Biology Open

Polarity is one of the fundamental properties of cells. It is characterised by the asymmetric distribution of lipids and proteins in the cell membrane and cortex, and organelles in the cell. Cell polarisation drives essential processes such as morphogenesis, cell migration, asymmetric cell division, directional transport of molecules across epithelium, nerve impulse transmission etc. Although, the importance of polarity proteins such as atypical protein kinase C (aPKC) and Lgl2 (lethal giant larvae 2) has been relatively well understood in the developing zebrafish epidermis, whether basal lamina components are important for polarity maintenance has remained unclear. I analysed the role of Laminin5 (basal lamina component) in the establishment and maintenance of apicobasal polarity, in developing bi layered zebrafish epidermis. I found that the loss of laminin 5 function results in reduced E cadherin localization and increased cell spreading along with dynamic cell boundaries and increased cell proliferation indicating acquisition of mesenchymal traits. A similar phenotype was observed in integrin 6b mutant, which did not exacerbate in the double mutant embryos indicating that laminin 5 and integrin 6b function in the same pathway. Interestingly, periderm, the cell layer above basal epidermis, maintains its apicobasal polarity and epithelial integrity presumably via reinforcing the localization of aPKC and Lgl. My work unravels the importance of Laminin 5 and Integrin 6b interaction in the maintenance of epithelial characteristics in the basal layer of the developing zebrafish epidermis.

Cutaneous wounds are common afflictions that follow a stereotypical healing process involving hemostasis, inflammation, proliferation, and remodeling phases. In the elderly or those suffering from vascular or metabolic diseases, poor healing following cutaneous injuries can lead to open chronic wounds susceptible to infection. The discovery of new therapeutic strategies to improve this defective wound healing requires a better understanding of the cellular behaviors and molecular mechanisms that drive the different phases of wound healing and how these are altered with age or disease. The zebrafish provides an ideal model for visualization and experimental manipulation of the cellular and molecular events during wound healing in the context of an intact, living animal. To facilitate studies of cutaneous wound healing in the zebrafish, we have developed an inexpensive, simple, and effective method for generating reproducible cutaneous injuries in adult zebrafish using a rotary tool. Using our injury system in combination with live imaging, we can monitor skin re-epithelialization, immune cell recruitment, and vessel regrowth and remodeling in the same animal over time. This injury system provides a valuable new experimental platform to study key cellular and molecular events during wound healing in vivo with unprecedented resolution.

Notch signalling is a critical regulator of multiple developmental processes through its ability to control gene expression and thereby influence cell fate specification and cell proliferation through direct cell-cell communication. Although Notch signalling has been implicated in myogenesis during late embryogenesis, its role in early mesoderm development has been largely unexplored. Endocytosis of the Notch ligand Delta and the Notch receptor extracellular domain, a critical step in Notch pathway activation, has been extensively observed in the ventral mesoderm of the early Drosophila embryo, indicating a potential for Notch signalling activity in this early germ layer. Here we present evidence that genes critical to mesoderm development require and are responsive to Notch signalling activity. Using a novel light-inducible Optogenetic variant of the Notch intracellular domain (OptoNotch), which affords precise spatial and temporal control over ectopic activation of Notch signalling, in combination with high-resolution fluorescent RNA in situ hybridization and qPCR, we identified a set of mesodermal genes whose expression is directly regulated by Notch signalling. We also provide evidence that Notch signalling indirectly regulates the dorsal-ventral patterning program mediated by the Toll signalling pathway through the Dorsal/ Twist/ Snail gene network. Our findings demonstrate that Notch signalling regulates ventral mesoderm patterning and is critical for establishing the mesoderm-mesectoderm-ectoderm boundary by regulating gene expression patterns and providing negative feedback on the upstream patterning network.

Zebrafish, a widely used model organism in developmental and biomedical research, offers several advantages such as external fertilization, embryonic transparency, and genetic similarity to humans. However, traditional methods for introducing exogenous genetic material into zebrafish embryos, particularly microinjection, pose significant technical challenges and limit throughput. To address this, we developed a novel approach utilizing Lipofectamine LTX for the efficient delivery of nucleic acids into zebrafish embryos by lipid-based transfection. Our protocol bypasses the need for microinjection, offering a cost-effective, high-throughput, and user-friendly alternative. This protocol out-lines new strategies for gene delivery in zebrafish to enhance the efficiency and scope of genetic studies in this model system.

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This protocol addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step methods specify reagents, equipment, acquisition parameters, and expected results. While recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, this protocol establishes a flexible approach to standardize organelle quantification to understand multiple organelles simultaneously in their cellular contexts. Basic Protocol 1: Mitochondrial Quantification Basic Protocol 2: Myofibril Quantification Basic Protocol 3: Golgi Apparatus Morphometry Basic Protocol 4: Endoplasmic Reticulum Network Analysis Alternate Protocol 1: Super-Resolution Imaging Protocol

The nucleoskeleton, which is comprised of Lamin A (stiffer), Lamin B, and Lamin interacting proteins, including Emerin, controls nuclear stiffness. Nuclear stiffness regulates 3D single cell migration, but its roles in collective cell migration remain unclear. To define the roles of the nucleoskeleton during collective migration we use Drosophila border cell migration. During migration the nucleoskeleton remodels. Throughout migration, Lamin A is predominantly in the nucleoskeleton of the polar cells, whereas Emerin is progressively reduced in the nucleoskeletons of both the border and polar cells, and Lamin B increases in the border cell nucleoskeleton. Further, the border cell nucleoskeleton is polarized; Lamin B is enriched in the front of the cluster while Emerin is enriched in the back. These nucleoskeletal changes require prostaglandin (PG) signaling. When PG signaling is lost, border cell migration is delayed, Lamin A and Emerin are prevalent within the border cell nucleoskeletons throughout migration and nucleoskeletal polarity is lost. Further, overexpression of Lamin A R237P in the border cells delays migration. These data reveal that border cell cluster nucleoskeletal remodeling requires PG signaling and support that this remodeling facilitates invasive, collective migration. Similar PG regulation of the nucleoskeleton likely promotes collective migration across organisms and contexts. Significance StatementO_LINucleoskeletal remodeling is critical for 3D single cell migration, but its roles in collective migration are poorly understood. C_LIO_LIDuring Drosophila border cell migration, the nucleoskeleton remodels and exhibits polarity that suggests the nuclei are softer in the front and stiffer in the back of the cluster. PG signaling is required for these nucleoskeletal changes and on-time border cell migration. Overexpression of Lamin A R237P in the border cells impairs migration. C_LIO_LIThese results demonstrate for the first time that nucleoskeletal remodeling occurs during an in vivo, collective cell migration and identify PG signaling as a novel regulator of the nucleoskeleton. C_LI

Mechanosensing and mechanotransduction are essential for all living cells. In mammals, Piezo1 and Piezo2 are two mechanically activated cation channels that serve as mechanosensors for a variety of physiological and pathological processes, ranging from touch sensing to sickle cell disease. These two channels are well evolutionarily conserved, and orthologous genes can be traced back to the origin of vertebrates, which underwent whole-genome duplications (WGDs). The number of paralogous genes originating from the vertebrate WGD varies across gene families. Thus, whether there are more PIEZO paralogous genes in vertebrates remains understudied. Here, we identified piezo3, a new paralog of the piezo gene family, and analyzed its evolutionary history using phylogenetic and synteny analyses. The piezo3 gene is present in most vertebrate lineages but absent in birds and most mammals, likely due to nonfunctionalization after WGDs. In addition, we demonstrated that this channel could mediate calcium flux in response to mechanical stimuli in HEK293T cells, suggesting that Piezo3 exhibits PIEZO1/2-like activation and conduction channel functions. Our CRISPR mutation analysis revealed that the zebrafish piezo3 gene is not developmentally essential, possibly because its expression overlaps with other PIEZO channels. Mutant zebrafish showed elevated sensitivity to mechanical force and increased locomotor activity under (photopic) light illumination. Our results suggest that this new mechanical-sensing Piezo channel is widespread in vertebrates and may be critical for vertebrate adaptation by modulating mechanical sensing and light responses during evolution. SIGNIFICANCEAll living cells must sense mechanical forces, whether endogenous or exogenous, and respond to them by transforming these forces into biological signals, which is essential to a wide range of cellular processes, including cell division, growth, and differentiation. PIEZO channels are well-characterized, critical, versatile mechanotransducers for touch and pain physiology and for human diseases. Currently, PIEZO1 and PIEZO2 are the only two known PIEZO channels in most vertebrates. In zebrafish, there are two Piezo2 channels (Piezo2a and Piezo2b) due to extra genome duplication in the ray-finned fishes. Here, we report Piezo3 channel, a long-missing paralog of Piezo1 and Piezo2, in most vertebrates. This channel is present in the majority of vertebrate lineages, except for most birds and mammals. The zebrafish piezo3 gene is expressed during early embryogenesis, and mutation of this gene leads to zebrafish larvae responding to tapping mechanical force and light with active movement. The widespread distribution of this Piezo3 channel across most vertebrate species, but its absence in birds and most mammals, suggests it may play important roles in vertebrate physiology and evolution.

Zebrafish is a powerful model organism for in vivo live imaging. However, protein visualization relies to this date on the overexpression of fluorescently tagged proteins from a specific promoter which very often does not recapitulate endogenous patterns of expression and dynamics. Tagging proteins in the endogenous locus is not widely used in the field due to its technical inefficiency and difficulty. Here we developed a knock-in reporter line for the Foxo1a transcription factor by inserting an EGFP-pA cassette in frame at the C-terminal generating a fusion protein. Foxo1a has been involved in the regulation of many biological processes regarding metabolism, stress response, longevity, cell differentiation and others and its functions are conserved from invertebrates to vertebrates. Using in-vivo live imaging at early developmental stages, we validated the expression in the cardiovascular network, CNS, olfactory epithelium, spinal cord, retina, skeletal muscle, and myocardium. Our line opens the way for imaging studies aiming to characterize the expression and localization of this transcription factor in a tissue and context specific manner. Also, the knock-in line can be used in combination with other modern techniques to determine the transcriptional targets of Foxo1a, many of which remain unknown.

The essential function of the Keap1-Nrf2 pathway in mediating transcriptional response to xenobiotic and oxidative stimuli has been well established. However, the mechanisms whereby Keap1 and Nrf2 regulate developmental genes remains unclear. We hypothesized that Drosophila Keap1 (dKeap1) and Nrf2 (CncC) proteins regulate transcription through controlling high-order chromatin structure. Here, we describe evidence supporting that dKeap1 can regulate chromatin through interaction with lamin, the intermediate filament proteins that form nuclear lamina and organize the overall chromatin architecture. dKeap1 and lamin Dm0, the B-type lamin in Drosophila, interact with each other and form complexes in the nucleus. Overexpression of dKeap1 resulted in a redistribution of lamin Dm0 to the intra-nuclear area and consistently, caused a spreading of the heterochromatin marker H3K9me2 from the pericentromeric region to chromosome arms. Overexpression of dKeap1 fusion proteins in the dKeap1 null background significantly disrupted the nuclear lamina morphology, indicating that dKeap1 is required for the maintenance of a normal nuclear lamina. Knock down of dKeap1 partially rescued the lethality caused by lamin Dm0 overexpression, suggesting that dKeap1 and lamin Dm0 function in the same pathway during development. Taken together, these results support a model where dKeap1 regulates chromatin structure and developmental transcription through interaction with lamin proteins, revealing a novel epigenetic function of the Keap1 oxidative/xenobiotic response factor.

Cardiovascular disease is characterized by aberrant and excessive extracellular matrix (ECM) remodelling, termed fibrosis. Fibrotic remodelling is typically triggered by inflammation, which occurs systemically in obesity. Despite the contribution of fibrosis to adverse clinical outcomes and disease progression, there are no available treatments for this condition. Developing therapeutics for chronic conditions requires an understanding of in vivo ECM regulation, and how the ECM responds to a systemic challenge. We have therefore developed a Drosophila model for obesity via chronic high fat diet feeding and evaluated the response of the cardiac ECM to this metabolic challenge. We found that this model displays a striking disorganization of the cardiac ECM, with corresponding deficits in heart function. Our study shows that different genotypes tolerate varying levels of high fat diets, and that some genotypes may require a different percentage of fat supplementation for achieving an optimal obesity phenotype.

Chemotherapy-related cognitive impairment (CRCI) is a common adverse effect of treatment and is characterized by deficits involving multiple cognitive domains including memory. Despite the significant morbidity of CRCI and the expected increase in cancer survivors over the coming decades, the pathophysiology of CRCI remains incompletely understood, highlighting the need for new model systems to study CRCI. Given the powerful array of genetic approaches and facile high throughput screening ability in Drosophila, our goal was to validate a Drosophila model of CRCI. We administered the chemotherapeutic agents cisplatin, cyclophosphamide, and doxorubicin to adult Drosophila. Neurocognitive deficits were observed with all tested chemotherapies, especially cisplatin. We then performed histologic and immunohistochemical analysis of cisplatin-treated Drosophila tissue, demonstrating neuropathologic evidence of increased neurodegeneration, DNA damage, and oxidative stress. Thus, our Drosophila model of CRCI recapitulates clinical, radiologic, and histologic alterations reported in chemotherapy patients. Our new Drosophila model can be used for mechanistic dissection of pathways contributing to CRCI and pharmacologic screens to identify novel therapies to ameliorate CRCI. Summary StatementWe present a Drosophila model of chemotherapy-related cognitive impairment, which recapitulates neurocognitive and neuropathologic changes observed in cancer patients treated with chemotherapy.

Energy storage and endocrine functions of the Drosophila fat body make it an excellent model for elucidating mechanisms that underlie physiological and pathophysiological organismal metabolism. Combined with Drosophilas robust genetic and immunofluorescence microscopy toolkits, studies of Drosophila fat body function are ripe for cell biological analysis. Unlike the larval fat body, which is easily removed as a single, cohesive sheet of tissue, isolating intact adult fat body proves to be more challenging, thus hindering consistent immunofluorescence labeling even within a single piece of adipose tissue. Here, we describe an improved approach to handling Drosophila abdomens that ensures full access of the adult fat body to solutions generally used in immunofluorescence labeling protocols. In addition, we assess the quality of fluorescence reporter expression and antibody immunoreactivity in response to variations in fixative type, fixation incubation time, and detergent used for cellular permeabilization. Overall, we provide several recommendations for steps in a whole mount staining protocol that results in consistent and robust immunofluorescence labeling of the adult Drosophila fat body. SUMMARY STATEMENTOptimization of adult Drosophila fat body fluorescence microscopy via modifications of tissue handling, fixation, and permeabilization steps in a whole mount immunolabeling protocol.

The cardiac extracellular matrix (ECM) is critical to defining the biophysical properties of the heart that adapt to changing stresses with growth or disease. The ECM is commonly dysregulated in chronic disease such as hypertension, diabetes, and cardiomyopathies, often leading to the development of fibrosis. There are no treatment options to address most ECM cardiomyopathies, but developing therapeutic targets necessitates an understanding of the regulation of ECM remodelling. Here, we employ a larval Drosophila overgrowth model ("giant larvae") to overload the heart and alter ECM remodelling in vivo. These larvae grow to immense sizes without exhibiting hallmarks of obesity. Remarkably, cardiac ECM organization scales allometrically despite overload. The main effect observed is a change in Collagen fibril thickness, possibly reflecting changes to tension in the system. Overgrowth-induced changes in gene expression similarly suggest changes in Collagen assembly, such as a dramatic increase in LOXL2, the main Collagen crosslinking enzyme. This could indicate that larvae may compensate for the stress of overgrowth by stabilizing the Collagen network. The enlarged hearts of giant larvae cannot contract fully at systole. Taken together, this reveals non-allometric changes to cardiac form and output with increasing body size. Overall, our overgrowth model presents an intriguing opportunity to examine the ability of a system to tolerate overgrowth without the metabolic inputs of obesity.

We present the BOOPTHAT (Batch-Operating Optically Powered Targeted Heater for Activating Transgenes) as a low-cost system for activating heat-shock inducible transgenes with spatiotemporal control in multiple zebrafish embryos at a time. Gaining finer spatiotemporal control over gene expression is critical for unraveling the regulatory networks that coordinate embryogenesis. While the heat-shock inducible gene expression system is a widely used tool for controlling temporal transgene expression, its applicability in spatiotemporal control is limited. By adding a level of spatial control onto this well-established system, we take advantage of the existing infrastructure surrounding the HS induction system and introduce new ways to use the multitude of existing lines. The BOOPTHAT system is built from 3D printed components and inexpensive consumer parts. Independent 3D printed micromanipulators are used to position optical fiber probes. When coupled to a light source, the probes are heated photothermally and are used to perform targeted gene activation in multiple samples at a time. We demonstrate the capabilities of our system and highlight some areas of research that stand to benefit from this frugal and effective system.

In this study we examined if the gene encoding Dendritic Cell-specific Six Transmembrane domain containing protein 2 (dcst2) plays a role in vertebrate muscle biology. Using the CRISPR/Cas9 mutagenic system we generated a 2 nucleotide deletion in exon 3 of the zebrafish ortholog dcst2 which resulted in a premature stop codon. Homozygous carriers of the mutation displayed reduced transcriptional expression of dcst2 suggesting that our mutation was indeed disrupting gene function. Mutant dcst2 zebrafish developed normally to adulthood and displayed no differences in motor function using a free-swim and swim tunnel assays. Furthermore, histological examination of muscle cells revealed no differences in slow-twitch or fast-twitch muscle cell cross-sectional area in our mutants. We did observe that dcst2-/- zebrafish were slightly heavier in weight and males were infertile. The data collected here, suggest that dcst2 does not play a role in zebrafish muscle cell biology.

Protein interaction network (PIN) or interactome has been mapped vigorously for the entire genome. We recognize, nonetheless, that such a map could illuminate profound insights had its context been revealed. We describe a scalable protein lableling method that could re-supply natural context back to the map of protein interactome. Genetically encoded fluorescent proteins, position-specific genomic integration and GAL4-responsive expression control enable labeling proteins A, B and C each with a either an eGFP, mCherry or NirFP in specified cells of optically transparent animals such as Drosophila embryos. While following multiple proteins through development and behavior, these labels offer separable pairs of Forster resonance energy transfer between proteins A and B and proteins B and C. We test and observe FRET interactions between specific protein pairs controlling cytoskeleton, nuclear signaling and cell polarity. By using our protein labeling method, it will be possible to map protein interaction network in situ -- isPIN.

Amyloid {beta} (A{beta}) is a peptide known for its characteristic aggregates in Alzheimers Disease and its ability to induce a wide range of detrimental effects in various model systems. However, A{beta} has also been shown to induce some beneficial effects, such as antimicrobial properties against pathogens. In this work, we explore the influence of A{beta} in stress resistance in a C. elegans model of Alzheimers Disease. We found that C. elegans that express human A{beta} exhibit increased resistance to heat and hypoxia, but not to oxidative stress. This beneficial effect of A{beta} was driven from A{beta} in neurons but not muscles, and the abundance of A{beta} in neurons correlated with stress resistance levels. Transcriptomic analysis revealed that this selective stress resistance was mediated by the Heat Shock Protein (HSPs) family of genes. Furthermore, neuropeptide signaling was necessary for A{beta} to induce stress resistance, suggesting neuroendocrine signaling plays a major role in activating organismal stress response pathways. These results highlight the potential beneficial role of A{beta} in cellular function, as well as its complex effects on cellular and organismal physiology that must be considered when using C. elegans as a model for Alzheimers Disease.

Craniofacial birth defects, such as cleft lip and palate, are among the most common congenital anomalies and often arise from disruptions in early facial patterning. Many of these defects are linked to environmental teratogens, yet such exposures cannot be directly tested in humans, making animal models essential for evaluating developmental risks. Xenopus laevis offers a powerful solution: its tadpoles develop externally, share deeply conserved craniofacial patterning mechanisms with humans, and provide an accessible platform for uncovering how environmental exposures reshape facial structures during development. Here, we present the XenCart Protocol, a reproducible workflow for Alcian Blue staining and quantitative morphometric analysis of Xenopus craniofacial cartilage. This method provides clear visualization of individual cartilage elements and can be readily applied to investigate genetic or environmental perturbations. The Xenopus craniofacial skeleton contains distinct cartilaginous structures that perform key biomechanical functions and share strong homology with regions of the human craniofacial skeleton. These similarities allow direct comparison of developmental outcomes across vertebrates. As part of a CURE-based undergraduate course, the XenCart Protocol was used to measure jaw cartilage dimensions in tadpoles exposed to an emerging teratogen, e-liquids used in vaping. E-liquid exposure caused consistent reductions across major craniofacial cartilages, including shorter Meckels cartilage, narrowed infrarostral width, decreased basihyobranchial and ceratohyal dimensions, and reduced suprarostral angles, reflecting an overall shift toward a smaller, more compact craniofacial morphology. These patterns suggest potential disruption of neural crest cell migration or signaling pathways for craniofacial cartilage development, mechanisms that, if similarly affected in humans, could contribute to midfacial narrowing, jaw underdevelopment, or increased vulnerability to conditions such as orofacial clefts. The ability to detect robust, structure-specific differences highlights the sensitivity of the protocol and its strong alignment with student-led research. These findings also pinpoint the precise regions of the jaw most affected by e-liquid exposure, providing a foundation for uncovering the developmental mechanisms driving these craniofacial changes. In summary, the XenCart Protocol provides a standardized, scalable method for quantifying craniofacial cartilage development and offers a powerful platform for both mechanistic research and undergraduate training in developmental biology and toxicology.

The Hippo signaling pathway has been shown to be involved in the regulation of cellular identity, cell/tissue size maintenance and mechanotransduction. The Hippo pathway consists of a kinase cascade which determines the nucleo-cytoplasmic localization of YAP in the cell. YAP is the effector protein in the Hippo pathway which acts as a transcriptional cofactor for TEAD. Phosphorylation of YAP upon activation of the Hippo pathway prevents it from entering the nucleus and hence abrogates its function in transcription of target genes. In Cnidaria, the information on the regulatory roles of the Hippo pathway is virtually lacking. Here, we report for the first time the existence of a complete set of Hippo pathway core components in Hydra. By studying their phylogeny and domain organization, we report evolutionary conservation of the components of the Hippo pathway. Protein modelling suggested conservation of YAP-TEAD interaction in Hydra. We also characterized the expression pattern of the homologs of yap, hippo, mob and sav in Hydra using whole mount RNA in situ hybridization and report their possible role in stem cell maintenance. Immunofluorescence assay revealed that Hvul_YAP expressing cells occur in clusters in the body column and are excluded in the terminally differentiated regions. The YAP expressing cells are recruited early during head regeneration and budding implicating the Hippo pathway in early response to injury or establishment of oral fate. These cells exhibit a non-clustered existence at the site of regeneration and budding, indicating the involvement of a new population of YAP expressing cells during oral fate specification. Collectively, we posit that the Hippo pathway is an important signaling system in Hydra, its components are ubiquitously expressed in the Hydra body column, and may play crucial role in Hydra oral fate specification.

Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, our understanding of its implications in multicellular organisms remains limited, largely due to the constraints of current techniques that hinder our ability to track and manipulate the ISR in vivo. To overcome these limitations, we have successfully developed an internal ribosome entry site (IRES)-based fluorescent reporter system. This innovative reporter enables us to label Drosophila cells, within the context of a living organism, that exhibit eIF2 phosphorylation-dependent translational shutoff - a characteristic feature of the ISR and viral infections. Through this methodology, we have unveiled tissue- and cell-specific regulation of stress response in Drosophila flies and have even been able to detect stressed tissues in vivo during virus and bacterial infections. To further validate the specificity of our reporter, we have engineered ISR-null eIF2S50A mutant flies for stress response analysis. Our results shed light on the tremendous potential of this technique for investigating a broad range of developmental, stress, and infection-related experimental conditions. Combining the reporter tool with ISR-null mutants establishes Drosophila as an exceptionally powerful model for studying the ISR in the context of multicellular organisms.