ABSTRACT Angelman syndrome (AS) is a rare neurogenetic disorder characterized by developmental delay, speech impairment, ataxia, epilepsy, and in some cases hyperphagic feeding behavior. AS is caused by loss of function mutations, loss of expression, or maternal allele deletion of the E3 ubiquitin ligase UBE3A. Recent work has identified a connection between UBE3A and the mechanosensitive ion channel PIEZO2, raising the possibility that UBE3A may regulate PIEZO-dependent satiety signaling. In this study, we investigated the role of the Drosophila UBE3A ortholog, Dube3a, in Piezo-associated feeding behaviors. Single-cell RNA-sequencing data revealed overlapping expression of Dube3a and Piezo within crop and enterocyte populations of the gut, identifying a relevant cellular context for this pathway to occur. We developed a novel feeding assay using GFP-expressing yeast to quantify food intake and gut distention in vivo. Dube3a loss-of-function (Dube3a 15b ) flies exhibited hyperphagia and gut distention nearly identical to Piezo knockout flies. Analysis of chromosomal deficiency lines spanning the Dube3a locus further supported a requirement for Dube3a in normal satiety signaling. Finally, biochemical analyses demonstrated that Dube3a knockdown results in decreased Piezo protein levels, consistent with an indirect regulatory relationship. Together, these findings identify Dube3a as a critical regulator of Piezo-dependent satiety pathways and suggest that dysregulation of mechanosensory signaling may contribute to hyperphagia observed in AS. Further work is needed to define the intermediate factors linking UBE3A activity to Piezo stability and function.

ABSTRACT Ribosomal protein (RP) gene haploinsufficiency is a conserved form of ribosome dysfunction across species and underlies a class of disorders known as ribosomopathies. In Drosophila, RP gene haploinsufficiency manifests as the Minute phenotype, characterized by thinner and shorter mechanosensory bristles. The development of both bristles and proprioceptive campaniform sensilla (CS) is initiated by the bHLH proneural proteins Achaete (Ac) and Scute (Sc). By analysing genetic interactions between ac sc mutants and Minute mutants of varying severity, we identified a novel bristle-promoting effect that occurs only in the strongly affected Minutes in which the average bristle length is shorter than a threshold. This threshold-dependent effect also promotes ectopic CS formation in the strong Minutes. Transcriptomic analyses comparing the sensory organ – promoting and non-promoting Minutes revealed significant differences in stress-response pathways, including differentially elevated expression of the Xrp1–Irbp18 transcriptional dimer. Notably, mutation of Xrp1 suppresses the ectopic CS phenotype, indicating a positive regulatory role. These findings reveal a previously unrecognized threshold effect in RP gene haploinsufficiency, in which excessive Xrp1 activity promotes supernumerary sensory organ formation, suggesting a compensatory mechanism that modulates neurogenesis under severe ribosomal stress.

ABSTRACT Neurodegenerative diseases are devastating conditions characterized by progressive cognitive decline with few available treatments. Neurodegeneration can be quantified in vertebrate and invertebrate models of disease by analysis of vacuolation – the formation of empty spaces within brain tissue. Previous approaches for quantifying this phenotype have required time-consuming methods such as manual counting and measuring of vacuole dimensions, which can be subjective. Here we describe VacQuant, a novel application that can be paired with existing machine learning software to automatically measure the area of vacuolation in brain tissue. Using Drosophila brain sections from tauopathy model flies, a well-described model of dementia-related neurodegeneration, we quantified a significant increase in brain vacuolation at several timepoints in adult flies with the aid of VacQuant. When compared with quantification by five blinded volunteers, the machine learning method positively correlated with their group average, confirming its accuracy and functionality. This automated method developed with VacQuant removes human bias and measurement variation, providing a consistent threshold for all brain sections and experiments. This automated pipeline will be particularly useful for high-throughput screening for genetic modifiers or therapeutic compounds in animal models of neurodegeneration.

ABSTRACT We are utilizing an adult penetrating traumatic brain injury (PTBI) model in Drosophila to investigate regenerative mechanisms after damage to the central brain. Here, we focus on cell proliferation as an early event in the regenerative process. To identify pathways that could trigger cell proliferation following PTBI, we utilized bulk RNA-Seq. We find that transcript levels for components of both Toll and Immune Deficiency (Imd) innate immunity pathways are rapidly and highly upregulated post-PTBI. We then tested mutants for the NF-κB transcription factors of the Toll and Imd pathways, Dorsal-related immunity factor (Dif) and Relish (Rel), respectively. We find that loss of either Dif or Rel results in loss of cell proliferation after injury and identify tissue-specific requirements for Dif and Rel. In addition, while the canonical downstream targets of Drosophila innate immune signalling, the antimicrobial peptides (AMPs), are upregulated following PTBI, their levels revert to near baseline within 24 hr. Taken together, these results indicate that the innate immunity pathways play an integral role in the regenerative response and that this response may not require the antimicrobial peptides. Innate immunity previously has been implicated as both a potentiator and an inhibitor of regenerative processes. Our work suggests that modulation of innate immunity may be essential to prevent adverse outcomes. Thus, this work is likely to inform future experiments to dissect regenerative mechanisms in higher organisms as well as in Drosophila.

ABSTRACT Drosophila melanogaster is an incredible model system, providing tools and technologies that allow careful, effective, and reproducible research. This experimental approach, and the genetic tools and techniques available in Drosophila are desperately needed for the study of other insects, a hugely diverse group of huge importance to natural and productive ecosystems. For those of you with the skills and ‘Drosophila mindset’, studying other insects may help us understand diversity, improve the security of food production, and help avoid the current, worrying, insect apocalypse.

ABSTRACT Three decades of research aimed at understanding the basis for autosomal recessive primary microcephaly (MCPH), a human clinical disorder defined by a significant reduction in head and brain size, has uncovered a suite of ~30 genes that participate in this process. Work in both vertebrate and invertebrate model systems have been instrumental in attempting to link MCPH gene function to the brain growth phenotype. However, we still lack definitive evidence as to what these functions are for many of these genes. In this review, we summarize recent work in Drosophila aimed at overcoming these limitations in our knowledge of MCPH gene function that may be applicable to humans. We discuss the clinical features of MCPH, parallels between human and Drosophila neurogenesis modes with a particular focus on the fly optic lobe, and highlight four of the most well-studied Drosophila MCPH orthologs: abnormal spindle (asp)/MCPH5, Microcephalin/MCPH1, WD Repeat-Containing Protein 62 (Wdr62)/MCPH2, and Ankryin Repeat-and LEM Domain- Containing Protein 2 (ANKLE2)/MCPH16. We focus on the multifunctional roles for these proteins that may underlie the microcephaly phenotype and advocate for the use of flies as a relevant model for human MCPH.

ABSTRACT UCH-L1 (Ubiquitin Carboxyl-terminal Hydrolase – L1) is a protein that plays a critical role in the ubiquitin-proteasome system. Previous studies have demonstrated a link between UCH-L1 and various diseases, including neurodegenerative disorders, diabetes, and cancer. However, the role of UCH-L1 in development remains unclear. To investigate the functions of UCH-L1 in a living organism, taking advantage of the Drosophila model, and to explore the correlation between Drosophila UCH (dUCH) and human UCH-L1, we established a GAL4/UAS-targeted expression system to examine the effect of dUCH on Drosophila eye development. We found that knockdown of dUCH resulted in a rough eye phenotype associated with the MAPK pathway. In this study, for the first time, we revealed that loss of dUCH function leads to a reduction in EGFR protein levels. Additionally, dUCH knockdown downregulated Spitz (spi), a ligand of EGFR, as well as Draf, a key component of the MAPK pathway. Furthermore, under dUCH knockdown conditions, several genes known to play critical roles in eye cell differentiation were affected, including the downregulation of sens, salm, lz, barth1/2, and salm, which are essential for the differentiation of R2/5, R3/4, and R1/6 photoreceptor cells. Interestingly, dUCH was found to be involved not only in the MAPK pathway but also in the regulation of pros, lz, barth1/2, and sev gene expression, suggesting its role in R7 photoreceptor differentiation. Taken together, these findings highlight the important role of dUCH in regulating genes associated with eye cell differentiation and its involvement in EGFR signalling in Drosophila melanogaster.

ABSTRACT One hundred years ago, two reports appeared of tetraploid D. melanogaster females – curiosities that had never been seen before. The authors, Calvin Bridges and Lilian Morgan, were among the famed founders of fly genetics in T.H. Morgan’s lab at Columbia University. Sadly, their findings have faded into the fog of ancient fly lore. This review exhumes those relics in order to offer modern fly-pushers some possible avenues for polyploid research. That subfield is undergoing a revival that may interest them.

ABSTRACT This study investigates the effects of polyglutamine (polyQ) expansions on the locomotion of Drosophila larvae, focusing on the role of class IV dendritic arborization (da) neurons. PolyQ expansions are associated with neurodegenerative diseases like Huntington’s disease, and Drosophila is a valuable model organism for studying these diseases due to its genetic tractability and short generation time. We found that expressing a polyQ protein in class IV da neurons caused significant locomotion deficits. Specifically, larvae with polyQ expression exhibited slower crawling speed and increased turn frequency, indicating impaired movement. The most intriguing finding of our study was that electrically silencing class IV da neurons completely rescued the locomotion deficits caused by polyQ expression. By expressing a potassium channel that makes the neurons less active, we effectively reversed the locomotion defects. This suggests that modulating the activity of these neurons could be a promising therapeutic approach for treating polyQ diseases. Our findings have significant implications for understanding polyQ diseases and developing new therapeutic approaches. By electrically silencing these neurons, we may be preventing the harmful effects of polyQ-induced cation channels, which are thought to disrupt cellular function. This opens up exciting possibilities for exploring electrical silencing as a potential treatment for polyQ diseases, offering hope for future therapies that target the underlying mechanisms of these devastating conditions.