A novel adipose loss-of-function mutant in Drosophila.

To identify genes required for brain growth, we took an RNAi knockdown reverse genetic approach in Drosophila. One potential candidate isolated from this effort is the anti-lipogenic gene adipose (adp). Adp has an established role in the negative regulation of lipogenesis in the fat body of the fly and adipose tissue in mammals. While fat is key to proper development in general, adp has not been investigated during brain development. Here, we found that RNAi knockdown of adp in neuronal stem cells and neurons results in reduced brain lobe volume and sought to replicate this with a mutant fly. We generated a novel adp mutant that acts as a loss-of-function mutant based on buoyancy assay results. We found that despite a change in fat content in the body overall and a decrease in the number of larger (>5 µm) brain lipid droplets, there was no change in the brain lobe volume of mutant larvae. Overall, our work describes a novel adp mutant that can functionally replace the long-standing adp60 mutant and shows that the adp gene has no obvious involvement in brain growth.

Molecular functions of ANKLE2 and its implications in human disease.

Ankyrin repeat and LEM domain-containing 2 (ANKLE2) is a scaffolding protein with established roles in cell division and development, the dysfunction of which is increasingly implicated in human disease. ANKLE2 regulates nuclear envelope disassembly at the onset of mitosis and its reassembly after chromosome segregation. ANKLE2 dysfunction is associated with abnormal nuclear morphology and cell division. It regulates the nuclear envelope by mediating protein-protein interactions with barrier to autointegration factor (BANF1; also known as BAF) and with the kinase and phosphatase that modulate the phosphorylation state of BAF. In brain development, ANKLE2 is crucial for proper asymmetric division of neural progenitor cells. In humans, pathogenic loss-of-function mutations in ANKLE2 are associated with primary congenital microcephaly, a condition in which the brain is not properly developed at birth. ANKLE2 is also linked to other disease pathologies, including congenital Zika syndrome, cancer and tauopathy. Here, we review the molecular roles of ANKLE2 and the recent literature on human diseases caused by its dysfunction.

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development.

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. Although adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of disease caused by this virus, which includes microcephaly. In this study, we generated a toolkit to ectopically express ZIKV proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We used this toolkit to identify phenotypes and potential host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins caused scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size and neuronal function defects. We further used this system to identify strain-dependent phenotypes that may have contributed to the increased pathogenesis associated with the outbreak of ZIKV in the Americas in 2015. Our work demonstrates the use of Drosophila as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

A Zika virus protein expression screen in Drosophila to investigate targeted host pathways during development.

In the past decade, Zika virus (ZIKV) emerged as a global public health concern. While adult infections are typically mild, maternal infection can lead to adverse fetal outcomes. Understanding how ZIKV proteins disrupt development can provide insights into the molecular mechanisms of symptoms caused by this virus including microcephaly. In this study, we generated a toolkit to ectopically express Zika viral proteins in vivo in Drosophila melanogaster in a tissue-specific manner using the GAL4/UAS system. We use this toolkit to identify phenotypes and host pathways targeted by the virus. Our work identified that expression of most ZIKV proteins cause scorable phenotypes, such as overall lethality, gross morphological defects, reduced brain size, and neuronal function defects. We further use this system to identify strain-dependent phenotypes that may contribute to the increased pathogenesis associated with the more recent outbreak of ZIKV in the Americas. Our work demonstrates Drosophila's use as an efficient in vivo model to rapidly decipher how pathogens cause disease and lays the groundwork for further molecular study of ZIKV pathogenesis in flies.

ANKLE2-related microcephaly: A variable microcephaly syndrome resembling Zika infection.

OBJECTIVE: This study delineates the clinical and molecular spectrum of ANKLE2-related microcephaly (MIC), as well as highlights shared pathological mechanisms between ANKLE2 and the Zika virus. METHODS: We identified 12 individuals with MIC and variants in ANKLE2 with a broad range of features. Probands underwent thorough phenotypic evaluations, developmental assessments, and anthropometric measurements. Brain imaging studies were systematically reviewed for developmental abnormalities. We functionally interrogated a subset of identified ANKLE2 variants in Drosophila melanogaster. RESULTS: All individuals had MIC (z-score ≤ -3), including nine with congenital MIC. We identified a broad range of brain abnormalities including simplified cortical gyral pattern, full or partial callosal agenesis, increased extra-axial spaces, hypomyelination, cerebellar vermis hypoplasia, and enlarged cisterna magna. All probands had developmental delays in at least one domain, with speech and language delays being the most common. Six probands had skin findings characteristic of ANKLE2 including hyper- and hypopigmented macules. Only one individual had scalp rugae. Functional characterization in Drosophila recapitulated the human MIC phenotype. Of the four variants tested, p.Val229Gly, p.Arg236*, and p.Arg536Cys acted as partial-loss-of-function variants, whereas the c.1421-1G>C splicing variant demonstrated a strong loss-of-function effect. INTERPRETATION: Deleterious variants in the ANKLE2 gene cause a unique MIC syndrome characterized by congenital or postnatal MIC, a broad range of structural brain abnormalities, and skin pigmentary changes. Thorough functional characterization has identified shared pathogenic mechanisms between ANKLE2-related MIC and congenital Zika virus infection. This study further highlights the importance of a thorough diagnostic evaluation including molecular diagnostic testing in individuals with MIC.

Drosophila as a Model for Infectious Diseases.

The fruit fly, Drosophila melanogaster, has been used to understand fundamental principles of genetics and biology for over a century. Drosophila is now also considered an essential tool to study mechanisms underlying numerous human genetic diseases. In this review, we will discuss how flies can be used to deepen our knowledge of infectious disease mechanisms in vivo. Flies make effective and applicable models for studying host-pathogen interactions thanks to their highly conserved innate immune systems and cellular processes commonly hijacked by pathogens. Drosophila researchers also possess the most powerful, rapid, and versatile tools for genetic manipulation in multicellular organisms. This allows for robust experiments in which specific pathogenic proteins can be expressed either one at a time or in conjunction with each other to dissect the molecular functions of each virulent factor in a cell-type-specific manner. Well documented phenotypes allow large genetic and pharmacological screens to be performed with relative ease using huge collections of mutant and transgenic strains that are publicly available. These factors combine to make Drosophila a powerful tool for dissecting out host-pathogen interactions as well as a tool to better understand how we can treat infectious diseases that pose risks to public health, including COVID-19, caused by SARS-CoV-2.

Using Drosophila to drive the diagnosis and understand the mechanisms of rare human diseases.

Next-generation sequencing has greatly accelerated the discovery of rare human genetic diseases. Nearly 45% of patients have variants associated with known diseases but the unsolved cases remain a conundrum. Moreover, causative mutations can be difficult to pinpoint because variants frequently map to genes with no previous disease associations and, often, only one or a few patients with variants in the same gene are identified. Model organisms, such as Drosophila, can help to identify and characterize these new disease-causing genes. Importantly, Drosophila allow quick and sophisticated genetic manipulations, permit functional testing of human variants, enable the characterization of pathogenic mechanisms and are amenable to drug tests. In this Spotlight, focusing on microcephaly as a case study, we highlight how studies of human genes in Drosophila have aided our understanding of human genetic disorders, allowing the identification of new genes in well-established signaling pathways.

Mutations in ANKLE2, a ZIKA Virus Target, Disrupt an Asymmetric Cell Division Pathway in Drosophila Neuroblasts to Cause Microcephaly.

The apical Par complex, which contains atypical protein kinase C (aPKC), Bazooka (Par-3), and Par-6, is required for establishing polarity during asymmetric division of neuroblasts in Drosophila, and its activity depends on L(2)gl. We show that loss of Ankle2, a protein associated with microcephaly in humans and known to interact with Zika protein NS4A, reduces brain volume in flies and impacts the function of the Par complex. Reducing Ankle2 levels disrupts endoplasmic reticulum (ER) and nuclear envelope morphology, releasing the kinase Ballchen-VRK1 into the cytosol. These defects are associated with reduced phosphorylation of aPKC, disruption of Par-complex localization, and spindle alignment defects. Importantly, removal of one copy of ballchen or l(2)gl suppresses Ankle2 mutant phenotypes and restores viability and brain size. Human mutational studies implicate the above-mentioned genes in microcephaly and motor neuron disease. We suggest that NS4A, ANKLE2, VRK1, and LLGL1 define a pathway impinging on asymmetric determinants of neural stem cell division.

Drosophila p53 directs nonapoptotic programs in postmitotic tissue.

TP53 is the most frequently mutated gene in human cancers, and despite intensive research efforts, genome-scale studies of p53 function in whole animal models are rare. The need for such in vivo studies is underscored by recent challenges to established paradigms, indicating that unappreciated p53 functions contribute to cancer prevention. Here we leveraged the Drosophila system to interrogate p53 function in a postmitotic context. In the developing embryo, p53 robustly activates important apoptotic genes in response to radiation-induced DNA damage. We recently showed that a p53 enhancer (p53RErpr) near the cell death gene reaper forms chromatin contacts and enables p53 target activation across long genomic distances. Interestingly, we found that this canonical p53 apoptotic program fails to activate in adult heads. Moreover, this failure to exhibit apoptotic responses was not associated with altered chromatin contacts. Instead, we determined that p53 does not occupy the p53RErpr enhancer in this postmitotic tissue as it does in embryos. Through comparative RNA-seq and chromatin immunoprecipitation-seq studies of developing and postmitotic tissues, we further determined that p53 regulates distinct transcriptional programs in adult heads, including DNA repair, metabolism, and proteolysis genes. Strikingly, in the postmitotic context, p53-binding landscapes were poorly correlated with nearby transcriptional effects, raising the possibility that p53 enhancers could be generally acting through long distances.

Comparative Flavivirus-Host Protein Interaction Mapping Reveals Mechanisms of Dengue and Zika Virus Pathogenesis.

Mosquito-borne flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), are a growing public health concern. Systems-level analysis of how flaviviruses hijack cellular processes through virus-host protein-protein interactions (PPIs) provides information about their replication and pathogenic mechanisms. We used affinity purification-mass spectrometry (AP-MS) to compare flavivirus-host interactions for two viruses (DENV and ZIKV) in two hosts (human and mosquito). Conserved virus-host PPIs revealed that the flavivirus NS5 protein suppresses interferon stimulated genes by inhibiting recruitment of the transcription complex PAF1C and that chemical modulation of SEC61 inhibits DENV and ZIKV replication in human and mosquito cells. Finally, we identified a ZIKV-specific interaction between NS4A and ANKLE2, a gene linked to hereditary microcephaly, and showed that ZIKV NS4A causes microcephaly in Drosophila in an ANKLE2-dependent manner. Thus, comparative flavivirus-host PPI mapping provides biological insights and, when coupled with in vivo models, can be used to unravel pathogenic mechanisms.

Developmental Expression of 4-Repeat-Tau Induces Neuronal Aneuploidy in Drosophila Tauopathy Models.

Tau-mediated neurodegeneration in Alzheimer's disease and tauopathies is generally assumed to start in a normally developed brain. However, several lines of evidence suggest that impaired Tau isoform expression during development could affect mitosis and ploidy in post-mitotic differentiated tissue. Interestingly, the relative expression levels of Tau isoforms containing either 3 (3R-Tau) or 4 repeats (4R-Tau) play an important role both during brain development and neurodegeneration. Here, we used genetic and cellular tools to study the link between 3R and 4R-Tau isoform expression, mitotic progression in neuronal progenitors and post-mitotic neuronal survival. Our results illustrated that the severity of Tau-induced adult phenotypes depends on 4R-Tau isoform expression during development. As recently described, we observed a mitotic delay in 4R-Tau expressing cells of larval eye discs and brains. Live imaging revealed that the spindle undergoes a cycle of collapse and recovery before proceeding to anaphase. Furthermore, we found a high level of aneuploidy in post-mitotic differentiated tissue. Finally, we showed that overexpression of wild type and mutant 4R-Tau isoform in neuroblastoma SH-SY5Y cell lines is sufficient to induce monopolar spindles. Taken together, our results suggested that neurodegeneration could be in part linked to neuronal aneuploidy caused by 4R-Tau expression during brain development.

Towards automatic image analysis and assessment of the multicellular apoptosis process.

Apoptotic programmed cell death (PCD) is a fundamental aspect of developmental maturation. However, the authors' understanding of apoptosis, especially in the multi-cell regime, is incomplete because of the difficulty of identifying dying cells by conventional strategies. Real-time in vivo microscopy of Drosophila, an excellent model system for studying the PCD during development, has been used to uncover plausible collective apoptosis at the tissue level, although the dynamic regulation of the process remains to be deciphered. In this work, the authors have developed an image-analysis program that can quantitatively analyse time-lapse microscopy of live tissues undergoing apoptosis with a fluorescent nuclear marker, and subsequently extract the spatiotemporal patterns of multicellular response. The program can process a large number of cells (>103) automatically tracked across sets of image frames. It is applied to characterise the apoptosis of Drosophila wing epithelium at eclosion. Using the natural anatomic structures as reference, the authors identify dynamic patterns in the progression of PCD within the Drosophila tissues. The results not only confirm the previously observed collective multi-cell behaviour from a quantitative perspective, but also reveal a plausible role played by the anatomic structures, such as the wing veins, in the PCD propagation across the Drosophila wing.

Hid arbitrates collective cell death in the Drosophila wing.

Elimination of cells and tissues by apoptosis is a highly conserved and tightly regulated process. In Drosophila, the entire wing epithelium is completely removed shortly after eclosion. The cells that make up this epithelium are collectively eliminated through a highly synchronized form of apoptotic cell death, involving canonical apoptosome genes. Here we present evidence that collective cell death does not require cell-cell contact and show that transcription of the IAP antagonist, head involution defective, is acutely induced in wing epithelial cells prior to this process. hid mRNAs accumulate to levels that exceed a component of the ribosome and likewise, Hid protein becomes highly abundant in these same cells. hid function is required for collective cell death, since loss of function mutants shows persisting wing epithelial cells and, furthermore, silencing of the hormone bursicon in the CNS produced collective cell death defective phenotypes manifested in the wing epithelium. Taken together, our observations suggest that acute induction of Hid primes wing epithelial cells for collective cell death and that Bursicon is a strong candidate to trigger this process, possibly by activating the abundant pool of Hid protein already present.

Analysis of p53 transactivation domain mutants reveals Acad11 as a metabolic target important for p53 pro-survival function.

The p53 tumor suppressor plays a key role in maintaining cellular integrity. In response to diverse stress signals, p53 can trigger apoptosis to eliminate damaged cells or cell-cycle arrest to enable cells to cope with stress and survive. However, the transcriptional networks underlying p53 pro-survival function are incompletely understood. Here, we show that in oncogenic-Ras-expressing cells, p53 promotes oxidative phosphorylation (OXPHOS) and cell survival upon glucose starvation. Analysis of p53 transcriptional activation domain mutants reveals that these responses depend on p53 transactivation function. Using gene expression profiling and ChIP-seq analysis, we identify several p53-inducible fatty acid metabolism-related genes. One such gene, Acad11, encoding a protein involved in fatty acid oxidation, is required for efficient OXPHOS and cell survival upon glucose starvation. This study provides new mechanistic insight into the pro-survival function of p53 and suggests that targeting this pathway may provide a strategy for therapeutic intervention based on metabolic perturbation.

A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases.

Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.

Automatic image analysis of multicellular apoptosis process.

Apoptotic programmed cell death (PCD) is a common and fundamental aspect of developmental maturation. Image processing techniques have been developed to detect apoptosis at the single-cell level in a single still image, while an efficient algorithm to automatically analyze the temporal progression of apoptosis in a large population of cells is unavailable. In this work, we have developed an ImageJ-based program that can quantitatively analyze time-lapse microscopy movies of live tissues undergoing apoptosis with a fluorescent cellular marker, and subsequently extract the temporospatial pattern of multicellular response. The protocol is applied to characterize apoptosis of Drosophila wing epithelium cells at eclosion. Using natural anatomic structures as reference, we identify dynamic patterns in the progression of apoptosis within the wing tissue, which not only confirms the previously observed collective cell behavior from a quantitative perspective for the first time, but also reveals a plausible role played by the anatomic structures in Drosophila apoptosis.

A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans.

We examined how a p53 enhancer transmits regulatory information in vivo. Using genetic ablation together with digital chromosome conformation capture and fluorescent in situ hybridization, we found that a Drosophila p53 enhancer region (referred to as the p53 response element [p53RE]) physically contacts targets in cis and across the centromere to control stress-responsive transcription at these sites. Furthermore, when placed at ectopic genomic positions, fragments spanning this element re-established chromatin contacts and partially restored target gene regulation to mutants lacking the native p53RE. Therefore, a defined p53 enhancer region is sufficient for long-range chromatin interactions that enable multigenic regulation.

Genome-wide silencing in Drosophila captures conserved apoptotic effectors.

Apoptosis is a conserved form of programmed cell death firmly established in the aetiology, pathogenesis and treatment of many human diseases. Central to the core machinery of apoptosis are the caspases and their proximal regulators. Current models for caspase control involve a balance of opposing elements, with variable contributions from positive and negative regulators among different cell types and species. To advance a comprehensive view of components that support caspase-dependent cell death, we conducted a genome-wide silencing screen in the Drosophila model. Our strategy used a library of double-stranded RNAs together with a chemical antagonist of Inhibitor of apoptosis proteins (IAPs) that simulates the action of native regulators in the Reaper and Smac (also known as Diablo) families. Here we present a highly validated set of targets that is necessary for death provoked by several stimuli. Among these, Tango7 is identified as a new effector. Cells depleted for this gene resisted apoptosis at a step before the induction of effector caspase activity, and the directed silencing of Tango7 in Drosophila prevented caspase-dependent programmed cell death. Unlike known apoptosis regulators in this model system, Tango7 activity did not influence stimulus-dependent loss of Drosophila DIAP1 (also known as th and IAP1), but instead regulated levels of the apical caspase Dronc (Nc). Similarly, the human Tango7 counterpart, PCID1 (also known as EIF3M), impinged on caspase 9, revealing a new regulatory axis affecting the apoptosome.

A collective form of cell death requires homeodomain interacting protein kinase.

We examined post-eclosion elimination of the Drosophila wing epithelium in vivo where collective "suicide waves" promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. We leveraged this phenotype in mosaic animals to discover relevant genes and establish here that homeodomain interacting protein kinase (HIPK) is required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers.