Project description:The eggshell of the fruit fly Drosophila melanogaster is a useful model for understanding the synthesis of a complex extracellular matrix. The eggshell is synthesized during mid-to-late oogenesis by the somatic follicle cells that surround the developing oocyte. We previously reported that female flies mutant for the gene drop-dead ( drd ) are sterile, but the underlying cause of the sterility remained unknown. In this study, we examined the role of drd in eggshell synthesis. We show that eggs laid by drd mutant females are fertilized but arrest early in embryogenesis, and that the innermost layer of the eggshell, the vitelline membrane, is abnormally permeable to dye in these eggs. In addition, the major vitelline membrane proteins fail to become crosslinked by nonreducible bonds, a process that normally occurs during egg activation following ovulation, as evidenced by their solubility and detection by Western blot in laid eggs. In contrast, the Cp36 protein, which is found in the outer chorion layers of the eggshell, becomes crosslinked normally. To link the drd expression pattern with these phenotypes, we show that drd is expressed in the ovarian follicle cells beginning in mid-oogenesis, and, importantly, that all drd mutant eggshell phenotypes could be recapitulated by selective knockdown of drd expression in the follicle cells. To determine whether drd expression was required for the crosslinking itself, we performed in vitro activation and crosslinking experiments. The vitelline membranes of control egg chambers could become crosslinked either by incubation in hyperosmotic medium, which activates the egg chambers, or by exogenous peroxidase and hydrogen peroxide. In contrast, neither treatment resulted in the crosslinking of the vitelline membrane in drd mutant egg chambers. These results indicate that drd expression in the follicle cells is necessary for vitelline membrane proteins to serve as substrates for peroxidase-mediated cross-linking at the end of oogenesis.
Project description:Bone Morphogenetic Proteins (BMPs) signal by activating Smad transcription factors to control a number of decisions during animal development. In Drosophila, signaling by the BMP ligand Decapentaplegic (Dpp) involves the activity of brinker (brk) which, in most contexts, is repressed by Dpp. Brk encodes a transcription factor which represses BMP signaling output by antagonizing Smad-dependent target gene activation. Here, we study BMP-dependent gene regulation during Drosophila oogenesis by following the signal transmission from Dpp to its target broad (br), a gene with a crucial function in eggshell patterning. We identify regulatory sequences that account for expression of both brk and br, and connect these to the transcription factors of the pathway. We show that Dpp directly regulates brk transcription through Smad- and Schnurri (Shn)-dependent repression. Brk is epistatic to Dpp in br expression and activates br indirectly, through removal of a repressor, which is yet to be identified. Our work provides first cis-regulatory insights into transcriptional interpretation of BMP signaling in eggshell morphogenesis and defines a transcriptional cascade that connects Dpp to target gene regulation.
Project description:Mutations in the gene drop-dead (drd) cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ) were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.
Project description:Telomeres are nucleoprotein complexes at the ends of linear chromosomes. These specialized structures ensure genome integrity and faithful chromosome inheritance. Recurrent addition of repetitive, telomere-specific DNA elements to chromosome ends combats end-attrition, while specialized telomere-associated proteins protect naked, double-stranded chromosome ends from promiscuous repair into end-to-end fusions. Although telomere length homeostasis and end-protection are ubiquitous across eukaryotes, there is sporadic but building evidence that the molecular machinery supporting these essential processes evolves rapidly. Nevertheless, no global analysis of the evolutionary forces that shape these fast-evolving proteins has been performed on any eukaryote. The abundant population and comparative genomic resources of Drosophila melanogaster and its close relatives offer us a unique opportunity to fill this gap. Here we leverage population genetics, molecular evolution, and phylogenomics to define the scope and evolutionary mechanisms driving fast evolution of genes required for telomere integrity. We uncover evidence of pervasive positive selection across multiple evolutionary timescales. We also document prolific expansion, turnover, and expression evolution in gene families founded by telomeric proteins. Motivated by the mutant phenotypes and molecular roles of these fast-evolving genes, we put forward four alternative, but not mutually exclusive, models of intra-genomic conflict that may play out at very termini of eukaryotic chromosomes. Our findings set the stage for investigating both the genetic causes and functional consequences of telomere protein evolution in Drosophila and beyond.
Project description:We report that the Drosophila mind bomb2 (mib2) gene is a novel regulator of muscle development. Unlike its paralogue, mib1, zygotic expression of mib2 is restricted to somatic and visceral muscle progenitors, and their respective differentiated musculatures. We demonstrate that in embryos that lack functional Mib2, muscle detachment is observed beginning in mid stage 15 and progresses rapidly, culminating in catastrophic degeneration and loss of most somatic muscles by stage 17. Notably, the degenerating muscles are positive for apoptosis markers, and inhibition of apoptosis in muscles prevents to a significant degree the muscle defects. Rescue experiments with Mib1 and Neuralized show further that these E3 ubiquitin ligases are not capable of ameliorating the muscle mutant phenotype of mib2. Our data suggest strongly that mib2 is involved in a novel Notch- and integrin-independent pathway that maintains the integrity of fully differentiated muscles and prevents their apoptotic degeneration.
Project description:Males' and females' reproductive strategies may differ, potentially leading to sexual conflict. Increased efforts by males (harassment, forced copulation, intimidation) to gain access to females could even negatively affect female survival and thus lead to reproductive failure for both individuals. In anurans, a higher mortality risk of mating females has been reported in explosive breeding species. During these mating events, several males cling to a female, which are mostly unable to get rid of the unwanted males. This can lead to the female's death. From the literature, it seems that females of explosive breeding frogs have no means to reject unwanted males. Here we describe female mate avoidance behaviours in the European common frog. We observed three female avoidance behaviours, namely 'rotation', 'release call(s)' and tonic immobility (death feigning). These behaviours were significantly associated with smaller female body size, and smaller females were more successful in escaping amplexus. Tonic immobility as a tactic to avoid mating or male harassment has only been observed in a handful of species and only in one other amphibian. Our observations show that females in explosive breeding frogs may not be as passive and helpless as previously thought.
Project description:DHR38 is the only Drosophila member of the NR4A subclass of vertebrate nuclear receptors, which have been implicated in multiple biological pathways, including neuronal function, apoptosis, and metabolism. Although an earlier study identified three point mutations in DHR38, none of these were shown to be a null allele for the locus, leaving it unclear whether a complete loss of DHR38 function might uncover novel roles for the receptor. Here we show that a specific DHR38 null allele, DHR38(Y214), leads to fully penetrant pharate adult lethality, similar to the most severe phenotype associated with the EMS-induced mutations. DHR38(Y214) mutants display minor effects on ecdysone-regulated transcription at the onset of metamorphosis. In contrast, cuticle gene expression is significantly reduced in DHR38(Y214) mutant pupae. These studies define the essential functions of DHR38 and provide a genetic context for further characterization of its roles during development.
Project description:How the nuclear lamina (NL) impacts on global chromatin architecture is poorly understood. Here, we show that NL disruption in Drosophila S2 cells leads to chromatin compaction and repositioning from the nuclear envelope. This increases the chromatin density in a fraction of topologically-associating domains (TADs) enriched in active chromatin and enhances interactions between active and inactive chromatin. Importantly, upon NL disruption the NL-associated TADs become more acetylated at histone H3 and less compact, while background transcription is derepressed. Two-colour FISH confirms that a TAD becomes less compact following its release from the NL. Finally, polymer simulations show that chromatin binding to the NL can per se compact attached TADs. Collectively, our findings demonstrate a dual function of the NL in shaping the 3D genome. Attachment of TADs to the NL makes them more condensed but decreases the overall chromatin density in the nucleus by stretching interphase chromosomes.
Project description:We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.
Project description:The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of the Drosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy.