality, and that escaping adult flies are characterized by a reduction in lifespan and general fitness. In situ hybridization reveals C3G expression in the developing embryonic somatic and visceral muscles, and indeed analysis of C3G mutants suggests essential functions of C3G for normal body wall muscle development during larval stages. C3G mutants display abnormal muscle morphology and attachment, as well as failure to properly localize βPS integrins to muscle attachment sites. Moreover, we show that C3G stimulates guanine nucleotide exchange on Drosophila Rap GTPases in vitro. Taken together, we conclude that Drosophila C3G is a Rap1-specific GEF with important functions in maintaining muscle integrity during larval stages