Larval Brain Research Articles

The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila central nervous system.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

Single dopaminergic neuron DAN-c1 in Drosophila larval brain mediates aversive olfactory learning through D2-like receptors.

The intricate relationship between the dopaminergic system and olfactory associative learning in Drosophila has been an intense scientific inquiry. Leveraging the formidable genetic tools, we conducted a screening of 57 dopaminergic drivers, leading to the discovery of DAN-c1 driver, uniquely targeting the single dopaminergic neuron (DAN) in each brain hemisphere. While the involvement of excitatory D1-like receptors is well-established, the role of D2-like receptors (D2Rs) remains underexplored. Our investigation reveals the expression of D2Rs in both DANs and the mushroom body (MB) of third instar larval brains. Silencing D2Rs in DAN-c1 via microRNA disrupts aversive learning, further supported by optogenetic activation of DAN-c1 during training, affirming the inhibitory role of D2R autoreceptor. Intriguingly, D2R knockdown in the MB impairs both appetitive and aversive learning. These findings elucidate the distinct contributions of D2Rs in diverse brain structures, providing novel insights into the molecular mechanisms governing associative learning in Drosophila larvae.

Darpp-32 is regulated by dopamine and is required for the formation of GABAergic neurons in the developing telencephalon

DARPP-32 (dopamine and cAMP-regulated phosphoprotein Mr. 32 kDa) is a phosphoprotein that is modulated by multiple receptors integrating intracellular pathways and playing roles in various physiological functions. It is regulated by dopaminergic receptors through the cAMP/protein kinase A (PKA) pathway, which modulates the phosphorylation of threonine 34 (Thr34). When phosphorylated at Thr34, DARPP-32 becomes a potent protein phosphatase-1 (PP1) inhibitor. Since dopamine is involved in the development of GABAergic neurons and DARPP-32 is expressed in the developing brain, it is possible that DARPP-32 has a role in GABAergic neuronal development. We cloned the zebrafish darpp-32 gene (ppp1r1b) gene and observed that it is evolutionarily conserved in its inhibitory domain (Thr34 and surrounding residues) and the docking motif (residues 7–11 (KKIQF)). We also characterized darpp-32 protein expression throughout the 5 days post-fertilization (dpf) zebrafish larval brain by immunofluorescence and demonstrated that darpp-32 is mainly expressed in regions that receive dopaminergic projections (pallium, subpallium, preoptic region, and hypothalamus). We demonstrated that dopamine acutely suppressed darpp-32 activity by reducing the levels of p-darpp-32 in the 5dpf zebrafish larval brain. In addition, the knockdown of darpp-32 resulted in a decrease in the number of GABAergic neurons in the subpallium of the 5dpf larval brain, with a concomitant increase in the number of DAergic neurons. Finally, we demonstrated that darpp-32 downregulation during development reduced the motor behavior of 5dpf zebrafish larvae. Thus, our observations suggest that darpp-32 is an evolutionarily conserved regulator of dopamine receptor signaling and is required for the formation of GABAergic neurons in the developing telencephalon.

Fruit ripening retardant Daminozide induces cognitive impairment, cell specific neurotoxicity, and genotoxicity in Drosophila melanogaster

BackgroundWe explored neurotoxic and genotoxic effects of Daminozide, a fruit ripening retardant, on the brain of Drosophila melanogaster, based on our previous finding of DNA fragmentation in larval brain cell in the flies experimentally exposed to this chemicals. MethodsAdult flies were subjected to two distinct concentrations of daminozide (200 mg/L and 400 mg/L) mixed in culture medium, followed by an examination of specific behaviors such as courtship conditioning and aversive phototaxis, which serve as indicators of cognitive functions. We investigated brain histology and histochemistry to assess the overall toxicity of daminozide, focusing on neuron type-specific effects. Additionally, we conducted studies on gene expression specific to neuronal function. Statistical comparisons were then made between the exposed and control flies across all tested attributes. ResultsThe outcome of behavioral assays suggested deleterious effects of Daminozide on learning, short term and long term memory function. Histological examination of brain sections revealed cellular degeneration, within Kenyon cell neuropiles in Daminozide-exposed flies. Neurone specific Immuno-histochemistry study revealed significant reduction of dopaminergic and glutaminergic neurones with discernible reduction in cellular counts, alteration in cell and nuclear morphology among daminozide exposed flies. Gene expression analyses demonstrated upregulation of rutabaga (rut), hb9 and down regulation of PKa- C1, CrebB, Ace and nAchRbeta-1 in exposed flies which suggest dysregulation of gene functions involved in motor neuron activity, learning, and memory. ConclusionTaken together, our findings suggests that Daminozide induces multifaceted harmful impacts on the neural terrain of Drosophila melanogaster, posing a threat to its cognitive abilities.

The FAcilitates Chromatin Transcription complex regulates the ratio of glycolysis to oxidative phosphorylation in neural stem cells.

Defects in the FAcilitates Chromatin Transcription (FACT) complex, a histone chaperone composed of SSRP1 and SUPT16H, are implicated in intellectual disability. Here, we reveal that the FACT complex promotes glycolysis and sustains the correct cell fate of neural stem cells/neuroblasts in the Drosophila 3rd instar larval central brain. We show that the FACT complex binds to the promoter region of the estrogen-related receptor (ERR) gene and positively regulates ERR expression. ERR is known to act as an aerobic glycolytic switch by upregulating the enzymes required for glycolysis. Dysfunction of the FACT complex leads to the downregulation of ERR transcription, resulting in a decreased ratio of glycolysis to oxidative phosphorylation (G/O) in neuroblasts. Consequently, neuroblasts exhibit smaller cell sizes, lower proliferation potential, and altered cell fates. Overexpression of ERR or suppression of mitochondrial oxidative phosphorylation in neuroblasts increases the relative G/O ratio and rescues defective phenotypes caused by dysfunction of the FACT complex. Thus, the G/O ratio, mediated by the FACT complex, plays a crucial role in neuroblast cell fate maintenance. Our study may shed light on the mechanism by which mutations in the FACT complex lead to intellectual disability in humans.

Aneuploidy is Linked to Neurological Phenotypes Through Oxidative Stress

Aneuploidy, having an aberrant genome, is gaining increasing attention in neurodegenerative diseases. It gives rise to proteotoxic stress as well as a stereotypical oxidative shift which makes these cells sensitive to internal and environmental stresses. A growing body of research from numerous laboratories suggests that many neurodegenerative disorders, especially Alzheimer’s disease and frontotemporal dementia, are characterised by neuronal aneuploidy and the ensuing apoptosis, which may contribute to neuronal loss. Using Drosophila as a model, we investigated the effect of induced aneuploidy in GABAergic neurons. We found an increased proportion of aneuploidy due to Mad2 depletion in the third-instar larval brain and increased cell death. Depletion of Mad2 in GABAergic neurons also gave a defective climbing and seizure phenotype. Feeding animals an antioxidant rescued the climbing and seizure phenotype. These findings suggest that increased aneuploidy leads to higher oxidative stress in GABAergic neurons which causes cell death, climbing defects, and seizure phenotype. Antioxidant feeding represents a potential therapy to reduce the aneuploidy-driven neurological phenotype.

Cognitive limits of larval Drosophila: testing for conditioned inhibition, sensory preconditioning, and second-order conditioning.

Drosophila larvae are an established model system for studying the mechanisms of innate and simple forms of learned behavior. They have about 10 times fewer neurons than adult flies, and it was the low total number of their neurons that allowed for an electron microscopic reconstruction of their brain at synaptic resolution. Regarding the mushroom body, a central brain structure for many forms of associative learning in insects, it turned out that more than half of the classes of synaptic connection had previously escaped attention. Understanding the function of these circuit motifs, subsequently confirmed in adult flies, is an important current research topic. In this context, we test larval Drosophila for their cognitive abilities in three tasks that are characteristically more complex than those previously studied. Our data provide evidence for (i) conditioned inhibition, as has previously been reported for adult flies and honeybees. Unlike what is described for adult flies and honeybees, however, our data do not provide evidence for (ii) sensory preconditioning or (iii) second-order conditioning in Drosophila larvae. We discuss the methodological features of our experiments as well as four specific aspects of the organization of the larval brain that may explain why these two forms of learning are observed in adult flies and honeybees, but not in larval Drosophila.

Pan-neuronal expression of human mutant SOD1 in Drosophila impairs survival and motor performance, induces early neuroinflammation and chromosome aberrations

Several mutations in the SOD1 gene encoding for the antioxidant enzyme Superoxide Dismutase 1, are associated with amyotrophic lateral sclerosis, a rare and devastating disease characterized by motor neuron degeneration and patients' death within 2–5 years from diagnosis. Motor neuron loss and related symptomatology manifest mostly in adult life and, to date, there is still a gap of knowledge on the precise cellular and molecular events preceding neurodegeneration.To deepen our awareness of the early phases of the disease, we leveraged two Drosophila melanogaster models pan-neuronally expressing either the mutation A4V or G85R of the human gene SOD1 (hSOD1A4V or hSOD1G85R).We demonstrate that pan-neuronal expression of the hSOD1A4V or hSOD1G85R pathogenic construct impairs survival and motor performance in transgenic flies. Moreover, protein and transcript analysis on fly heads indicates that mutant hSOD1 induction stimulates the glial marker Repo, up-regulates the IMD/Toll immune pathways through antimicrobial peptides and interferes with oxidative metabolism. Finally, cytological analysis of larval brains demonstrates hSOD1-induced chromosome aberrations. Of note, these parameters are found modulated in a timeframe when neurodegeneration is not detected.The novelty of our work is twofold: we have expressed for the first time hSOD1 mutations in all neurons of Drosophila and confirmed some ALS-related pathological phenotypes in these flies, confirming the power of SOD1 mutations in generating ALS-like phenotypes. Moreover, we have related SOD1 pathogenesis to chromosome aberrations and antimicrobial peptides up-regulation. These findings were unexplored in the SOD1-ALS field.

Condensin-mediated restriction of retrotransposable elements facilitates brain development in Drosophila melanogaster

Neural stem and progenitor cell (NSPC) maintenance is essential for ensuring that organisms are born with proper brain volumes and head sizes. Microcephaly is a disorder in which babies are born with significantly smaller head sizes and cortical volumes. Mutations in subunits of the DNA organizing complex condensin have been identified in microcephaly patients. However, the molecular mechanisms by which condensin insufficiency causes microcephaly remain elusive. We previously identified conserved roles for condensins in repression of retrotransposable elements (RTEs). Here, we show that condensin subunit knockdown in NSPCs of the Drosophila larval central brain increases RTE expression and mobility which causes cell death, and significantly decreases adult head sizes and brain volumes. These findings suggest that unrestricted RTE expression and activity may lead to improper brain development in condensin insufficient organisms, and lay the foundation for future exploration of causative roles for RTEs in other microcephaly models.

Single-Nucleus Sequencing of Silkworm Larval Brain Reveals the Key Role of Lysozyme in the Antiviral Immune Response in Brain Hemocytes

Introduction: The brain is considered as an immune-privileged organ, yet innate immune reactions can occur in the central nervous system of vertebrates and invertebrates. Silkworm (Bombyx mori) is an economically important insect and a lepidopteran model species. The diversity of cell types in the silkworm brain, and how these cell subsets produce an immune response to virus infection, remains largely unknown. Methods: Single-nucleus RNA sequencing (snRNA-seq), bioinformatics analysis, RNAi, and other methods were mainly used to analyze the cell types and gene functions of the silkworm brain. Results: We used snRNA-seq to identify 19 distinct clusters representing Kenyon cell, glial cell, olfactory projection neuron, optic lobes neuron, hemocyte-like cell, and muscle cell types in the B. mori nucleopolyhedrovirus (BmNPV)-infected and BmNPV-uninfected silkworm larvae brain at the late stage of infection. Further, we found that the cell subset that exerts an antiviral function in the silkworm larvae brain corresponds to hemocytes. Specifically, antimicrobial peptides were significantly induced by BmNPV infection in the hemocytes, especially lysozyme, exerting antiviral effects. Conclusion: Our single-cell dataset reveals the diversity of silkworm larvae brain cells, and the transcriptome analysis provides insights into the immune response following virus infection at the single-cell level.

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates axon guidance genes in Drosophila.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

Seven-up acts in neuroblasts to specify adult central complex neuron identity and initiate neuroblast decommissioning.

An unanswered question in neurobiology is how are diverse neuron cell types generated from a small number of neural stem cells? In the Drosophila larval central brain, there are eight bilateral Type 2 neuroblast (T2NB) lineages that express a suite of early temporal factors followed by a different set of late temporal factors and generate the majority of the central complex (CX) neurons. The early-to-late switch is triggered by the orphan nuclear hormone receptor Seven-up (Svp), yet little is known about how this Svp-dependent switch is involved in specifying CX neuron identities. Here, we: (1) birth date the CX neurons P-EN and P-FN (early and late, respectively); (2) show that Svp is transiently expressed in all early T2NBs; and (3) show that loss of Svp expands the population of early born P-EN neurons at the expense of late born P-FN neurons. Furthermore, in the absence of Svp, T2NBs fail decommissioning and abnormally extend their lineage into week-old adults. We conclude that Svp is required to specify CX neuron identity, as well as to initiate T2NB decommissioning.

The insc-GAL4 driver marks distinct cell types in Drosophila midgut

Drosophila geneticists frequently employ the binary GAL4-UAS system of conditional gene expression to direct expression of the desired transgene in tissues of interest. The inscuteable -GAL4 driver (insc-GAL4) expresses in the type 1 and type 2 neuroblasts of Drosophila larval brain, a frequent target tissue in many investigations. This GAL4 line additionally displayed its expression in the midgut. In this study, we examined the expression of the UAS-mCD8GFP reporter under the command of the insc-GAL4 driver and observed that this driver expresses exclusively to intestinal stem cells (ISCs) of the Drosophila adult midgut as well as adult midgut precursors (AMPs) of the larval midgut besides its expression in larval brain. Additionally, using the G-TRACE method, it was observed that AMPs in the larval midgut consistently expressed insc-GAL4 in real-time, and the lineage expression of this GAL4 was observed in the enterocyte cells. This study reveals for the first time that insc-GAL4 is specific to larval AMPs and adult ISCs of the midgut.

Angiopoietin 1 and integrin beta 1b are vital for zebrafish brain development.

Angiopoietin 1 (angpt1) is essential for angiogenesis. However, its role in neurogenesis is largely undiscovered. This study aimed to identify the role of angpt1 in brain development, the mode of action of angpt1, and its prime targets in the zebrafish brain. We investigated the effects of embryonic brain angiogenesis and neural development using qPCR, in situ hybridization, microangiography, retrograde labeling, and immunostaining in the angpt1sa14264, itgb1bmi371, tekhu1667 mutant fish and transgenic overexpression of angpt1 in the zebrafish larval brains. We showed the co-localization of angpt1 with notch, delta, and nestin in the proliferation zone in the larval brain. Additionally, lack of angpt1 was associated with downregulation of TEK tyrosine kinase, endothelial (tek), and several neurogenic factors despite upregulation of integrin beta 1b (itgb1b), angpt2a, vascular endothelial growth factor aa (vegfaa), and glial markers. We further demonstrated that the targeted angpt1sa14264 and itgb1bmi371 mutant fish showed severely irregular cerebrovascular development, aberrant hindbrain patterning, expansion of the radial glial progenitors, downregulation of cell proliferation, deficiencies of dopaminergic, histaminergic, and GABAergic populations in the caudal hypothalamus. In contrast to angpt1sa14264 and itgb1bmi371 mutants, the tekhu1667 mutant fish regularly grew with no apparent phenotypes. Notably, the neural-specific angpt1 overexpression driven by the elavl3 (HuC) promoter significantly increased cell proliferation and neuronal progenitor cells but decreased GABAergic neurons, and this neurogenic activity was independent of its typical receptor tek. Our results prove that angpt1 and itgb1b, besides regulating vascular development, act as a neurogenic factor via notch and wnt signaling pathways in the neural proliferation zone in the developing brain, indicating a novel role of dual regulation of angpt1 in embryonic neurogenesis that supports the concept of angiopoietin-based therapeutics in neurological disorders.

Brain size scaling through development in the whitelined sphinx moth (Hyles lineata) shows mass and cell number comparable to flies, bees, and wasps

Factors regulating larval growth and determinants of adult body size are described for several holometabolous insects, but less is known about brain size scaling through development. Here we use the isotropic fractionation (“brain soup”) method to estimate the number of brain cells and cell density for the whitelined sphinx moth (Lepidoptera: Hyles lineata) from the first instar through the adult stage. We measure mass and brain cell number and find that, during the larval stages, body mass shows an exponential relationship with head width, while the total number of brain cells increases asymptotically. Larval brain cell number increases by a factor of ten from nearly 8000 in the first instar to over 80,000 in the fifth instar. Brain cell number increases by another factor of 10 during metamorphosis, with the adult brain containing more than 900,000 cells. This is similar to increases during development in the vinegar fly (Drosophila melanogaster) and the black soldier fly (Hermetia illucens). The adult brain falls slightly below the brain-to-body allometry for wasps and bees but is comparable in the number of cells per unit brain mass, indicating a general conservation of brain cell density across these divergent lineages.

Generation of glial cell-derived neurotrophic factor (gdnf) morphants in zebrafish larvae by cerebroventricular microinjection of vivo morpholino.

Dopaminergic neurons in the brain are an important source of dopamine, which is a crucial neurotransmitter for wellbeing, memory, reward, and motor control. Deficiency of dopamine due to advanced age and accumulative dopaminergic neuron defects can lead to movement disorders such as Parkinson's disease. Glial cell-derived neurotrophic factor (GDNF) is one of many factors involved in dopaminergic neuron development and/or survival. However, other endogenous GDNF functions in the brain await further investigation. Zebrafish is a well-established genetic model for neurodevelopment and neurodegeneration studies. Importantly, zebrafish shares approximately 70% functional orthologs with human genes including GDNF. To gain a better understanding on the precise functional role of gdnf in dopaminergic neurons, our laboratory devised a targeted knockdown of gdnf in the zebrafish larval brain using vivo morpholino. Here, detailed protocols on the generation of gdnf morphants using vivo morpholino are outlined. This method can be applied for targeting of genes in the brain to determine specific spatiotemporal gene function in situ.

Aneuploidy during development in facultative parthenogenetic Drosophila

From concatenated chromosomes to polyploidization, large-scale genome changes are known to occur in parthenogenetic animals. Here, we report mosaic aneuploidy in larval brains of facultatively parthenogenetic Drosophila. We identified a background of aneuploidy in D. mercatorum strains and found increased levels of aneuploidy in the larval brain tissue of animals arising parthenogenetically versus those arising from sexual reproduction. There is also intra-individual variation in germline-derived aneuploidy within the same strain. To determine if this is a general feature of facultative parthenogenesis in drosophilids, we compared sexually reproduced and parthenogenetic offspring from an engineered facultative parthenogenetic strain of D. melanogaster. In addition to germline-derived aneuploidy, this revealed somatic aneuploidy that increased by up to fourfold in parthenogens compared to sexually reproduced offspring. Therefore, the genetic combination identified in D. mercatorum that causes facultative parthenogenesis in D. melanogaster results in aneuploidy, which indicates that the loss of mitotic control resulting in parthenogenesis causes subsequent genome variation within the parthenogenetic offspring. Our findings challenge the assumption that parthenogenetic offspring are near genetic replicas of their mothers.

4-Methylbenzylidene camphor induced neurobehavioral toxicity in zebrafish (Danio rerio) embryos

4-Methylbenzylidene camphor (4-MBC) is a widely used organic UV filter in personal care products. Extensive use of 4-MBC and its frequent detection in aquatic ecosystems defile the biota with muscular and neuronal impairments. This study investigates the neurobehavioral toxicity of 4-MBC using Danio rerio as a model organism. Embryos were exposed semi-statically to 4-MBC at 5, 50, and 500 μg/L concentrations for 10-day post fertilization (dpf). Embryos exhibited a significant thigmotaxis and decreased startle touch response with altered expression of nervous system mRNA transcripts on 5 & 10 dpf. Compared to the sham-exposed group, 4-MBC treated larvae exhibited changes in the expression of shha, ngn1, mbp, elavl3, α1-tubulin, syn2a, and gap43 genes. Since ngn1 induction is mediated by shh signaling during sensory neuron specification, the elevated protein expression of NGN1 indicates 4-MBC interference in the sonic hedgehog signaling pathway. This leads to sensory neuron impairment and function such as ‘sense’ as evident from reduced touch response. In addition, larval brain histology with a reduced number of cells in the Purkinje layer emblazing the defunct motor coordination. Predictive toxicity study also showed a higher affinity of 4-MBC to modeled Shh protein. Thus, the findings of the present work highlighted that 4-MBC is potential to induce developmental neurotoxicity at both behavioral and molecular functional perspectives, and developing D. rerio larvae could be considered as a suitable alternate animal model to assess the neurological dysfunction of organic UV filters.

Single-cell transcriptomes provide insights into expansion of glial cells in Bombyx mori.

The diversity of cell types in the brain and how these change during different developmental stages, remains largely unknown. The life cycle of insects is short and goes through 4 distinct stages including embryonic, larval, pupal, and adult stages. During postembryonic life, the larval brain transforms into a mature adult version after metamorphosis. The silkworm, Bombyx mori, is a lepidopteran model insect. Here, we characterized the brain cell repertoire of larval and adult B. mori by obtaining 50708 single-cell transcriptomes. Seventeen and 12 cell clusters from larval and adult brains were assigned based on marker genes, respectively. Identified cell types include Kenyon cells, optic lobe cells, monoaminergic neurons, surface glia, and astrocyte glia. We further assessed the cell type compositions of larval and adult brains. We found that the transition from larva to adult resulted in great expansion of glial cells. The glial cell accounted for 49.8% of adult midbrain cells. Compared to flies and ants, the mushroom body kenyon cell is insufficient in B. mori, which accounts for 5.4% and 3.6% in larval and adult brains, respectively. Analysis of neuropeptide expression showed that the abundance and specificity of expression varied among individual neuropeptides. Intriguingly, we found that ion transport peptide was specifically expressed in glial cells of larval and adult brains. The cell atlas dataset provides an important resource to explore cell diversity, neural circuits and genetic profiles.

Imaging the radioprotective effect of amifostine in the developing brain using an apoptosis-reporting transgenic zebrafish

Purpose Normal tissue radioprotectants alleviate radiation-induced damages and preserve critical organ functions. Investigating their efficacy in vivo remains challenging, especially in enclosed organs like the brain. An animal model that enables direct visualization of radiation-induced apoptosis while possessing the structural complexity of a vertebrate brain facilitates these studies in a precise and effective manner. Materials and methods We employed a secA5 transgenic zebrafish expressing secreted Annexin V fused with a yellow fluorescent protein to visualize radiation-induced apoptosis in vivo. We developed a semi-automated imaging method for standardized acquisition of apoptosis signals in batches of zebrafish larvae. Using these approaches, we studied the protective effect of amifostine (WR-2721) in the irradiated zebrafish larval brain. Results Upon 2 Gy total-body 137Cs irradiation, increased apoptosis could be visualized at high resolution in the secA5 brain at 2, 24, and 48 hour post irradiation (hpi). Amifostine treatment (4 mM) during irradiation reduced apoptosis significantly at 24 hpi and preserved Wnt active cells in the larval brain. When the 2 Gy irradiation was delivered in combination with cisplatin treatment (0.1 mM), the radioprotective effect of amifostine was also observed. Conclusions Our study reveals the radioprotective effect of amifostine in the developing zebrafish larval brain, and highlights the utility of secA5 transgenic zebrafish as a novel system for investigating normal tissue radioprotectants in vivo.