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In situ hybridization techniques are powerful methods for exploring gene expression in a wide range of biological contexts, providing spatial information that is most often lost in traditional biochemical techniques. However, many in situ hybridization methods are costly and time-inefficient, particularly for screening-based projects that follow on from single-cell RNA sequencing data, which rely on of tens of custom-synthetized probes against each specific RNA of interest. Here we provide an optimized pipeline for Hybridization Chain Reaction (HCR)-based RNA visualization, including an open-source code for optimized probe design. Our method achieves high specificity and sensitivity with the option of multiplexing using only five pairs of probes, which greatly lowers the cost and time of the experiment. These features of our HCR protocol are particularly useful and convenient for projects involving screening several genes at medium throughput, especially as the method include an amplification step, which makes the signal readily visible at low magnification imaging.

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.

Adenosine-to-inosine (A-to-I) RNA editing recodes the genome and confers flexibility for the organisms to adapt to the environment. It is believed that RNA recoding sites are well suited for facilitating adaptive evolution by increasing the proteomic diversity in a temporal-spatial manner. The function and essentiality of a few conserved recoding sites are recognized. However, the experimentally discovered functional sites only make up a small corner of the total sites, and there is still the need to expand the repertoire of such functional sites with bioinformatic approaches. In this study, we define a new category of RNA editing sites termed 'conserved editing with non-conserved recoding' and systematically identify such sites in Drosophila editomes, figuring out their selection pressure and signals of adaptation at inter-species and intra-species levels. Surprisingly, conserved editing sites with non-conserved recoding are not suppressed and are even slightly overrepresented in Drosophila. DNA mutations leading to such cases are also favoured during evolution, suggesting that the function of those recoding events in different species might be diverged, specialized, and maintained. Finally, structural prediction suggests that such recoding in potassium channel Shab might increase ion permeability and compensate the effect of low temperature. In conclusion, conserved editing with non-conserved recoding might be functional as well. Our study provides novel aspects in considering the adaptive evolution of RNA editing sites and meanwhile expands the candidates of functional recoding sites for future validation.

The Drosophila melanogaster brain is a complex organ with various cell types, orchestrating the development, physiology, and behaviors of the fly. While each cell type in Drosophila brain is known to express a unique gene set, their complete genetic profile is still unknown. Advances in the RNA sequencing techniques at single-cell resolution facilitate identifying novel cell type markers and/or re-examining the specificity of the available ones. In this study, exploiting a single-cell RNA sequencing data of Drosophila optic lobe, we categorized the cells based on their expression pattern for known markers, then the genes with enriched expression in astrocytes were identified. CG11000 was identified as a gene with a comparable expression profile to the Eaat1 gene, an astrocyte marker, in every individual cell inside the Drosophila optic lobe and midbrain, as well as in the entire Drosophila brain throughout its development. Consistent with our bioinformatics data, immunostaining of the brains dissected from transgenic adult flies showed co-expression of CG11000 with Eaat1 in a set of single cells corresponding to the astrocytes in the Drosophila brain. Physiologically, inhibiting CG11000 through RNA interference disrupted the normal development of male D. melanogaster, while having no impact on females. Expression suppression of CG11000 in adult flies led to decreased locomotion activity and also shortened lifespan specifically in astrocytes, indicating the gene's significance in astrocytes. We designated this gene as 'deathstar' due to its crucial role in maintaining the star-like shape of glial cells, astrocytes, throughout their development into adult stage.

Parkinson's disease (PD), the most prevalent type of parkinsonism, is a progressive neurodegenerative condition marked by several non-motor and motor symptoms. PD is thought to have a complex aetiology that includes a combination of age, genetic predisposition, and environmental factors. Increased expression of α-synuclein (α-Syn) protein is central to the evolvement of neuropathology in this devastating disorder, but the potential of ribose-cysteine and levodopa in abating pathophysiologic changes in PD model is unknown. Crosses were set up between flies conditionally expressing a pathological variant of human α-Syn (UAS-α-Syn) and those expressing GAL4 in neurons (elav-GAL4) to generate offspring referred to as PD flies. Flies were randomly assigned to five groups (n = 40) from the total population of flies, with each group having five replicates. Groups of PD flies were treated with either 500 mg/kg ribose-cysteine diet, 250 mg/kg levodopa diet, or a combination of the two compounds for 21 days, whereas the control group (w¹¹¹⁸) and the PD group were exposed to a diet without ribose-cysteine or levodopa. In addition to various biochemical and neurochemical assays, longevity, larval motility, and gravitaxis assays were carried out. Locomotive capability, lifespan, fecundity, antioxidant state, and neurotransmitter systems were all significantly (p < 0.05) compromised by overexpression of α-Syn. However, flies treated both ribose cysteine and levodopa showed an overall marked improvement in motor functions, lifespan, fecundity, antioxidant status, and neurotransmitter system functions. In conclusion, ribose-cysteine and levodopa, both singly and in combination, potentiated a therapeutic effect on alpha-synuclein transgenic Drosophila melanogaster models of Parkinsonism.

Amino acyl-tRNA synthetases perform diverse non-canonical functions aside from their essential role in charging tRNAs with their cognate amino acid. The phenylalanyl-tRNA synthetase (PheRS/FARS) is an α₂β₂ tetramer that is needed for charging the tRNA^Phe for its translation activity. Fragments of the α-subunit have been shown to display an additional, translation-independent, function that activates growth and proliferation and counteracts Notch signalling. Here we show in Drosophila that overexpressing the β-subunit in the context of the complete PheRS leads to larval roaming, food avoidance, slow growth, and a developmental delay that can last several days and even prevents pupation. These behavioural and developmental phenotypes are induced by PheRS expression in CCHa2⁺ and Pros⁺ cells. Simultaneous expression of β-PheRS, α-PheRS, and the appetite-inducing CCHa2 peptide rescued these phenotypes, linking this β-PheRS activity to the appetite-controlling pathway. The fragmentation dynamic of the excessive β-PheRS points to β-PheRS fragments as possible candidate inducers of these phenotypes. Because fragmentation of human FARS has also been observed in human cells and mutations in human β-PheRS (FARSB) can lead to problems in gaining weight, Drosophila β-PheRS can also serve as a model for the human phenotype and possibly also for obesity.

Chromatin accessibility, histone modifications, and transcription factor binding are highly dynamic during Drosophila metamorphosis and drive global changes in gene expression as larval tissues differentiate into adult structures. Unfortunately, the presence of pupa cuticle on many Drosophila tissues during metamorphosis prevents enzyme access to cells and has limited the use of enzymatic in situ methods for assessing chromatin accessibility and histone modifications. Here, we present a dissociation method for cuticle-bound pupal tissues that is compatible for use with ATAC-Seq and CUT&RUN to interrogate chromatin accessibility and histone modifications. We show this method provides comparable chromatin accessibility data to the non-enzymatic approach FAIRE-seq, with only a fraction of the amount of input tissue required. This approach is also compatible with CUT&RUN, which allows genome-wide mapping of histone modifications with less than 1/10th of the tissue input required for more conventional approaches such as Chromatin Immunoprecipitation Sequencing (ChIP-seq). Our protocol makes it possible to use newer, more sensitive enzymatic in situ approaches to interrogate gene regulatory networks during Drosophila metamorphosis.

Parkinson's disease (PD) is the second most common neurodegenerative disorder, afflicting over 1% of the population of age 60 y and above. The loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) is the primary cause of its characteristic motor symptoms. Studies using Drosophila melanogaster and other model systems have provided much insight into the pathogenesis of PD. However, little is known why certain cell types are selectively susceptible to degeneration in PD. Here, we describe an approach to identify vulnerable subpopulations of neurons in the genetic background linked to PD in Drosophila, using the split-GAL4 drivers that enable genetic manipulation of a small number of defined cell populations. We identify split-GAL4 lines that target neurons selectively vulnerable in a model of leucine-rich repeat kinase 2 (LRRK2)-linked familial PD, demonstrating the utility of this approach. We also show an unexpected caveat of the split-GAL4 system in ageing-related research: an age-dependent increase in the number of GAL4-labelled cells.

Dendritic morphogenesis requires dynamic microtubules (MTs) to form a coordinated cytoskeletal network during development. Dynamic MTs are characterized by their number, polarity and speed of polymerization. Previous studies described a correlation between anterograde MT growth and terminal branch extension in Drosophila dendritic arborization (da) neurons, suggesting a model that anterograde MT polymerization provides a driving force for dendritic branching. We recently found that the Ste20-like kinase Tao specifically regulates dendritic branching by controlling the number of dynamic MTs in a kinase activity-dependent fashion, without affecting MT polarity or speed. This finding raises the interesting question of how MT dynamics affects dendritic morphogenesis, and if Tao kinase activity is developmentally regulated to coordinate MT dynamics and dendritic morphogenesis. We explored the possible correlation between MT dynamics and dendritic morphogenesis together with the activity changes of Tao kinase in C1da and C4da neurons during larval development. Our data show that spatiotemporal changes in the number of dynamic MTs, but not polarity or polymerization speed, correlate with dendritic branching and Tao kinase activity. Our findings suggest that Tao kinase limits dendritic branching by controlling the abundance of dynamic MTs and we propose a novel model on how regulation of MT dynamics might influence dendritic morphogenesis.

Genetic screens are used in Drosophila melanogaster to identify genes key in the regulation of organismal development and growth. These screens have defined signalling pathways necessary for tissue and organismal development, which are evolutionarily conserved across species, including Drosophila. Here, we have used an FLP/FRT mosaic system to screen for conditional regulators of cell growth and cell division in the Drosophila eye. The conditional nature of this screen utilizes a block in the apoptotic pathway to prohibit the mosaic mutant cells from dying via apoptosis. From this screen, we identified two different mutants that mapped to the Hedgehog signalling pathway. Previously, we described a novel Ptc mutation and here we add to the understanding of disrupting the Hh pathway with a novel allele of Cos2. Both of these Hh components are negative regulators of the pathway, yet they depict mutant differences in the type of overgrowth created. Ptc mutations lead to overgrowth consisting of almost entirely wild-type tissue (non-autonomous overgrowth), while the Cos2 mutation results in tissue that is overgrown in both the mutant and wild-type clones (both autonomous and non-autonomous). These differences in tissue overgrowth are consistent in the Drosophila eye and wing. The observed difference is correlated with different deregulation patterns of pMad, the downstream effector of DPP signalling. This finding provides insight into pathway-specific differences that help to better understand intricacies of developmental processes and human diseases that result from deregulated Hedgehog signalling, such as basal cell carcinoma.