The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion

Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.     

Drosophila ELMO/CED-12 interacts with Myoblast city to direct myoblast fusion and ommatidial organization

Members of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors function in diverse processes that include cell migration and myoblast fusion. Previous studies have shown that the SH3, DHR1 and DHR2 domains of Myoblast city (MBC) are essential for it to direct myoblast fusion in the Drosophila embryo, while the conserved DCrk-binding proline rich region is expendable. Herein, we describe the isolation of Drosophila ELMO/CED-12, an ∼ 82 kDa protein with a pleckstrin homology (PH) and proline-rich domain, by interaction with the MBC SH3 domain. Mass spectrometry confirms the presence of an MBC/ELMO complex within the embryonic musculature at the time of myoblast fusion and embryos maternally and/or zygotically mutant for elmo exhibit defects in myoblast fusion. Overexpression of MBC and ELMO in the embryonic mesoderm causes defects in myoblast fusion reminiscent of those seen with constitutively-activated Rac1, supporting the previous finding that both the absence of and an excess of Rac activity are deleterious to myoblast fusion. Overexpression of MBC and ELMO/CED-12 in the eye causes perturbations in ommatidial organization that are suppressed by mutations in Rac1 and Rac2, demonstrating genetically that MBC and ELMO/CED-12 cooperate to activate these small GTPases in Drosophila.     

3D analysis of founder cell and fusion competent myoblast arrangements outlines a new model of myoblast fusion

Formation of the Drosophila larval body wall muscles requires the specification, coordinated cellular behaviors and fusion of two cell types: Founder Cells (FCs) that control the identity of the individual muscle and Fusion Competent Myoblasts (FCMs) that provide mass. These two cell types come together to control the final size, shape and attachment of individual muscles. However, the spatial arrangement of these cells over time, the sequence of fusion events and the contribution of these cellular relationships to the fusion process have not been addressed. We analyzed the three-dimensional arrangements of FCs and FCMs over the course of myoblast fusion and assayed whether these issues impact the process of myoblast fusion. We examined the timing of the fusion process by analyzing the fusion profile of individual muscles in wild type and fusion mutants. We showed that there are two temporal phases of myoblast fusion in wild type embryos. Limited fusion events occur during the first 3 h of fusion, while the majority of fusion events occur in the remaining 2.5 h. Altogether, our data have led us to propose a new model of myoblast fusion where the frequency of myoblast fusion events may be influenced by the spatial arrangements of FCs and FCMs.     

Formin3 is required for assembly of the F-actin structure that mediates tracheal fusion in Drosophila

During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion.     

Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. Here, we show a novel striking feature of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, we found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, we did a detailed analysis of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1 and found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles.     

The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis

Centrosomes consist of a pair of centrioles surrounded by an amorphous pericentriolar material (PCM). Proteins that contain a Pericentrin/AKAP450 centrosomal targeting (PACT) domain have been implicated in recruiting several proteins to the PCM. We show that the only PACT domain protein in Drosophila (the Drosophila pericentrin-like protein [D-PLP]) is associated with both the centrioles and the PCM, and is essential for the efficient centrosomal recruitment of all six PCM components that we tested. Surprisingly, however, all six PCM components are eventually recruited to centrosomes during mitosis in d-plp mutant cells, and mitosis is not dramatically perturbed. Although viable, d-plp mutant flies are severely uncoordinated, a phenotype usually associated with defects in mechanosensory neuron function. We show that the sensory cilia of these neurons are malformed and the neurons are nonfunctional in d-plp mutants. Moreover, the flagella in mutant sperm are nonmotile. Thus, D-PLP is essential for the formation of functional cilia and flagella in flies.     

Abdominal-B is essential for proper sexually dimorphic development of the Drosophila gonad

Sexual dimorphism requires the integration of positional information in the embryo with the sex determination pathway. Homeotic genes are a major source of positional information responsible for patterning along the anterior-posterior axis in embryonic development, and are likely to play a critical role in sexual dimorphism. Here, we investigate the role of homeotic genes in the sexually dimorphic development of the gonad in Drosophila. We have found that Abdominal-B (ABD-B) is expressed in a sexually dimorphic manner in the embryonic gonad. Furthermore, Abd-B is necessary and sufficient for specification of a sexually dimorphic cell type, the male-specific somatic gonadal precursors (msSGPs). In Abd-B mutants, the msSGPs are not specified and male gonads now resemble female gonads with respect to these cells. Ectopic expression of Abd-B is sufficient to induce formation of extra msSGPs in additional segments of the embryo. Abd-B works together with abdominal-A to pattern the non-sexually dimorphic somatic gonad in both sexes, while Abd-B alone specifies the msSGPs. Our results indicate that Abd-B acts at multiple levels to regulate gonad development and that Abd-B class homeotic genes are conserved factors in establishing gonad sexual dimorphism in diverse species.     

Syntrophin-2 is required for eye development in Drosophila

Syntrophins are components of the dystrophin glycoprotein complex (DGC), which is encoded by causative genes of muscular dystrophies. The DGC is thought to play roles not only in linking the actin cytoskeleton to the extracellular matrix, providing stability to the cell membrane, but also in signal transduction. Because of their binding to a variety of different molecules, it has been suggested that syntrophins are adaptor proteins recruiting signaling proteins to membranes and the DGC. However, critical roles in vivo remain elusive. Drosophila Syntrophin-2 (Syn2) is an orthologue of human γ1/γ2-syntrophins. Western immunoblot analysis here showed Syn2 to be expressed throughout development, with especially high levels in the adult head. Morphological aberrations were observed in Syn2 knockdown adult flies, with lack of retinal elongation and malformation of rhabdomeres. Furthermore, Syn2 knockdown flies exhibited excessive apoptosis in third instar larvae and alterations in the actin localization in the pupal retinae. Genetic crosses with a collection of Drosophila deficiency stocks allowed us to identify seven genomic regions, deletions of which caused enhancement of the rough eye phenotype induced by Syn2 knockdown. This information should facilitate identification of Syn2 regulators in Drosophila and clarification of roles of Syn2 in eye development.     

Drosophila homologue of the Rothmund-Thomson syndrome gene: essential function in DNA replication during development

Members of the RecQ family play critical roles in maintaining genome integrity. Mutations in human RecQL4 cause a rare genetic disorder, Rothmund-Thomson syndrome. Transgenic mice experiments showed that the RecQ4 null mutant causes embryonic lethality. Although biochemical evidence suggests that the Xenopus RecQ4 is required for the initiation of DNA replication in the oocyte extract, its biological functions during development remain to be elucidated. We present here our results in establishing the use of Drosophila as a model system to probe RecQ4 functions. Immunofluorescence experiments monitoring the cellular distribution of RecQ4 demonstrated that RecQ4 expression peaks during S phase, and RecQ4 is expressed only in tissues active in DNA replication, but not in quiescent cells. We have isolated Drosophila RecQ4 hypomorphic mutants, recq^EP and recq4²³, which specifically reduce chorion gene amplification of follicle cells by 4-5 fold, resulting in thin and fragile eggshells, and female sterility. Quantitative analysis on amplification defects over a 14-kb domain in chorion gene cluster suggests that RecQ4 may have a specific function at or near the origin of replication. A null allele recq4¹⁹ causes a failure in cell proliferation, decrease in DNA replication, chromosomal fragmentation, and lethality at the stage of first instar larvae. The mosaic analysis indicates that cell clones with homozygous recq4¹⁹ fail to proliferate. These results indicate that RecQ4 is essential for viability and fertility, and is required for most aspects of DNA replication during development.     

Conserved interactions with cytoskeletal but not signaling elements are an essential aspect of Drosophila WASp function

Wiskott-Aldrich Syndrome proteins (WASp) serve as important regulators of cytoskeletal organization and function. These modular proteins, which are well-conserved among eukaryotic species, act to promote actin filament assembly in response to cues from various signal transduction pathways. Genetic analysis has revealed a requirement for the single Drosophila homolog, Wasp (Wsp), in cell-fate decisions governing specific neuronal lineages. We have used this unique developmental context to assess the contributions of established signaling and cytoskeletal partners of WASp. We present biochemical and genetic evidence that, as expected, Drosophila Wsp performs its developmental role via the Arp2/3 complex, indicating conservation of the cytoskeletal aspect of Wsp function in vivo. In contrast, we find that association with the key signaling molecules CDC42 and PIP2 is not an essential requirement, implying that activation of Wsp function in vivo depends on additional or alternative signaling pathways.     

A new Drosophila gene wh (wuho) with WD40 repeats is essential for spermatogenesis and has maximal expression in hub cells

Through mutagenesis by P-element transposition, we identified a series of mutants with deletions in topoisomerase 3beta gene (top3beta) and an adjacent, previously uncharacterized gene CG15897, here named wuho (wh). Whereas top3beta truncation does not affect viability or fertility, wh null mutants display male sterile and female semi-sterile phenotypes. Furthermore, wh mutants can be fully rescued by wh transgenes, but not by top3beta transgenes, suggesting that the fertility phenotypes are caused by wh deletion. The alignment of WH protein sequence with other eukaryotic putative homologues shows they are evolutionarily conserved proteins with 5 WD40 repeats in the middle portion of the protein, and a bipartite nuclear localization signal at the carboxyl terminus. Yeast homologue with 5 WD40 repeats, Trm82, is the non-catalytic subunit of a tRNA methylase. Immunostaining shows that WH has the highest expression in hub cells, a niche for germline stem cells of testis. However, WH is not required for the maintenance of hub cells or the germline stem cells. In wh mutant males, spermatogenesis is arrested at the elongating stage of the developing spermatids, resulting in an absence of mature sperms in the seminal vesicles. The decreased fertility in wh mutant females is mostly due to defects in oogenesis. There are abnormal egg chambers present in the mutant females, in which the cystocytes fail to arrest their cell division at the fourth mitotic cycle, resulting in more than 16 cells in a single egg chamber. Additionally, these abnormal cystocytes do not undergo multiple rounds of endoreplication as the nurse cells do in a normal egg chamber. Therefore, the cytological analyses demonstrate that wh has a critical function in cellular differentiation for germline cells during gametogenesis.     

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.     

Drosophila Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis

The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. We have determined that the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, "dumping" of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis.     

The Drosophila parkin homologue is required for normal mitochondrial dynamics during spermiogenesis

Drosophila parkin, the ortholog of the human parkin gene, responsible for a familiar form of autosomal recessive juvenile parkinsonism, has been shown previously to be involved in Drosophila male fertility. Loss-of-function mutations in the parkin gene cause failure of spermatid individualization by affecting the proper progression of the actin-based investment cones that assemble in the nuclear region, but fail to translocate in synchrony down the cyst. In parkin mutants, the investment cones are scattered along the post-elongated spermatid bundles and fail to act properly in the process of sperm individualization. Using phase-contrast and electron microscopy analysis, we demonstrate that the parkin spermatids assemble a seemingly normal onion-stage nebenkern, but when the axoneme elongates only one mitochondrial derivative unfurls from the nebenkern. This unique mitochondrial derivative undergoes abnormal shaping and condensation during spermatid elongation. Our results indicate that parkin gene function is necessary for mitochondrial morphogenesis during earlier and later phases of spermiogenesis. The failure of cyst individualization may be due to the sensitivity of investment cone movement to the perturbation of mitochondrial morphology during spermatid elongation.     

The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann–Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.     

Cofilin/ADF is required for retinal elongation and morphogenesis of the Drosophila rhabdomere

Drosophila photoreceptors undergo marked changes in their morphology during pupal development. These changes include a five-fold elongation of the retinal cell body and the morphogenesis of the rhabdomere, the light sensing structure of the cell. Here we show that twinstar (tsr), which encodes Drosophila cofilin/ADF (actin-depolymerizing factor), is required for both of these processes. In tsr mutants, the retina is shorter than normal, the result of a lack of retinal elongation. In addition, in a strong tsr mutant, the rhabdomere structure is disorganized and the microvilli are short and occasionally unraveled. In an intermediate tsr mutant, the rhabdomeres are not disorganized but have a wider than normal structure. The adherens junctions connecting photoreceptor cells to each other are also found to be wider than normal. We propose, and provide data supporting, that these wide rhabdomeres and adherens junctions are secondary events caused by the inhibition of retinal elongation. These results provide insight into the functions of the actin cytoskeleton during morphogenesis of the Drosophila eye.     

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

Rab11 is essential for fertility in Drosophila

Rab11, a small GTP binding protein involved in vesicular trafficking, has emerged as a key player in regulating various cellular events during Drosophila development and differentiation. In our earlier study a P-insertion line, Rab11(mo), was established as a new hypomorphic allele of Rab11 gene, showing degenerated eye phenotype, bristle abnormalities and sterility. We show here that Rab11 is expressed in the entire testis, more prominently in the secretory cells, and in ovary it is localized at the posterior pole. Rab11(mo) males and females are sterile. The sterility in males has been attributed to defects in the sperm individualization process, while in females, cytoskeleton disruption and reduction/loss of the posteriorly localized protein, Vasa, as a consequence of loss/mislocalization of Rab11 might be the cause of sterility. Fertility as well as the posterior localization of Rab11 and Vasa or cytoskeleton integrity was restored in pCaSpeR4-Rab11/+; Rab11(mo)/Rab11(mo) egg chambers, confirming the requirement of Rab11 in these events.